One-generation Reproductive Toxicity Study Research Articles

Perinatal exposure to the immune-suppressant di-n-octyltin dichloride affects brain development in rats

Disruption of the immune system during embryonic brain development by environmental chemicals was proposed as a possible cause of neurodevelopmental disorders. We previously found adverse effects of di-n-octyltin dichloride (DOTC) on maternal and developing immune systems of rats in an extended one-generation reproductive toxicity study according to the OECD 443 test guideline. We hypothesize that the DOTC-induced changes in the immune system can affect neurodevelopment. Therefore, we used in-vivo MRI and PET imaging and genomics, in addition to behavioral testing and neuropathology as proposed in OECD test guideline 443, to investigate the effect of DOTC on structural and functional brain development. Male rats were exposed to DOTC (0, 3, 10, or 30 mg/kg of diet) from 2 weeks prior to mating of the F0-generation until sacrifice of F1-animals. The brains of rats, exposed to DOTC showed a transiently enlarged volume of specific brain regions (MRI), altered specific gravity, and transient hyper-metabolism ([18F]FDG PET). The alterations in brain development concurred with hyper-responsiveness in auditory startle response and slight hyperactivity in young adult animals. Genomics identified altered transcription of key regulators involved in neurodevelopment and neural function (e.g. Nrgrn, Shank3, Igf1r, Cck, Apba2, Foxp2); and regulators involved in cell size, cell proliferation, and organ development, especially immune system development and functioning (e.g. LOC679869, Itga11, Arhgap5, Cd47, Dlg1, Gas6, Cml5, Mef2c). The results suggest the involvement of immunotoxicity in the impairment of the nervous system by DOTC and support the hypothesis of a close connection between the immune and nervous systems in brain development.

Is there any added value of the second generation in the Extended One-Generation Reproductive Toxicity Study (EOGRTS)? A retrospective analysis of 24 EOGRTS

The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is a standard information requirement under Registration, Evaluation, Authorization and restriction of Chemicals (REACH) in Europe. Inclusion of an F2 generation is considered useful if this impacts hazard identification or when it provides additional information altering the reproductive or developmental No Observed Adverse Effect Level (NOAEL) and subsequent risk assessment. In this retrospective analysis the added value of the F2 generation in 24 EOGRTS was evaluated. Our retrospective analysis showed an added value of the second generation in 1/24 EOGRTS only, where extending with the F2 generation lowered both the reproductive and developmental NOAEL. For this substance, extension with the F2 generation led to classification for effects on fertility. Hazard identification for developmental effects was not impacted based on clear developmental toxic effects already observed in the F1 generation and in previous studies with this substance. Risk assessment may be impacted if the developmental toxic effects are the most critical effects observed for this substance. For 23/24 studies included in this retrospective analysis, hazard and risk assessment were unaffected by inclusion of the F2 generation. Moreover, for 13/13 studies with external triggers for inclusion of the F2 generation hazard and risk assessment were unaffected. By decreasing the number of EOGRTS that are extended with an F2 generation, the number of animals used in regulatory developmental and reproductive toxicity studies may be significantly reduced in the future without negatively affecting the ability to identify potentially harmful chemicals.

S03-02: Extended One-Generation Reproductive Toxicity study (EOGRTS) in the EU regulatory context

S03-02: Extended One-Generation Reproductive Toxicity study (EOGRTS) in the EU regulatory context

Extended one-generation reproductive toxicity study evaluating gardenia blue in Sprague Dawley rats

Gardenia blue powder was administered at 0.5%, 2.5%, or 5.0% in feed to male and female Sprague Dawley rats in an Extended One-Generation Reproductive Toxicity Study (OECD Test Guideline 443). The dosed diet began 14 days before mating and was continued at the same concentration level for the entire study for all parental animals (P0) and offspring (F1). At weaning, offspring were allocated into one of 5 cohorts for different endpoints. P0 and F1 animals had blue urine, blue or black feces, and blue discolorations in gastrointestinal organs, mesenteric lymph nodes, and kidneys. This treatment-related finding was not considered adverse as there were no histopathologic correlates. There was a dose-related increase in sperm concentration in P0 and F1 males. There were dose-related increases in heart weights of F1 postnatal day (PND) 21 males, male and female thyroid weights, and female TSH levels of PND 91 F1 offspring, with no histopathological correlate. There were no consistent treatment-related adverse effects on any other parameters evaluated for general toxicity, reproductive toxicity, developmental neurotoxicity, or developmental immunotoxicity. The highest dietary concentration (5.0%) of gardenia blue powder was the no observed adverse effect level (NOAEL) for male and female rats at all life stages evaluated.

Extended one generation reproductive toxicity study and effect on gut flora of genetically modified rice rich in β-carotene in wistar rats

To evaluate the reproductive toxicity of gene modified rice generated by introducing phytoene synthase (Psy) and bacterial phytoene desaturase (CrtI) from maize and Erwinia uredovora, Wistar rats were allocated into 3 groups and fed with Psy and CrtI gene modified rice mixture diet (GM group), non-gene modified rice mixture diet (non-GM group), and AIN-93 diet (Blank control group) from parental generation (F0) to the offsprings (F1). GM rice, Heijinmi (HJM) and Non-GM rice, Heishuai (HS), were both formulated into diets at ratios of 73.5% and 75.5% according to the AIN93 diet for rodent animals, respectively. Relative to the non-GM group, no biologically relevant differences were observed in GM group rats concerning reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, and reproductive organ pathology. The developmental parameters results were not significantly different from the non-GM group such as body weight, food consumption, developmental neurotoxicity, behavior, hematology, and serum chemistry. In terms of immunotoxicity, the IgG indicators of offspring from the GM group improved in contrast with the non-GM group. Additional gut flora analysis of F0 generation rats resulted as that the treatment elicited an increased gut microflora diversity of F0 rats. And no horizontal gene transfer of Psy and CrtI genes in rats fed a GM rice HJM diet. In conclusion, we found no adverse effects related to GM rice in the extended one-generation reproductive toxicity study, indicating that GM rice is a safe alternative for its counterpart rice regarding reproductive toxicity.

Safety assessment of 2-methyloxolane as a food extraction solvent.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of 2‐methyloxolane as an extraction solvent under the intended conditions of use and the maximum residue limits (MRLs) proposed by the applicant. 2‐Methyloxolane is intended to be used in processes currently applying hexane for oil and protein extraction from plant sources or for extraction of food additives. The proposed MRLs for the following uses are: (i) 1 mg/kg in fat, oil or butter; (ii) 10 mg/kg in defatted protein products, defatted flour and other defatted solid ingredients; (iii) 1 mg/kg in food category 13 (foods intended for particular nutritional uses as defined by Directive 2009/39/EC); and (iv) 1 mg/kg for the extraction of food additives. The Panel calculated the dietary exposure with the highest potential maximum (95th percentile) for toddlers as 0.32 mg/kg body weight (bw) per day. Based on the available toxicological data, the Panel concluded that 2‐methyloxolane was rapidly metabolised with a low bioaccumulation potential and does not raise a concern for genotoxicity. The Panel identified different no observed adverse effect levels (NOAELs) in a subchronic oral toxicity study in rats, an oral developmental toxicity study and an extended one‐generation reproductive toxicity study, and a TDI of 1 mg/kg bw per day for 2‐methyloxolane was derived based on the lowest identified NOAEL (100 mg/kg bw per day) for reproductive and developmental toxicity. This TDI was not exceeded in any of the population groups at the mean and 95th percentile exposure. The Panel concluded that the extraction solvent 2‐methyloxolane does not raise a safety concern when used according to the intended conditions and at the proposed MRLs in the extracted foods or food ingredients.

Adverse postnatal developmental effects in offspring from humidifier disinfectant biocide inhaled pregnant rats

Inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-P), one of the primary biocides used in humidifier disinfectants, caused a fatal pulmonary disease in Korea. Pregnant women were also exposed to PHMG-P, and subsequent studies showed that PHMG-P inhalation during pregnancy adversely affects their health and embryo-fetal development. However, the postnatal developmental effects after birth on prenatally PHMG-P-exposed offspring have not yet been investigated. Therefore, in this study, we aimed to examine the postnatal development of prenatally PHMG-P-exposed offspring. Pregnant rats (22 or 24 females per group) were exposed to PHMG-P during pregnancy in a whole-body inhalation chamber at the target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3. After parturition, the prenatally exposed offspring were transferred to non-exposed surrogate mothers to minimize the secondary effects of severe maternal toxicities. Postnatal development of offspring was then examined with a modified extended one-generation reproductive toxicity study design. At 3.20 mg/m3 PHMG-P, increased perinatal death rates and decreased viability index (postnatal survival of offspring between birth and postnatal day 4) were observed. In addition, F1 offspring had lower body weight at birth that persisted throughout the study. PHMG-P-exposed pregnant rats also had severe systemic toxicities and increased gestation period. At 1.60 mg/m3 PHMG-P, a decreased viability index was also observed with systemic toxicities of PHMG-P-exposed pregnant rats. These results indicate that prenatal PHMG-P exposure adversely affects the offspring's future health and could be used for human risk assessment.

A one-generation reproductive toxicity study of the mycotoxin ochratoxin A in Fischer rats

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium molds. Grain-based foods account for most human dietary exposures to OTA. OTA is a teratogen, but its reproductive and developmental effects are poorly understood. A one-generation reproductive toxicity study was conducted with groups of 16 male and 16 female Fischer rats exposed to 0, 0.026, 0.064, 0.16, 0.4 or 1.0 mg OTA/kg in diet. Dams exposed to 1.0 mg OTA/kg diet had statistically significant F1 pup losses between implantation and postnatal day (PND 4). Delays in preputial separation (PPS) and vaginal opening (VO) were indicative of delayed puberty in F1 rats. Mild renal lesions in nursing pups indicated that exposure prior to weaning impacted the kidneys. The developing kidney was more susceptible to OTA than the adult kidney. Significant increases in multi-oocyte follicles (MOFs) and proportional changes in resting and growing follicles were observed in F1 female ovaries. Plasma testosterone was reduced in F0 males, and there were negative effects on sperm quality in F0 and F1 male rats. The results confirm that continuous dietary exposure to OTA causes post-implantation fetotoxicity in dams, and renal and reproductive toxicity in their male and female offspring.

Extended One-Generation Reproductive Toxicity (EOGRT) study of benzoic acid in Sprague Dawley rats

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary concentrations were adjusted (based on historical control body weight and food consumption data) to maintain the target mg/kg bw/day dose levels during those life periods when food intake per unit of body weight was increased to support milk production by females (gestation and lactation) and rapid pup growth (post-weaning).In the parental (F0) generation, survival, clinical observations, organ weights, pathology, hematology, serum chemistry, urinalysis, and bile acids were unaffected by BA administration. Reproductive parameters were also unaffected by BA administration.In the F1 generation, survival, growth and developmental landmarks, organ weights, pathology, immunotoxicity assessment, and neurotoxicity and neurobehavioral parameters such as auditory startle response, locomotor activity, learning and memory assessments were unaffected by BA administration, as were clinical pathology (hematology, serum chemistry, urinalysis, bile acids and thyroid hormones) and reproductive performance. Similarly, no adverse effects or systemic toxicity were observed in the F2 generation.Overall, the highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

Effects of endocrine disruptor furan on reproductive physiology of Sprague Dawley rats: An F1 Extended One-Generation Reproductive Toxicity Study (EOGRTS).

The present study investigated the reproductive toxicity of furan in an Extended One-Generation Reproductive Toxicity Study in rats. Sprague Dawley F0 weaning rats (30 per sex per group) were exposed to furan orally at 0, 1, 2.5, 5, and 10 mg kg-1 for 10 weeks (males) and 2 weeks (females) and then mated. Results of F0 indicated that in the furan-treated groups (5 mg kg-1 and 10 mg kg-1), body weight (bw) gain decreased during prebreed and gestational period while increased during lactation periods. F0 animals prebreeding exposure resulted in head tilt and foot splay at 10 mg kg-1. Number of live pups at birth were decreased (p < 0.001) at 10 mg kg-1. At postnatal day (PND) 70, a significant (p = 0.03) decrease in testosterone levels of male rats and estrogen levels of female rats (p = 0.05) was observed in 10 mg kg-1 furan-treated group in F1 generation. Luteinizing hormone, follicle-stimulating hormone, and progesterone levels were also reduced, but their reduction was not statistically significant in all groups. In higher dose furan group (10 mg kg-1), testicular and ovarian weights were reduced in F1 generation at PND 70, with decreased daily sperm production (p = 0.01) and disturbed estrous cyclicity (p < 0.01). Some histopathological changes were also observed in testis and ovaries in groups whose parents were previously exposed to 10 mg kg-1 bw of furan group. Based on the above results, it is suggested that exposure to food-based contaminant furan induced remarkable changes in the F0 (parental stage) and F1 (offspring, pubertal, and adult stage) generations of Sprague Dawley rats.

Flubenziamide (Pesticides).

The Food Safety Commission of Japan (FSCJ) conducted a risk assessment of flubendiamide (CAS No. 272451-65-7), an iodophthalimide insecticide for the setting of an acceptable daily intake (ADI) in 2006. FSCJ now has assessed this insecticide for the setting of an acute reference dose (ARfD). Data including fate in animals (rats and mice) and residues in crops (burdock roots, pumpkins and others) were newly submitted. Major adverse effects of flubendiamide include hepatocellular hypertrophy, fatty changes in hepatocytes, follicular epithelial cell hypertrophy in thyroid and ocular enlarged eye in rats. No neurotoxicity, carcinogenicity, reproductive toxicity, teratogenicity, neurodevelopmental toxicity and genotoxicity were observed. The lowest no-observed-adverse-effect level (NOAEL) in the toxicological studies was 1.70 mg/kg body weight/day in a two-year carcinogenicity study in rats. FSCJ confirmed an ADI of 0.017 mg/kg bw/day after applying a safety factor of 100 to the NOAEL. Adverse effects elicited by a single oral administration of flubendiamide would be abnormalities in eyes such as ocular hypertrophy and iris adhesion in offspring, which were obtained in a two-generation reproductive toxicity study, a one-generation reproductive toxicity study and a neurodevelopmental toxicity study in rats. FSCJ judged that these studies may be applicable to set the ARfD for lactating women in relation to the exposure of flubendiamide to offspring after the birth through breast milk. By taking into account the overall evaluations of the two-generation reproductive toxicity study, one-generation reproductive toxicity study and neurodevelopmental toxicity study in rats, FSCJ judged NOAEL of 15.0 mg/kg bw/day as for an overall NOAEL, and consequently specified an ARfD of 0.15 mg/kg bw/day for lactating women by applying a safety factor of 100 to the NOAEL.

Flubenziamide (Pesticides)

The Food Safety Commission of Japan (FSCJ) conducted a risk assessment of flubendiamide (CAS No. 272451-65-7), an iodophthalimide insecticide for the setting of an acceptable daily intake (ADI) in 2006. FSCJ now has assessed this insecticide for the setting of an acute reference dose (ARfD). Data including fate in animals (rats and mice) and residues in crops (burdock roots, pumpkins and others) were newly submitted. Major adverse effects of flubendiamide include hepatocellular hypertrophy, fatty changes in hepatocytes, follicular epithelial cell hypertrophy in thyroid and ocular enlarged eye in rats. No neurotoxicity, carcinogenicity, reproductive toxicity, teratogenicity, neurodevelopmental toxicity and genotoxicity were observed. The lowest no-observed-adverse-effect level (NOAEL) in the toxicological studies was 1.70 mg/kg body weight/day in a two-year carcinogenicity study in rats. FSCJ confirmed an ADI of 0.017 mg/kg bw/day after applying a safety factor of 100 to the NOAEL. Adverse effects elicited by a single oral administration of flubendiamide would be abnormalities in eyes such as ocular hypertrophy and iris adhesion in offspring, which were obtained in a two-generation reproductive toxicity study, a one-generation reproductive toxicity study and a neurodevelopmental toxicity study in rats. FSCJ judged that these studies may be applicable to set the ARfD for lactating women in relation to the exposure of flubendiamide to offspring after the birth through breast milk. By taking into account the overall evaluations of the two-generation reproductive toxicity study, one-generation reproductive toxicity study and neurodevelopmental toxicity study in rats, FSCJ judged NOAEL of 15.0 mg/kg bw/day as for an overall NOAEL, and consequently specified an ARfD of 0.15 mg/kg bw/day for lactating women by applying a safety factor of 100 to the NOAEL.

Suggestions for the OECD's extended one-generation reproductive toxicity study to further understand the temporality of endocrine-disrupting effects from chemical exposure: example using bisphenol A

Suggestions for the OECD's extended one-generation reproductive toxicity study to further understand the temporality of endocrine-disrupting effects from chemical exposure: example using bisphenol A

Regulatory aspects under REACH relating to the developmental immunotoxicity (DIT) cohort in the extended one-generation reproductive toxicity study (EOGRTS)

Regulatory aspects under REACH relating to the developmental immunotoxicity (DIT) cohort in the extended one-generation reproductive toxicity study (EOGRTS)

An extended one-generation reproductive toxicity study of plant protection product containing glyphosate on rats – Androgen- and estrogen-dependent endpoints

Roundup Transorb® (RT) is a glyphosate-based herbicide and despite its wide use around the world there are few studies comparing the effects of the active ingredient with the formulated product. In this context the purpose of this study was to compare the genotoxicity of the active ingredient glyphosate with the formulated product RT in order to clarify whether the active ingredient and the surfactant of the RT formula may exert toxic effects on the DNA molecule in juveniles of fish Prochilodus lineatus. Erythrocytes and gill cells of fish exposed to glyphosate and to RT showed DNA damage scores significantly higher than control animals. These results revealed that both glyphosate itself and RT were genotoxic to gill cells and erythrocytes of P. lineatus, suggesting that their use should be carefully monitored considering their potential impact on tropical aquatic biota.

Re-evaluation of pectin (E440i) and amidated pectin (E440ii) as food additives.

Following a request from the European Commission, the EFSA Panel on Food Additives and Nutrient sources added to Food (ANS) was asked to deliver a scientific opinion on the re‐evaluation of pectin (E 440i) and amidated pectin (E 440ii) as food additives. An acceptable daily intake (ADI) ‘not specified’ was allocated by the Scientific Committee for Food (SCF) for E 440i and E 440ii. Pectin and amidated pectin would not be absorbed intact, but extensively fermented by intestinal microbiota in animals and humans; products formed from pectins in the gastrointestinal tract are similar to manufactured pectin‐derived acidic oligosaccharides (pAOS). There is no indication of genotoxicity for pectin and amidated pectin, although the available data were limited. No adverse effects were reported in a chronic toxicity study in rats at levels up to 5,000 mg pectin/kg bw per day, the highest dose tested. No treatment‐related effects were observed in a dietary one‐generation reproductive toxicity study with pAOS in rats at up to 6,200 mg/kg body weight (bw) per day, the highest dose tested. The Panel did not consider E 440i and E 440ii as having allergenic potential. A dose of 36 g/day (equivalent to 515 mg/kg bw per day) for 6 weeks in humans was without adverse effects. Exposure to pectins from their use as food additives ranged up to 442 mg/kg bw per day for toddlers at the 95th percentile (brand‐loyal scenario). The Panel concluded that there is no safety concern for the use of pectin (E 440i) and amidated pectin (E 440ii) as food additives for the general population and that there is no need for a numerical ADI.

Route-to-route extrapolation of 1,2-dichloroethane studies from the oral route to inhalation using physiologically based pharmacokinetic models

To help develop a comprehensive, quantitative understanding of the hazards of 1,2-dichloroethane (ethylene dichloride, EDC, CAS No. 107-06-2) exposure by the inhalation route, the results of existing subchronic studies and an extended one-generation reproductive toxicity (EOGRT) study recently conducted by the oral route in rats were extrapolated using a physiologically based pharmacokinetic (PBPK) model. The no observed adverse effects levels (NOAELs) for the endpoints of neurotoxicity and reproductive/developmental toxicity were the highest tested doses of 169 and 155 mg/kg-day, respectively. These NOAELs were equivalent to continuous exposure of rats to minimums of 76 ppm and 62 ppm EDC, respectively, using total metabolism of EDC as the dose metric that is equivalent in the oral and inhalation scenarios. In contrast, the subchronic study NOAEL of 37.5 mg/kg-day corresponded to continuous inhalation of 4.4 ppm EDC, based on equivalent extrahepatic metabolism. The selection of the internal metric which serves to establish route-to-route equivalency was found to profoundly influence the NOAEL-equivalent inhalation exposure concentration and thus will be a key determinant of inhalation toxicity reference criteria developed on the basis of EDC studies conducted by the oral route.

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443)

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 443) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.

Reproductive and developmental toxicity testing: Examination of the extended one-generation reproductive toxicity study guideline

An important aspect of safety assessment of chemicals (industrial and agricultural chemicals and pharmaceuticals) is determining their potential reproductive and developmental toxicity. A number of guidelines have outlined a series of separate reproductive and developmental toxicity studies from fertilization through adulthood and in some cases to second generation. The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the most recent and comprehensive guideline in this series. EOGRTS design makes toxicity testing progressive, comprehensive, and efficient by assessing key endpoints across multiple life-stages at relevant doses using a minimum number of animals, combining studies/evaluations and proposing tiered-testing approaches based on outcomes. EOGRTS determines toxicity during preconception, development of embryo/fetus and newborn, adolescence, and adults, with specific emphasis on the nervous, immunological, and endocrine systems, EOGRTS also assesses maternal and paternal toxicity. However, EOGRTS guideline is complex, criteria for selecting doses is unclear, and monitoring systemic dose during the course of the study for better interpretation and human relevance is not clear. This paper discusses potential simplification of EOGRTS, suggests procedures for relevant dose selection and monitors systemic dose at multiple life-stages for better interpretation of data and human relevance.

2. Regulation of Chemicals

This year, some of the annexes to EC Regulation 1907/2006 Concerning the Regulation, Evaluation, and Authorization of Chemicals (REACH Regulation) were amended, and case law emerged. Outside of the REACH Regulation, a certain substance was approved as an active substance for use in biocidal products, and the commission decided on its position to be taken in negotiations about the Stockholm Convention on Persistent Organic Pollutants (Stockholm Convention). EU Regulation 2015/282 amends Annexes VIII, IX and X to the Reach Regulation, specifying the scope of a new modular test method called the extended one-generation reproductive toxicity study (EOGRTS), which was adopted by the Organisation for Economic Co-operation and Development in 2011. According to the current amendment, a two-generation reproductive toxicity study is regularly required to fulfil the standard information requirements in point 8.7.3 of Annexes IX and X. However, under EOGRTS, breeding and assessment of a second filial generation and testing for developmental neurotoxicity and developmental immunotoxicity constitute distinct and independent modules (Commission Regulation (EU) 2015/282, preamble, para. 3). EOGRTS focuses on the analysis of the first filial generation and addresses additional parameters in the hope of improvements in sensitivity and the level of information that can be gained from the tests.