Contents lists available at ScienceDirect Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488 Francesco Marchettia,⁎, Marilyn Aardemab, Carol Beeversc, Jan van Benthemd, George R. Douglasa, Roger Godschalke, Carole L. Yauka, Robert Youngf, Andrew Williamsa a Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON, Canada bMarilyn Aardema Consulting, LLC USA c Exponent International Limited, Harrogate, United Kingdom dNational Institute of Public Health and the Environment, Bilthoven, The Netherlands e Department of Pharmagology and Toxicology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, The Netherlands fMilliporeSigma, Bioreliance® Testing Services, Rockville, MD, USA A R T I C L E I N F O Keywords: Mutations Germ cells Transgene Test guideline Rodent A B S T R A C T The Organisation for Economic Co-operation and Development Test Guideline (TG) 488 for the transgenic rodent (TGR) mutation assay recommends two sampling times for assessing germ cell mutagenicity following the re- quired 28-day exposure period: 28+ > 49 days for mouse sperm and 28+ >70 days for rat sperm from the cauda epididymis, or three days (i.e., 28+3d) for germ cells from seminiferous tubules (hereafter, tubule germ cells) plus caudal sperm for mouse and rat. Although the latter protocol is commonly used for mutagenicity testing in somatic tissues, it has several shortcomings for germ cell testing because it provides limited exposure of the proliferating phase of spermatogenesis when mutations are fixed in the transgene. Indeed, analysis of sperm at 28+ 3d has generated negative results with established germ cell mutagens, while the analysis of tubule germ cells has generated both positive and either negative or equivocal results. The Germ Cell workgroup of the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute modelled mouse and rat spermatogenesis to better define the exposure history of the cell population collected from seminiferous tubules. The modelling showed that mouse tubule germ cells at 28+3d receive, as a whole, 42% of the total exposure during the proliferating phase. This percentage increases to 99% at 28+ 28d and reaches 100% at 28+30d. In the rat, these percentages are 22% and 80% at 28+3d and 28+28d, reaching 100% at 28+44d. These results show that analysis of tubule germ cells at 28+ 28d may be an effective protocol for assessing germ cell mutagenicity in mice and rats using TG 488. Since TG 488 recommends the 28+ 28d protocol for slow dividing somatic tissues, this appears to be a better compromise than 28+ 3d when slow dividing somatic tissues or germ cells are the critical tissues of interest. 1. Introduction The Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 488 entitled “Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays” [1] provides recommendations for mutagenicity testing in somatic tissues and germ cells using trans- genic rodent (TGR) models. These TGR models, which include mouse and rat, allow the detection of mutations induced in vivo using a bac- terial in vitro assay with a variety of reporter genes such as lacZ, lacI and cII [2,3]. There is international consensus that the protocol for somatic tissue testing works well and over 220 chemicals have been tested for mutagenicity in somatic tissues using TGR models [3]. Only 45 of these chemicals have been tested for germ cell effects and further con- sideration is needed to improve the ability to assess mutagenic effects in germ cells using TGR models [4]. The recommended protocol for mutagenicity assessment in TG 488 requires 28 daily exposures followed by tissue sampling three days after the last exposure (i.e., 28+ 3d) for somatic tissues. TG 488 further states that collection 28 days after the last exposure (i.e., 28+28d) should be considered when slow dividing tissues, such as the liver, are of particular interest. TG 488 also recommends two protocols specifi- cally for germ cell testing: analysis of sperm from the cauda epididymis at> 49 days (28+ 49d) post-exposure for the mouse and>70 days for the rat (28+70d); or analysis of germ cells from seminiferous tubules https://doi.org/10.1016/j.mrgentox.2018.05.020 Received 25 April 2018; Received in revised form 28 May 2018; Accepted 28 May 2018 ⁎ Corresponding author at: Environmental Health Science and Research Bureau, Health Canada, 50 Colombine Driveway, Ottawa, ON, K1A 0K9, Canada. E-mail address: francesco.marchetti@canada.ca (F. Marchetti). Mutat Res Gen Tox En 832–833 (2018) 19–28 Available online 01 June 2018 1383-5718/ Crown Copyright © 2018 Published by Elsevier B.V. All rights reserved. T i An update to this article is included at the end http://www.sciencedirect.com/science/journal/13835718 https://www.elsevier.com/locate/gentox https://doi.org/10.1016/j.mrgentox.2018.05.020 https://doi.org/10.1016/j.mrgentox.2018.05.020 mailto:francesco.marchetti@canada.ca https://doi.org/10.1016/j.mrgentox.2018.05.020 http://crossmark.crossref.org/dialog/?doi=10.1016/j.mrgentox.2018.05.020&domain=pdf (hereafter, tubule germ cells) and caudal sperm at 28+3d for both mouse and rat. The analysis of mouse sperm at +49d and rat sperm at +70d allows the assessment of the effects of exposing spermatogonial stem cells and takes into account differences in the duration of sper- matogenesis between mouse and rat [5]. Although this approach is considered the gold standard for determining whether an exposure af- fects spermatogonial stem cells, it requires an additional set of animals than those used for somatic tissue testing. The 28+3d protocol allows integration of germ cell and somatic tissue testing, providing a direct comparison of the response in the two tissues and minimizing the number of animals needed for testing. However, the 28+ 3d protocol is suboptimal for germ cells, because it does not adequately evaluate cells exposed during the proliferating phase of spermatogenesis. Specificially, sperm collected from the cauda at 28+ 3d represent a population exposed mostly during the latter part of spermatogenesis that lacks DNA replication and cell proliferation and undergoes progressive loss of DNA repair capacity [6]. These processes are necessary for fixing mutations in the reporter genes and analyses of caudal sperm at this time point fail to detect established germ cell mutagens, such as benzo(a)pyrene (BaP) [7] and, more importantly, N- ethyl-N-nitrosourea (ENU; O’Brien et al., in preparation), the proto- typical germ cell mutagen. Tubule germ cells collected at 28+3d comprise a mixed population of developing germ cells that is enriched in postmeiotic cells (i.e., where there is little possibility of fixing mu- tations in the transgene). Data with six established germ cell mutagens (following with acute dosing of 5 days or less) show significant in- creases in mutant frequencies only after collecting mouse tubule germ cells at least a week after the end of the exposure [4]. Furthermore, a TG 488-compliant study with BaP produced a non-statistically sig- nificant increase in mutant frequencies in tubule germ cells at 28+3d while significant increases were detected at later time points [7]. Thus, false negative results may be obtained with this cell population. Assessment of germ cell mutagenicity is most often conducted in tubule germ cells sampled at 28+3d when mutagenicity in somatic tissues is normally evaluated. However, there are very few chemicals that have been assessed for germ cell mutagenicity using OECD-com- pliant regimens. An accurate characterization of the composition of the tubule germ cell population and the exposure history of each cell type is critical to properly evaluate the suitability of the 28+ 3d protocol for detecting an increase in mutant frequencies in germ cells using TGR models. Toward this objective, the Germ Cell workgroup of Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC) used current knowledge to generate a model of both mouse and rat spermatogenesis to guide genotoxicity testing in germ cells using TG 488. The timing of spermatogenesis is well-established in several mam- malian species and is characterized by three distinct phases: mitotic, meiotic, and postmeiotic [5]. Spermatogonia represent the mitotic phase of spermatogenesis and include: spermatogonial stem cells, pro- liferating spermatogonia, and differentiating spermatogonia. Pro- liferating spermatogonia include A pair (Apr) and A aligned (Aal), while differentiating spermatogonia includes A1, A2, A3, A4, Inter- mediate (In), and B spermatogonia [8,9]. Overall, spermatogonia re- present ∼2% of the germ cell population in the rodent testis. In both mouse and rat, there are ∼10 mitotic divisions that occur over ∼2 weeks before differentiating spermatogonia commit to meiosis [8]. Of critical importance to the detection of mutations in germ cells with TGR models, the mitotic phase of spermatogenesis represents the only phase where there is active DNA replication and cell proliferation. The process of meiosis takes approximately 2 and 3 weeks in the mouse [10] and the rat [11], respectively. More than 50% of meiosis is represented by the pachytene phase, during which recombination between homologous chromosomes occurs. It is estimated that a mouse testis contains about 25 million spermatocytes [12], while there are more than 79 million in the rat [13]. Finally, completion of the two meiotic divisions creates haploid spermatids that, during the postmeiotic phase, undergo major morphological changes involving the replacements of somatic histones with protamines [14]. Round (RD), elongating (EL), and elongated (ED) spermatids are recognized in the haploid population, with RD sper- matids representing the longest period of this phase. After about 2 weeks in the mouse, or 3 weeks in the rat, spermatids leave the testis and complete sperm maturation while transiting through the epidi- dymis, ultimately accumulating in the cauda prior to ejaculation. Overall, the duration of spermatogenesis from the beginning of meiosis to accumulation of sperm in the cauda takes about 35 and 50 days in the mouse [10] and rat [11], respectively. The strictly controlled progression of germ cell development during spermatogenesis has been critical for germ cell mutagenesis studies that have used the timing between exposure to genotoxic agents and col- lection of sperm (or mating with untreated females) to characterize the phases of spermatogenesis most sensitive to a specific exposure [15,16]. Because of the progressive loss of DNA repair, exposures occurring during the postmeiotic phase tend to result in the highest increase in embryo dominant lethality, while this effect largely disappears when the exposure targets spermatogonia [16]. Similarly, results of the spe- cific locus test (SLT) show that the majority of the genotoxic agents produce maximal mutation induction with postmeiotic exposures, al- though significant increases in SLT mutations are also induced in spermatogonial stem cells by select agents [17]. Post-meiotic effects are thought to arise primarily after fertilization by heavily damaged sperm, with mutation fixation during the first embryonic divisions. Once ex- posed postmeiotic cells are replaced by newly produced germ cells there is no evidence of the exposure [15,16]. However, mutations oc- curring in stem cell populations are expected to persist throughout the lifetime of the exposed individual and produce mutated sperm at each cycle of spermatogenesis. The latter forms the basis for the re- commendation in TG 488 to analyze mouse sperm at> 49 days and rat sperm at> 70 days. Knowledge of the timing of spermatogenesis can be used to model the progression of each spermatogenic cell type during the 28+3d study design to evaluate its effectiveness for assessing germ cell muta- genicity. At the same time, this allows the characterization of the ex- posure history of each cell type during the mitotic phase when muta- tions can be induced in the transgenes. In this analysis, we generated simulated mouse and rat testes to model the cyclic waves of sperma- togenesis over the 28 days of exposure and four different sampling times. This allowed us to characterize the exposure history of the po- pulation of tubule germ cells and demonstrate that collection of tubule germ cells in a 28+ 28d protocol, the alternative design in TG 488 for somatic tissue testing, provides better exposure coverage of the mitotic phase of spermatogenesis than the 28+3d protocol, for both mouse and rat. 2. Methods To model the progression of spermatogenesis during the 28 days of exposure recommended in TG 488, we first created simulated mouse and rat testes using the equilibrium population model [18]. This model considers two main variables: the number of each germ cell type pre- sent during spermatogenesis and the duration (in days) of the differ- entiation phase of each cell type. Because the number of cell divisions that Apr and Aal can undergo before becoming A1 spermatogonia is variable within and between different cycles of spermatogenesis [8], they were not modelled here and were combined with stem cells. Furthermore, we assumed that there was no spontaneous nor chemi- cally-induced germ cell apoptosis or toxicity-associated cell cycle delay. Thus, we started simulating the rodent testis by considering one A1 spermatogonia and doubling the number at each cell division that oc- curs during spermatogenesis (i.e, the six mitotic divisions of differ- entiating spermatogonia and the two meiotic divisions). For the dura- tion of the differentiation phases, we used data from de Rooij [8] for mouse spermatogonia, and from Oakberg [10] for mouse meiosis and F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 20 post-meiosis. For the rat, the differentiation times were taken from Huckins [19,20] for spermatogonia, and from Clermont et al. [11] for meiosis and postmeiosis. The composition of the simulated mouse and rat testes are shown in Table 1. 2.1. Simulation of the progression of spermatogenesis using the R statistical environment To estimate the number of days of exposure during the mitotic phase that each spermatogenic cell type received, we simulated the progression of spermatogenesis using the R statistical environment [21]. This was achieved by estimating the distributions of the number of days of exposure for the 16 spermatogenic cell types in the testis (A1, A2, A3, A4, In and B spermatogonia; Pr, L, Z, P, Dp, Dk, and MII spermatocytes; R, EL, and ED spermatids; Supplementary Fig. 1). The data in Table 1 were used as the starting point to estimate these dis- tributions. Four protocols were simulated: 28+3 d, the recommended protocol in TG 488 for somatic tissues; 28+ 28 d, the alternative pro- tocol in TG 488 for slowly dividing somatic tissues; 28+14 d, an in- termediate time point between the two protocols in TG 488; and 28+49 d for the mouse (28+ 70 d for the rat), the germ cell specific protocol in TG 488. For each spermatogenic cell type, a p dimensional square matrix was generated (a graphical representation of the matrix is shown in Supplementary Fig. 1). In this matrix, p represents the total number of days, including both exposure and sampling time. For example, p is 31 for the 28+ 3d protocol. In each matrix, the column and row dimen- sions represent days. The value in the [1,1] position of the matrix re- presents the starting value for that spermatogenic cell type and de- termines all other values in the matrix based on the developmental days in Table 1. To complete each matrix, the column entries for the first row were calculated as follows. The value in position [1,2] was defined as the value at [1, 1] plus 1 day. This is repeated for the p columns where the value in position [1,p] is the value at [1,p-1] plus 1. The rows of the matrix are generated by subtracting 1 from the entries in the previous row. That is, the values for row 2 were defined as the values in row 1 subtracted by 1. This was repeated for all q rows where the values in row q were equaled to the value in row q-1 minus 1. This was done for all 16 cell types of spermatogenesis. For these matrices, the pth column represents the spermatogenic cell type reached at the time when the cells are sampled. The first 28 columns of the matrix represent the exposure history of the population of cells. Using these columns in conjugation with values in column p, the distributions for the number of days of exposure were estimated using kernel density estimation [22]. The total number of days of exposure as stem cells, spermatogonia, spermatocytes, and spermatids were also compiled as well as for each exposure protocol. 2.2. Expected number of days of exposure during the mitotic phase The expected number of days of exposure was estimated as the weighted average of the statistical expectation [23] for the number of days of exposure during the mitotic phase for all spermatogenic cell types. Statistical expectation is written as a weighted average of the statistical expectations for each cell type as: ∫∑ ∑ ∑ = ≈ = ∞ > Expected Number of Days Exposed w xf x dx w P X x ( ) ˆ ˆ ( ). s s s s s x o s 0 Here, w represents the weight for each of the cell types s, and fs re- presents the probability density function for that cell type. The weights were determined based on the number of cells at each sub-stage esti- mated according to the equilibrium population model [18]. In the equation above, =P̂ X x( )s is the relative frequency of cell type s being exposed x days. An overall average, and the averages of the four cell types of spermatogenesis (stem cells, spermatogonia, spermatocytes, and spermatids), were also estimated. The R script, and its codes, used to model spermatogenesis are found in the Supplementary Materials. 3. Results 3.1. Generation of simulated mouse and rat testes We applied the equilibrium population model [18] to simulate Table 1 Equilibrium population models for mouse and rat spermatogenesis. Mouse Rat Cell type Starting number Duration of differentiation phase (days)a Cell x Duration % of population Total (%)b Duration of differentiation phase (days)c Cell x Duration % of population Total (%)b Spermatogonia 2.00 1.50 A1 1 2.4 2.4 0.052 2.3 2.3 0.031 A2 2 1.25 2.5 0.054 1.7 3.4 0.045 A2 4 1.25 5 0.109 1.8 7.2 0.096 A4 8 1.25 10 0.217 1.7 13.6 0.181 Intermediate (In) 16 1.5 24 0.521 1.8 28.8 0.383 B 32 1.5 48 1.043 1.8 57.6 0.765 Spermatocytes 22.94 17.22 Preleptotene (Pr) 64 2.5 160 3.475 3.5 224 2.976 Leptotene (L) 64 2.0 128 2.780 0.9 57.6 0.765 Zygotene (Z) 64 2.2 140.8 3.058 2.1 134.4 1.876 Pachytene (P) 64 7.7 492.8 10.704 11.25 720 9.565 Diplotene (Dp) 64 0.9 57.6 1.251 0.7 44.8 0.595 Diakinesis (Dk) 64 0.4 25.6 0.556 0.6 38.4 0.510 Secondary (MII) 128 0.4 51.2 1.112 0.6 76.8 1.020 Spermatids 75.07 81.28 Round (R) 256 6.4 1638.4 35.587 7.5 1920 25.507 Elongating (EL) 256 2.2 563.2 12.233 4.4 1162.4 14.964 Elongated (ED) 256 4.9 1254.4 27.246 12 3072 40.811 a Data from Oakberg (1956) for spermatocytes and spermatids, and from de Rooji (2001) for spermatogonia. b % of spermatogonia, spermatocytes, and spermatids in the simulated testis. c Data from Clermont at al (1959) for spermatocytes and spermatids, and from Huckins (1971a;b) for spermatogonia. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 21 mouse and rat spermatogenesis as described in Methods. This approach generated a simulated mouse testis that contains approximately 2% spermatogonia, 22.9% spermatocytes, and 75.1% spermatids (Table 1). These estimates are in line with those reported using a histological examination of the mouse testis [12]. Similarly, we generated a simu- lated rat testis that contains approximately 1.5% spermatogonia, 17.2% spermatocytes, and 81.3% spermatids (Table 1). We used these simulated mouse and rat testes to model the pro- gression of spermatogenesis over the 28 days of exposure and four different sampling times (i.e., +3d, +14d, +28, and +>49d) to estimate the totality of ‘effective exposure’ of the cell population col- lected from seminiferous tubules at each sampling time. Effective ex- posure refers to an exposure that occurred during the mitotic phase of spermatogenesis when damage to the DNA can be induced in a cell that undergoes DNA replication and cell division thereby fixing mutations in the transgene. Maximum effective exposure is 28 days. Table 2 shows the progression of the various germ cell types over the 28 days of ex- posure and three days of sampling times, as well as the number of days of effective exposure that each one received. For simplicity, only the fate of a cell that has just entered that differentiation phase on the first day of exposure is shown here. As indicated in Table 2, the progeny from a cell that was a mouse Pr spermatocyte, or later, on the first day of exposure has moved out of the testis by the time the seminiferous tubules are collected at 28+3 d; whereas, the progeny from mouse B spermatogonia and earlier are still recovered as spermatids at 28+3 d, and have received increasing days of effective exposure depending on whether they were A4, A3, A2, etc. on the first day of exposure. A si- milar exercise for the rat shows that the progeny from cells that were rat Dp spermatocytes, or later, on the first day of exposure have moved out of the testis by the time tubule germ cells are collected; whereas, the progeny from rat P spermatocytes and earlier are still recovered as spermatids at 28+3 d. Tables 3 and 4 show the results of modelling all cell types found at any given day in the testis, the cyclical waves of spermatogenesis that occur over the exposure period, and the various sampling times for the mouse and rat, respectively. These tables report the weighted average of days of effective exposure, the range of days of effective exposure within each cell type, and whether the exposure occurred during the stem cell phase or spermatogonia phase for the four protocols that were modelled. The data is presented separately for each of the major cell types collected from seminiferous tubules (i.e., spermatogonia, sper- matocytes, and spermatids). Note that the maximum number of days of exposure that a cell can receive while undergoing the differentiating phase of spermatogonia is ∼10 and 12 days for the mouse and rat, respectively. The optimum scenario for accurate assessment of germ cell mutagenicity is that all spermatogonia, spermatocytes and sper- matids collected from seminiferous tubules received the entire 28 days of exposure during the mitotic phase. 3.2. Modelling of mouse spermatogenesis As shown in Table 3, the sampling time does not change the ex- posure history of mouse spermatogonia, which represent 2% of the tubule germ cell population (Table 1), as they always receive 28 days of effective exposure as stem cells, except for some at 28 +3d that re- ceived an average of 4.41 days as differentiating spermatogonia. Even at 28+14d, all spermatogonia recovered from seminiferous tubules received 28 days of effective exposure as stem cells because this sam- pling time is longer than the time necessary for a cell to complete the differentiating spermatogonia phase (∼10 days). Therefore, all sper- matogonia collected at this time point were still stem cells on the last day of exposure. In contrast, the modelling shows that it is necessary to use at least the 28+ 28 d design to ensure that all mouse spermato- cytes, which represent 23% of the tubule germ cell population (Table 1), received 28 days of effective exposure. In fact, mouse sper- matocytes collected from seminiferous tubules at 28+3 d received an average of 12.3 days (range 5–24 days) of exposure as stem cells, and 9.1 days (range 4–10 days) as spermatogonia; while spermatocytes collected at 28+ 14 d received an average of 22.9 days (range 16–28 days) of exposure as stem cells, and 5.1 days (range 0–10 days) as spermatogonia. More relevant to the question of whether the analysis of cells from seminiferous tubules at 28+ 3 d can provide a suitable approach to detect mutations is the exposure history of mouse spermatids, which represent ∼75% of the cells collected from the tubules. As shown in Table 3, mouse spermatids collected at 28+3 d were exposed only for Table 2 Fate of germ cell types present in the mouse and rat testes on the first day of exposure when seminiferous tubules are collected at 28+ 3d. Mouse Rat Cell type Daysa Presentb Cell typec Days of exposured Daysa Presentb Cell typec Days of exposured Spermatogonia A1 38.25 Yes R spermatid 10 54.65 Yes R spermatid 11 A2 35.85 Yes EL spermatid 7 52.35 Yes R spermatid 9 A3 34.6 Yes EL spermatid 6 50.65 Yes R spermatid 7 A4 33.35 Yes ED spermatid 5 48.85 Yes R spermatid 5 Intermediate (In) 32.1 Yes ED spermatid 4 47.15 Yes EL spermatid 4 B 30.85 Yes ED spermatid 2 45.35 Yes EL spermatid 2 Spermatocytes Preleptotene (Pr) 29.6 No 43.55 Yes EL spermatid 0 Leptotene (L) 27.1 No 40.05 Yes ED spermatid 0 Zygotene (Z) 25.1 No 39.15 Yes ED spermatid 0 Pachytene (P) 22.9 No 37.05 Yes ED spermatid 0 Diplotene (Dp) 15.2 No 25.8 No Diakinesis (Dk) 14.3 No 25.1 No Secondary (MII) 13.9 No 24.5 No Spermatids Round (R) 13.5 No 23.9 No Elongating (EL) 7.1 No 16.4 No Elongated (ED) 4.9 No 12 No a Length of time (in days) it would take for this cell type to mature and move out of the testis, beginning on day one of an exposure. For simplicity, only one cell per cell type is modelled. For example, although the mouse pachytene phase lasts 7.7 days, only a cell that is on the first day of the pachytene phase is modelled here. b Presence of progeny cell type in the seminiferous tubules at 28+ 3? c Differentiation phase reached on sampling day. d Number of days of effective exposure during the mitotic phase. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 22 an average of 1.3 days (range 0–5 days) as stem cells and 7.2 days (range 1–10 days) as spermatogonia. Even with the 28+28 d design, some mouse spermatids did not receive 28 days of effective exposure (24.4 days as stem cells; 3.5 days as spermatogonia). Among the four protocols modelled, only the 28+ 49 d design ensures that all mouse spermatids collected from the seminiferous tubules received 28 days of effective exposure. Daily modelling of sampling times longer than +28 d indicated that complete exposure during the mouse mitotic phase is reached with the 28+30 d protocol. Overall, our modelling indicates that collection of tubule germ cells from the mouse testis results in the analysis of a cell population that has received as a whole 42.2%, 76.4%, 99.6% and 100% of the maximum effective exposure at 28+ 3d, 28+ 14d, 28+28d and 28+49 d, re- spectively (Table 3). At all four time points modelled, there are no mouse tubule germ cells that received zero days of effective exposure. 3.3. Modelling of rat spermatogenesis The data for the rat are shown in Table 4 and indicate that the re- sults for rat spermatogonia and spermatocytes are consistent with those in the mouse. In fact, except for some cells sampled at 28+ 3 d, rat spermatogonia collected from the seminiferous tubules always received 28 days of effective exposure as stem cells. Similarly, the modelling shows that the 28+28d design achieves almost 28 days of effective exposure for rat spermatocytes collected from seminiferous tubules. At 28+3 d, rat spermatocytes collected from the seminiferous tubules received an average of 8.3 days (range 0–23 days) of exposure as stem cells and 11.0 days (range 5–12 days) as spermatogonia; while sper- matocytes collected at 28+14 d received an average of 19.3 days (range 11–28 days) of exposure as stem cells, and 7.9 days (range 0–12 days) as spermatogonia. The major difference between the simulated mouse and rat sper- matogenesis models involves the exposure history of rat spermatids, which represent over 80% of the cell population collected from rat seminiferous tubules. As shown in Table 4, rat spermatids collected at 28+ 3d received no exposure as stem cells and only an average of 2.8 days (range 0–11 days) as spermatogonia. At 28+14 d, rat sper- matids received an average of 2.3 days (range 0–11 days) of exposure as stem cells and 7.2 days (range 0–12 days) as spermatogonia. Even the 28+ 28 d design does not ensure 28 days of effective exposure, with rat spermatids receiving an average of 11.1 days (range 1–25 days) of ex- posure as stem cells and 10.1 days (range 3–12 days) as spermatogonia. As it is for the mouse, among the four protocols modelled, only the 28+ 70d design ensures that all rat spermatids collected from the seminiferous tubules received 28 days of effective exposure. However, daily modelling of sampling times longer than 28 days indicated that complete exposure during the rat mitotic phase is reached with the 28+ 44d protocol. Our modelling indicates that collection of germ cells from the seminiferous tubules in the rat testis results in the analysis of a cell population that has received as a whole 21.6%, 45.9%, 80.3% and 100% of the maximum effective exposure at 28+ 3d, 28+ 14 d, 28+ 28 d and 28+ 70 d, respectively (Table 4). Unlike in the mouse, 46% and 7% of the rat tubule germ cells collected at 28+ 3 d and 28+ 14 d received zero days of effective exposure, respectively. 3.4. Distribution of the seminiferous tubule population according to the number of days of exposure during the mitotic phase The data in Tables 3 and 4 provide a summary of the exposure Table 3 Summary of effective exposure received by the cell types collected from seminiferous tubules of the mouse testis at various time points after the end of exposure. Experimental Effective days of exposureb designa Exposure as stem cell Exposure as spermatogonia Total effective exposure Weighted Daysc Rangec Daysc Rangec Days %d total dayse 28+3 Spermatogonia 23.59 21–28 4.41 0–7 28 100 0.56 Spermatocytes 12.31 5–24 9.10 4–10 21.41 76.5 4.91 Spermatids 1.27 0–5 7.19 1–10 8.46 30.2 6.35 Total: 11.82 days (42.2%) 28+14 Spermatogonia 28 28–28 0 0–0 28 100 0.56 Spermatocytes 22.90 16–28 5.03 0–10 27.93 99.8 6.40 Spermatids 9.95 3–16 9.26 9–10 19.21 68.6 14.42 Total: 21.39 days (76.4%) 28+28 Spermatogonia 28 28–28 0 0–0 28 100 0.56 Spermatocytes 28 28–28 0 0–0 28 100 6.42 Spermatids 24.43 17–28 3.46 0–10 27.89 99.6 20.90 Total: 27.88 days (99.6%) 28+49 Spermatogonia 28 28–28 0 0–0 28 100 0.56 Spermatocytes 28 28–28 0 0–0 28 100 6.42 Spermatids 28 28–28 0 0–0 28 100 21.02 Total: 28 days (100%)f a Days of exposure plus fixation (sampling) time. Indicated are the major cell types that can be isolated from the seminiferous tubules. b Effective exposure is defined as the number of days of exposure that occurred during the mitotic phase of spermatogenesis. Maximum effective exposure is 28 days. c Numbers report the days of exposure that each cell type collected from the seminiferous tubules experienced as stem cell or as spermatogonia. The range shows the minimum and maximum number of days of exposure within each cell type population. d Percent of maximum effective exposure by cell type. e Total number of days multiplied by the proportion of each cell type that can be collected from seminiferous tubules (i.e., 2% for spermatogonia, 22.9% for spermatocytes and 75.1% for spermatids). For each sampling time, the total number of days (and its percentage with respect to the maximum effective exposure) is shown. f First day of complete effective exposure: 28+ 30d. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 23 history of the cell population collected from the seminiferous tubules at the four modelled protocols. It is also useful to analyze the distribution of the cell population based on the days of effective exposure. This analysis is shown in Fig. 1 (data in Supplementary Tables S1–S4) and 2 (data in Supplementary tables S5–S8) for the mouse and rat, respec- tively. For the mouse, Fig. 1D shows the optimal scenario at 28+49d where all cells collected from seminiferous tubules received 28 days of effective exposure. By contrast, Fig. 1A shows that only ∼6% (4% re- presented by spermatocytes and 2% represented by spermatogonia) of the population collected from seminiferous tubules at 28+ 3d received 28 days of effective exposure (Supplementary Table S1). At the same time, all cells collected from seminiferous tubules at this sampling time received at least one day of exposure during the mitotic phase. In fact, the mouse spermatid population is composed of subpopulations that received increasing days of effective exposure (range 1–15 days) each representing between ∼3.2% and 5.6% of the entire population of germ cells collected at this time point (Supplementary Table S1). The mouse spermatocyte population at 28+3d is composed of sub- populations that received between 14 and 28 days of effective exposure. Finally, all mouse spermatogonia collected at 28+ 3d received the 28 days of effective exposure. At 28+ 14d (Fig. 1B), 21.4% of the cell population collected from the mouse seminiferous tubules received 28 days of effective exposure (Supplementary Table S2) while sperma- tids received between 12 and 25 days of effective exposure. Finally, the data in Fig. 1C (Supplementary Tables S3) show that all mouse germ cells collected from seminiferous tubules at 28+ 28 d received 28 days of effective exposure except of two subpopulations of mouse sperma- tids, each representing ∼3.6% and 5.7% of the total population, that received 26 and 27 days of effective exposure, respectively (Supplementary Table S3). Overall, Figs. 1A–D (and Supplementary Tables S1–S4) show that the percentages of the cell population that received 28 days of effective exposure are 6.1%, 21.4%, 90.7% and 100% at 28+3d, 28+14 d, 28+28 d and 28+49 d, respectively. As shown in Fig. 2A–D (Supplementary Tables S5–S8), collection of germ cells from rat seminiferous tubules at 28+ 3 d provides even less effective exposure than in the mouse due to the longer duration of spermatogenesis. Only ∼4% of the population collected from the seminiferous tubules at this time point received 28 days of effective exposure (Fig. 2A), which is not much different from the 6% observed in the mouse. However, in the rat, almost 46% (versus none in the mouse) of the cells collected at 28+3d (represented by the entire ED spermatid subpopulation and a small fraction of the EL subpopulation) did not receive exposure during the mitotic phase (Supplementary Table S5). The rat spermatocyte subpopulation is composed of sub- populations that received between 11 and 28 days of effective exposure, while all rat spermatogonia collected received the 28 days of effective exposure. At 28+14 d, only 13.3% of the cell population collected from the rat seminiferous tubules received 28 days of effective exposure (Fig. 2B and Supplementary Table S6). Finally, the data in Fig. 2C (Supplementary Table S7) show that at 28+ 28 d, 47.0% (90.7% in the mouse) of rat germ cells collected from seminiferous tubules received 28 days of effective exposure and that spermatids receiving between 12 and 27 days of effective exposure represented the other 53%. Overall, Figs. 2A–D (and Supplementary Tables S5–S8) show that the percen- tages of the cell population that received 28 days of effective exposure are 4.0%, 13.4%, 47.0% and 100% at 28+3d, 28+14 d, 28+28 d and 28+49 d, respectively. Thus, the alternative design for somatic tissues in TG 488 (i.e., 28+ 28 d) achieves an excellent exposure of the mitotic phase of Table 4 Summary of effective exposure received by the cell types collected from seminiferous tubules of the rat testis at various time points after the end of exposure. Experimental Effective days of exposureb designa Exposure as stem cell Exposure as spermatogonia Total effective exposure Weighted Daysc Rangec Daysc Rangec Days %d total dayse 28+3 Spermatogonia 21.89 19–28 6.11 0–9 28 100 0.42 Spermatocytes 8.43 0–23 10.99 5–12 19.44 69.4 3.35 Spermatids 0 0–0 2.79 0–11 2.79 10.0 2.27 Total: 6.04 days (21.6%) 28+14 Spermatogonia 28 28–28 0 0–0 28 100 0.42 Spermatocytes 19.31 11–28 7.92 0–12 27.23 97.3 4.69 Spermatids 2.28 0–11 7.23 0–12 9.51 34.0 7.73 Total: 12.84 days (45.9%) 28+28 Spermatogonia 28 28–28 0 0–0 28 100 0.42 Spermatocytes 27.90 25–28 0.10 0–3 28 100 4.82 Spermatids 11.11 1–25 10.09 3–12 21.20 75.7 17.23 Total: 22.47 days (80.3%) 28+70 Spermatogonia 28 28–28 0 0–0 28 100 0.42 Spermatocytes 28 28–28 0 0–0 28 100 4.82 Spermatids 28 28–28 0 0–0 28 100 22.72 Total: 28 days (100%)f a Days of exposure plus fixation (sampling) time. Indicated are the various cell types that can be isolated from the seminiferous tubules. b Effective exposure is defined as the number of days of exposure that occurred during the mitotic phase of spermatogenesis. Maximum effective exposure is 28 days. c Numbers report the days of exposure that each cell type collected from the seminiferous tubules experienced as stem cell or as spermatogonia. The range shows the minimum and maximum number of days of exposure within each cell type population. d Percent of maximum effective exposure by cell type. e Total number of days multiplied by the proportion of each cell type that can be collected from seminiferous tubules (i.e., 1.5% for spermatogonia, 17.2% for spermatocytes and 81.3% for spermatids). For each sampling time the total number of days (and its percentage with respect to the maximum effective exposure) is shown. f First day of complete effective exposure: 28+ 44 d. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 24 spermatogenesis for the mouse and greatly improves exposure during the proliferating phase for rat germ cells collected from the semi- niferous tubules. 4. Discussion We developed simulated mouse and rat testes to model the pro- gression of spermatogenesis during the course of the recommended protocols in TG 488. The models were used to determine the exposure history of the populations of cells that are collected from seminiferous tubules to inform on the most appropriate sampling time for in- vestigating the induction of mutations in male germ cells using TGR models. Our results show that sampling of tubule germ cells at 28+ 3 d captures a cell population that has received only ∼42% and ∼21% in the mouse and rat, respectively, of the maximum effective exposure. At the same time, 100% and 54% of tubule germ cells in mouse and rat, respectively, received at least one day of effective exposure. The per- centages of cells receiving 28 days of effective exposure increase to ∼99% and 80% (mouse and rat, respectively) when the alternative design in TG 488 (i.e., 28+28 d) recommended for assessing muta- tions in slowly dividing somatic tissues is used. These results demon- strate that the 28+28 d protocol provides more complete exposure of the proliferating phase of spermatogenesis than the 28+3 d protocol and represents a better approach for properly characterizing the mu- tagenicity of chemicals in tubule germ cells using TG 488. Any assay that investigates the genotoxicity of chemicals has an optimal experimental design, based on knowledge of cell proliferation and DNA damage/repair, that maximizes the chance of detecting an effect. Since it is not practical to design an optimal experiment for every type of cell and type of chemical, all OECD test guidelines for geno- toxicity testing recommend appropriate sampling times to allow suffi- cient time for the type of genotoxic damage that is measured to be expressed [24]. The recommended sampling times for OECD geno- toxicity tests are therefore a combination of background knowledge of cell proliferation/DNA damage/repair, and are supported by a broad range of chemicals that have been evaluated. This was the case for selecting the 28+3 d protocol as the default sampling time for testing somatic tissues in TG 488 and was based on the understanding that the +3d sampling time, while an adequate compromise for the purpose of safety assessment for all somatic tissues, may not be optimal for some tissues (e.g., slow proliferative tissues such as liver). The +28d sam- pling time may not be optimal for fast dividing tissues (e.g., bone marrow, small intestine, etc). However, multiple somatic tissues can be analyzed at a given time point so that the proper call on the mutagenic potential of the chemical being tested can rely on effects measured in both slow and fast dividing tissues. Determination of germ cell mutagenicity can be conducted only using testis and it is paramount that the time point selected for analysis has the optimal sensitivity to detect an effect. The recommendation in TG 488 to conduct germ cell testing in tubule germ cells at 28+3 d was dictated by an effort to harmonize mutagenicity testing between so- matic tissues and germ cells, even though it was recognized that the optimal tissue and sampling time for germ cell mutagenicity testing required the analysis of caudal sperm at much longer sampling times [1]. The selection of an appropriate sampling time(s) to collect germ cells in both mice and rats is particularly complex and requires critical Fig. 1. Distribution of the cell population collected from mouse seminiferous tubules at different sampling times according to the number of days of exposure during the mitotic phase (effective exposure on x-axis). (A) 28+ 3d; (B) 28+ 14d; (C) 28+ 28d; and (D) 28+ 49d. Spermatogonia, spermatocyte, and spermatid po- pulations are shown in yellow, orange, and red, respectively. Note that the Y axes are on a different scale. The 28+49d design (D) represents the optimum scenario when all cells recovered from the seminiferous tubules have received 28 days of effective exposure. At 28+3d (A), only 6% of the cells have received 28 days of effective exposure and the spermatid population is composed by subpopulations that have received between 1 and 14 of relevant exposure. At 28+ 14d (B), ∼22% of the cells recovered from the seminiferous tubules have received 28 days of effective exposure and still no spermatids are included in this subpopulation. At 28+28d (C), ∼90% of the cells recovered from the seminiferous tubules have received 28 days of effective exposure and there are two spermatid subpopulations that have received 26 and 27 days of effective exposure. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 25 considerations of the long time that is needed to generate mature sperm from stem cells, and the changing physiological and cellular char- acteristics that male germ cells undergo as they progress through spermatogenesis [25]. Because of these differences in germ cell char- acteristics and known vulnerabilities to toxicants during spermatogen- esis [15,16], it is critical to consider the cell population that is treated, as well as the population that is sampled when designing and inter- preting the results of a germ cell mutagenicity study. For the induction of mutations in the transgene using TGR models, DNA replication and cell proliferation are essential [26]. In our model, we considered only an exposure occurring during the mitotic phase of spermatogenesis as being capable of inducing mutations that can be detected with TGR models. Although DNA synthesis is occurring in preleptonene spermatocytes [9], cell division does not take place for at least two weeks after its completion and we considered exposure during meiosis irrelevant in our modelling approach. This is supported by the results obtained in mouse sperm collected from the cauda within a 28+3 d protocol showing no induction of lacZ mutations when es- tablished germ cell mutagens such as ENU (O’Brien et al., in prepara- tion) and BaP [7] were used. With this experimental protocol, mouse sperm collected from the cauda at 28+3 d have received ∼10 daily exposures during meiosis (representing over 60% of the meiotic phase). Thus, it is unlikely that one day of exposure in Pr spermatocytes would be sufficient to increase the incidence of mutations. The remarkable conclusion is that on the first day of exposure only 2% of the germ cells present in the testis (i.e., stem cells and spermatogonia) are susceptible to the induction of mutations that can be detected with TGR models. Even more remarkable is the fact that 98% of the germ cell population present in the mouse testis (88% in the rat) on the first day of exposure is no longer recoverable at 28+3 d because they have completed the maturation process and moved out of the testis. Extension of the sam- pling time to +28d is therefore absolutely critical for optimally asses- sing mutagenicity in tubule germ cells because it allows the semi- niferous tubules to be populated by cells that experienced the majority of exposure during the proliferative phase of spermatogenesis. A critical variable for our modelling is the timing of spermatogen- esis, which has been established for many decades following the pio- neering work of Oakberg [10] for the mouse, and Clermont et al. [11] and Huckins [19,20] for the rat. Since then, our understanding of the mitotic phase of spermatogenesis has evolved and four types of A spermatogonia are now recognized [8,9] instead of the original three. However, knowledge of the timing of the meiotic and postmeiotic phases has remained virtually unchanged. The duration of these two phases determines the fraction of the tubule germ cell population that receive an effective exposure at 28+3d. This is clearly demonstrated in Table 2, which shows that there are 29.6 days and 43.5 days left for a cell that has just entered the Pr spermatocyte phase in the mouse and rat, respectively, to complete the developmental phase of spermato- genesis in the testis. The longer duration of the meiotic and post meiotic phases in the rat means that some of the cells that are collected at 28+ 3d have received no effective exposure, while this is not the case in the mouse. The results in Table 2 also serve as a confirmation of the findings from our modelling, which indicated that ∼45% of cells (versus 0% in the mouse) collected from the rat seminiferous tubules at 28+ 3d received no effective exposure (Figs. 1A and 2A). The equilibrium population model (Table 1) is a simplified Fig. 2. Distribution of the cell population collected from rat seminiferous tubules at different sampling times according to the number of days of exposure during the mitotic phase (effective exposure on x-axis). (A) 28+ 3d; (B) 28+ 14d; (C) 28+ 28d; and (D) 28+ 49d. Spermatogonia, spermatocyte, and spermatid populations are shown in yellow, orange, and red, respectively. Note that the Y axes are on a different scale. The 28+ 70d design (D) represents the optimum scenario when all cells recovered from the seminiferous tubules have received 28 days of effective exposure. At 28+ 3d (A), only 4% of the cells have received 28 days of effective exposure. More importantly, ∼45% of the population (composed entirely by spermatids) have received zero days of effective exposure. At 28+ 14d (B), ∼13% of the cells recovered from the seminiferous tubules have received 28 days of effective exposure and still no spermatids are included in this subpopulation. At 28+ 28d (C),∼45% of the cells recovered from the seminiferous tubules have received 28 days of effective exposure and there several spermatid subpopulations have received between 12 and 27 days of effective exposure. F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 26 approach to model spermatogenesis in vivo. Some level of germ cell degeneration is present even in the unexposed testis and the number of postmeiotic cells is always less than the number that would be expected if there was no cell loss during the replicative phase of spermatogenesis [27–29]. For example, Tegelenbosch and de Rooij [12] reported that there are ∼8500 mouse A1 spermatogonia and that the numbers (and fold increase with respect to the preceding cell type) of A2, A3, A4, In, and B spermatogonia are approximately: 13,100 (1.54 fold); 19,300 (1.47 fold); 30,600 (1.58 fold); 59,600 (1.94 fold); and 117,200 (1.90 fold), respectively. Modelling of spermatogenesis using these sperma- togonia numbers does not appreciably change the result that the germ cell population collected from mouse seminiferous tubules at 28+3d received less than 45% of the maximum effective exposure as a whole (Supplementary Table S9). In contrast, the population collected at 28+28d received almost 100% of the maximum effective exposure. Therefore, the conclusion that the 28+28d protocol provides a more complete exposure of the mitotic phase than the 28+3d protocol is not a consequence of the simplified approach that was used to create the simulated testis. Two important assumptions in our model were that: 1) all cell types are equally recoverable when collecting tubule germ cells; and 2) the exposure does not produce a significant induction of germ cell apoptosis or delays in the progression of spermatogenesis. This is clearly an oversimplification as the efficiency of recovering the various germ cell types progressively declines for spermatids, spermatocytes, and sper- matogonia, with the latter being the most difficult population to com- pletely isolate and recover from the tubules. Also, cytotoxic effects of the tested agents are more likely to affect spermatogonia and sperma- tocytes than spermatids [30–32], resulting in a further decline in the proportion of these two cell types in the population that is collected from the seminiferous tubules at 28+3d. Therefore, the proportions of spermatogonia and spermatocytes that can be collected from semi- niferous tubules in our current model represent the best-case scenario for detecting germ cell mutations. In real life experiments, uneven re- covery of spermatogonia, spermatocytes, and spermatids together with toxic effects predominantly in spermatogonia and spermatocytes, most likely reduce the proportion of tubule germ cells that received the majority of the effective exposure. This raises the issue that a potential increase in mutations in those cells that were exposed during the mi- totic phase may be masked by the presence of an excess of spermatids that received limited days of effective exposure. Indeed, there are several published examples where significant induction of mutations in tubule germ cells were observed at later sampling times but not at +3d [4]. Sampling at 28+28d would circumvent this issue because, as shown in Tables 3 and 4, the great majority of the cells collected at this time point, including spermatids, would have been exposed for longer times during the mitotic phase. The modelling results indicate that the 28+28d design, which is already permitted under TG 488, may provide a satisfactory compro- mise for integrating somatic tissue and germ cell testing using TG 488, especially when slow dividing somatic tissues or germ cells are the tissue of interest. Germ cells and somatic tissues have both similar and different factors that affect a cell’s ability to express a mutant pheno- type in the TGR assay that must be considered in any harmonized study design. In both tissue types, a cell must sustain DNA damage that is not repaired and be viable enough to replicate thereby allowing the fixation of a mutation in a daughter cell. In slow dividing somatic tissues, large fractions of cells, while exposed for 28 days, may not divide and would benefit from longer sampling times to express the mutation. Longer sampling times may allow fast dividing tissues to undergo clonal am- plification with adverse effects on quantitation of mutations. Thus, extension of the sampling time to +28d will have varying effects on different types of somatic tissues and more work is needed to assess its impact on somatic tissue mutagenicity testing. Based on the results of our spermatogenesis modelling and the limited available data, described in more detail in the companion manuscript [4], the workgroup considers the analysis of tubule germ cells at 28+28d for germ cell mutagenicity testing using TG 488 to be preferable to the analysis at 28+3d. Our modelling provides several arguments supporting this. Specifically: 1) the 28+ 28d protocol is superior with respect to the 28+3d pro- tocol because the extra 25 days of sampling time allow the testis to be populated by germ cells that received almost 28 days of effective exposure. This is particularly important for the rat because it elim- inates the ∼45% of the seminiferous tubule population that re- ceived no effective exposure at 28+ 3d and provides ∼80% of maximum effective exposure (versus ∼22% at 28+3d). In the mouse, the 28+28d protocol provides 99% of the maximum ef- fective exposure versus ∼42% at 28+3d. 2) It is more easily integrated with somatic tissue testing than the analysis of sperm at 28+49d (mouse) and 28+70d (rat) since 28+28d is already a recommended somatic tissue protocol in TG 488 for both mice and rats. Importantly, despite providing only 80% of the maximum exposure in the rat, the 28+28 d protocol has been demonstrated to detect significant increases in mutant fre- quencies in tubule germ cells after exposure to weak mutagens such as acrylamide and glycidamide [33]. 3) It allows the assessment of effects in differentiating spermatogonia and not just in spermatogonial stem cells as it is with the analysis of sperm at 28+49 d (mouse) and 28+ 70 d (rat). Differentiating spermatogonia divide more quickly than spermatogonial stem cells [8] and are known to be more sensitive to the mutagenic effects of chemicals. For example, BaP induced a 2.5-fold increase in lacZ mutant frequency in sperm derived from exposed spermatogonial stem cells and differentiating spermatogonia versus sperm derived from exposed spermatogonial stem cells only [7]. Thus, mutation assessment in a population that has sustained part of the exposure as differentiating spermatogonia may provide higher sensitivity to detect chemically-induced increases in mutations than a population that has experienced the exposure as spermatogonial stem cells only. Finally, the workgroup notes that the current recommendation in TG 488 of analyzing sperm at 28+ 49d (for the mouse) and 28+70d (for the rat) should be considered in those cases where it is important to know whether the chemical being tested affects spermatogonial stem cells. In conclusion, our modelling of spermatogenesis suggests that col- lection of tubule germ cells in both mouse and rat testes at 28+ 28d should be the recommended protocol for properly identifying chemicals that induce germ cell mutagenicity using TGR models. We suggest that the 28+ 28d protocol could represent a unifying protocol for si- multaneously assessing the potential of chemicals to induce mutations in somatic tissues and in germ cells using TGR models. Work is ongoing to demonstrate the suitability of this sampling time for rapidly dividing somatic tissues and to generate tubule germ cell data at 28+ 28d. Conflict of Interest Robert Young is employed by MilliporeSigma, BioReliance® Testing Services, a contract research laboratory that performs transgenic rodent mutation assays. Acknowledgments Supported in part by the Health Canada Chemicals Management Plan. The authors wish to acknowledge the support provided by Health and Environmental Sciences Institute. Washington, DC, USA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the F. Marchetti et al. Mutat Res Gen Tox En 832–833 (2018) 19–28 27 online version, at https://doi.org/10.1016/j.mrgentox.2018.05.020. 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Res. 832–833 (2018) 19–28] Francesco Marchettia,⁎, Marilyn J. Aardemab, Carol Beeversc, Jan van Benthemd, George R. Douglasa, Roger Godschalke, Carole L. Yauka, Robert Youngf, Andrew Williamsa a Environmental Health Science Research Bureau, Health Canada, Ottawa, ON, Canada bMarilyn Aardema Consulting, LLC, USA c Exponent International Limited, Harrogate, United Kingdom dNational Institute for Public Health and the Environment, Bilthoven, the Netherlands e Depertment of Pharmacology and Toxicology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Maastricht, the Netherlands fMilliporeSigma, BioReliance®Testing Services, Rockville, MD, USA We have noted that the study of Wang et al. 2010 (reference 33 in the article), which investigated the induction of mutations in the testis after exposure to either acrylamide or glycidamide, is erroneously mentioned as having used Big Blue® rats, when instead Big Blue® mice were used. Thus, the second sentence in the second bullet on page 27 of the article stating “Importantly, despite providing only 80% of the max- imum exposure in the rat, the 28+ 28d protocol has been demonstrated to detect significant increases in mutant frequencies in tubule germ cells after exposure to weak mutagens such as acrylamide and glycidamide [33].” is incorrect and should be deleted. Reference 33 should also be deleted from the reference list. As the data contained in reference 33 was not used in the simulation of spermatogenesis, there is no impact on the results of the modelling of rat spermatogenesis. However, we apologize for this error and for any inconvenience that this may have caused to the readership of this ar- ticle. https://doi.org/10.1016/j.mrgentox.2019.05.019 DOI of original article: https://doi.org/10.1016/j.mrgentox.2018.05.020 ⁎ Corresponding author. E-mail address: francesco.marchetti@canada.ca (Marchetti). Mutat Res Gen Tox En 844 (2019) 69 Available online 12 June 2019 1383-5718/ © 2019 Health Canada. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T http://www.sciencedirect.com/science/journal/13835718 https://www.elsevier.com/locate/gentox https://doi.org/10.1016/j.mrgentox.2019.05.019 https://doi.org/10.1016/j.mrgentox.2019.05.019 https://doi.org/10.1016/j.mrgentox.2018.05.020 mailto:francesco.marchetti@canada.ca https://doi.org/10.1016/j.mrgentox.2019.05.019 http://crossmark.crossref.org/dialog/?doi=10.1016/j.mrgentox.2019.05.019&domain=pdf Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488 Introduction Methods Simulation of the progression of spermatogenesis using the R statistical environment Expected number of days of exposure during the mitotic phase Results Generation of simulated mouse and rat testes Modelling of mouse spermatogenesis Modelling of rat spermatogenesis Distribution of the seminiferous tubule population according to the number of days of exposure during the mitotic phase Discussion Conflict of Interest Acknowledgments Supplementary data References Update Corrigendum to “Simulation of mouse and rat spermatogenesis to inform genotoxicity testing using OECD test guideline 488” [Mutat. Res. 832–833 (2018) 19–28]