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Key value for chemical safety assessment

Effects on fertility

Description of key information

Weight of Evidence for Section 8.7.3 Extended One-Generation Reproductive Toxicity Study (EOGRTS) of Annex X of the REACH Regulation


 


1. Requirement for an Extended One-Generation Reproductive Toxicity Study (EOGRTS)


The Extended One-Generation Reproductive Toxicity Study (EOGRTS) (OECD TG443, EU B.56) in its basic test design is a standard information requirement at REACH Annex IX (where concerns regarding reproductive toxicity are evident) and X levels. EOGRTS was introduced under Commission Regulation (EU) 2015/282 of 20 February 2015 amending Annexes VIII, IX and X to the REACH Regulation (EC No. 1907/2006). It is a test method developed to assess the reproductive toxicity of chemical substances and is the preferred test method to address the standard information requirement defined in Column 1 of Point 8.7.3 of Annexes IX and X to the REACH Regulation instead of the Two-Generation Reproductive Toxicity study. Standard information requirement for reproduction toxicity related to EOGRTS in Annexes IX and X is limited to the basic configuration of EOGRTS, which includes only cohorts 1A and IB without extension to include a F2 generation and developmental neurotoxicity and developmental immunotoxicity cohorts.


There are no Specific Rules for Adaptation from Column 1 in Annex X Section 8.7 of the REACH Regulation, but these are in place for the test at Annex IX. However, adaptions pursuant to Annex XI (General Rules for Adaptation of the Standard Testing Regime set out in Annexes VII to X) and a recent Judgement of the General Court in Case T-755/17 (September 2019) in relation to the Principle of Proportionality are considered relevant to the potential for adaption of this test requirement in appropriate instances. Paragraph 287 of the Judgement states that:


The relevant criterion relating to the principle of proportionality is the result of balancing the different objectives pursued by Regulation No 1907/2006 and the application of the precautionary principle. In accordance with that criterion, in order to justify a request to conduct testing, the ECHA must not only demonstrate the existence of a potential risk for human health and the environment, and the necessity to clarify that risk, but also establish that there is a realistic likelihood that the information requested would allow improved risk management measures to be taken”.


The requirement to conduct complex mammalian toxicity tests at Annex X which potentially involves the use of hundreds of vertebrates (normally rats) needs to be carefully considered to ensure that it satisfies Article 25(1) of the REACH Regulation that any testing on vertebrate animals is undertaken only as a last resort. Furthermore, any testing carried out needs to satisfy Recital 63 of the REACH Regulation which sets out a general principle according to which ‘[i]t is also necessary to ensure that generation of information is tailored to real information needs…’. Where testing is proposed it is also important to ensure that in the studies conducted the fewest number of animals possible are used to satisfy the objective pursued as required by Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33). Furthermore, the extent to which the test information will improve the quantification and management of risks to human health and the environment based on the Risk Characterisation Exercise needs to be demonstrated. Therefore, an implicit presumption underlying the requirement to provide data from an EOGRTS study on Technical Grade is that without the requested information it will not be possible to verify whether there remains an uncontrolled risk with the substance that should be subject to further risk management measures.


The OECD TG443 Test Guideline indicates that “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”. With regard to other related toxicity studies the Test Guideline states that “For reproductive endpoints, it is envisaged that, as a first step and when available, information from repeat-dose studies (including screening reproductive toxicity studies, e.g. TG 422 (32)), or short term endocrine disrupter screening assays, (e.g. Uterotrophic assay - TG 440 (36); and Hershberger assay - TG 441 (37)) is used to detect effects on reproductive organs for males and females. This might include spermatogenesis (testicular histopathology) for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females”


In its basic design EOGRTS provides 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. The test chemical is administered continuously (on a once a day basis) 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 chemical, but for a minimum of two weeks) and a two-week mating period. Parental generation males are further treated at least until weaning of the F1 offspring, which involves treatment for a minimum of 10 weeks. 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. after 8-10 weeks of treatment). The F1 offspring receive further treatment with the test chemical from weaning to adulthood. In the basic study design, 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 chemical 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.


REACH Annex XI (General Rules for Adaptation of the Standard Testing Regime set out in Annexes VII to X) indicates that there are a number of approaches by which a registrant may adapt the standard testing regime in accordance with the general rules set out in Section 1 of the Annex. Section 1.2 relates to the use of a weight of evidence approach and states that “There may be sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion”. This approach has been used to address the data requirement for the Extended One-Generation Reproductive Toxicity Study (EOGRTS) for Technical Grade at Annex X.


Although there is no defined rationale for adapting the EOGRTS test given in Annex X this is not the case for Annex IX. This Annex allows for the possibility of not conducting the basic text design and Column 1 of Section 8.7.3 states that the test is required “if the available repeated dose toxicity studies (e.g. 28- or 90-days studies or OECD TGs 421 or 422 screening tests) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. Information from non-animal approaches are thus not listed as triggers for this study at REACH Annex IX level in the REACH Annex text. However, if there is a serious concern based on available information from non-animal approaches or structurally analogous substances, the study may be triggered” (ECHA, 2017). The inference from this tonnage-based information strategy is that there could be no circumstances at Annex X where the test would not need to be conducted and the test should be triggered irrespective of the existing evidence regarding the extent of the effects of the test substance on reproductive and developmental endpoints. However, this is contrary to the view expressed in Recital 63 of the REACH Regulation and the Judgement of the General Court in Case T-755/17. This should mean that the test does not need to be carried out if there is sufficient reliable data available to assess the potential hazards and risks posed by the substance to pregnant females and their offspring and provide a weight of evidence that further testing is not required as the potential risks are adequately controlled.


This principle has been confirmed by an ECHA Board of Appeal ruling on 25 September 2018 in Case A-008-2017. This annulled the Agency’s decision of 23 March 2017 on the substance evaluation of 2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol pursuant to Article 46 of REACH. The Appellants claimed that the Agency failed to fulfil the conditions for imposing further information requirements, including the need for EOGRTS, under Article 46 of the REACH Regulation and failed to provide an adequate statement of reasons, in particular regarding the potential risk perceived by the Agency. In addition, the Appellants claimed that the Agency failed to provide information or explanations on how the information required in the Contested Decision would lead to an improvement of the risk management measures in place.


In their judgement the Board of Appeal upheld the Appellant’s view that the Agency had made no evaluation whatsoever of whether the available information showed 'serious concerns about the potential for adverse effects on fertility or development' within the meaning of Column 2 of Section 8.7.1. of Annex VIII of the REACH Regulation. Although this judgement relates to a substance at a lower tonnage band than that relevant to Technical Grade and relates to substance evaluation rather than compliance checking it establishes principles requiring ECHA to evaluate the existing data to demonstrate that the conduct of an EOGRTS can be justified to further assess the perceived risks from a test substance, particularly given the large numbers of mammals that would be used. This weight of evidence document reviews the available relevant mammalian reproductive and developmental data for Technical Grade and provides arguments for not conducting the EOGRTS.


 


2. Weight of evidence approach for the EOGRTS


 


The weight of evidence approach for addressing the EOGRTS endpoint for Technical Grade is based on two elements:


 



  • The absence of evidence of reproductive and developmental toxicity in an already extensive dataset.


 



  • The control of risks to human health in all identified exposure scenarios


 


Information relevant to each of these elements is provided in the following sections.


The weight of evidence approach for addressing the EOGRTS endpoint for Technical Grade is based on the absence of reproductive and developmental toxicity in an extensive dataset for the test substance and other members of the category. The Final Decisions for the Annex IX and X testing of the three grades of processed Cashew (Anacardium occidentale) Nutshell Extract that constitute a group or category (namely Distilled, Distillation Residue and Technical Grades) specify that the tests are carried out on the source substances Distilled and Distillation Residue Grades. The Final Decision for the target substance Technical Grade specified that information for the required environmental fate, mammalian toxicology and ecotoxicological Annex IX and X endpoints should be generated by interpolation from tests on the source substances Distilled and Distillation Residue Grades. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”.


 


It should be noted that the dermal route of exposure is the most relevant in terms of potential human exposure to three grades of CNSL rather than the oral route based on normal use patterns. However, all the grades are classified as a Category 2 Skin Irritant (H315) and it was not considered appropriate from an animal welfare viewpoint to conduct any studies using this route of exposure. Therefore, all the existing data assessing reproductive and developmental toxicity has been generated in tests using the oral route.


 


 


2.1 Absence of evidence of reproductive and developmental toxicity in studies on the three grades of CNSL representing a category


 


2.1.1 Absence of evidence of reproductive and developmental toxicity in studies on Technical Grade


 


The potential reproductive and developmental toxicity of Technical Grade has been evaluated in a series of studies by Kaneda and Mochizuk (2016). These have investigated the potential to which feeding cattle with pellets containing unprocessed Cashew Nutshell Extract and Technical Grade (which is compositionally similar to Distilled Grade) results in changes in reproductivity (with regard to conception and pregnancy) as well as changes in milk yield and/or milk quality and the incidence of perinatal disease (which accounts for more than half the deaths in cattle and results in the disposal of huge quantities of milk). The studies have been classified as reliable with restriction (Klimisch Code 2). Although they were not conducted to Good Laboratory Practice (GLP) they satisfy the requirements given in Klimisch et al. (1999) for classification as reliable with restriction, namely: “Studies or data (mostly not performed according to GLP) in which the test parameters documented do not comply totally with the specific testing guideline, but are sufficient to accept the data or in which investigations are described that cannot be subsumed under a testing guideline, but which are nevertheless well-documented and scientifically acceptable. They form part of a research study on the potential use of cashew nutshell liquid as an alternative to current registered antibiotics given the substances known antibacterial action and relieving action against parasitic disease of the intestinal tract of animals caused by coccidian protozoa.


In the study evaluating the effect of feeding Technical Grade on reproductivity, adult female Holstein cattle were used with the mature cows (i.e. those having had at least one calf) weighing between 450 kg (1,000 pounds) and 680 kg (1,500 pounds) depending on their age. Individuals which had calved were divided at random into a test group and control group. Technical Grade was fed to the individuals in the test group in pellets at an amount of 100 g (10 g in terms of CNSL) per day for 5 days from the date of calving. The feeding rate is equivalent to approximately 20 mg/kg bw/day of CNSL. The Technical Grade used largely comprised 55-80% cardanol and 5-30% cardol, which is consistent with the substance identity profile of the registered substance. The test group, which were fed Technical Grade, consisted of 9 cows (2 cows having had two calves, 4 cows having had three calves, 1 cow having had four calves, 1 cow having had five calves and 1 cow having had six calves). Five cows (all of which had had three calves) were allocated to control group and were not fed Technical Grade containing-pellets. Although the feed was modified depending on the condition of the cows, it was designed so that 7 kg of timothy hay (which is high in fibre and energy content but low in protein content) and 2 to 3 kg of a concentrate feed were fed on average per day.


The effect of ingestion of Technical Grade on bovine reproductivity was evaluated based on a number of indices namely: the number of artificial inseminations required for pregnancy, the number of non-pregnant days, the actual number of non-pregnant days, days to first service, pregnancy rate at first service, pregnancy rate, and the detection rate of oestrus. For the non-pregnant days, the percentages of individuals with non-pregnant days of 90 days or less (evaluated as good) and individuals with non-pregnant days of 120 days or more (evaluated as poor) were also calculated.


 


The test results from the reproductivity study are shown in Table 1 and indicate that the feeding of the Technical Grade-containing pellets was found to result in improvements in almost all the endpoints for the test group compared with control group. In particular, significant effects in terms of reducing the number of inseminations, decreasing the number of non-pregnant days and actual non-pregnant days and improving the pregnancy rate were measured. Further, the feeding of Technical Grade was able to achieve a conception rate exceeding 50-60% and an oestrus detection rate exceeding 70% or more of target levels, respectively. No change was noted in the days of first insemination between the groups. Overall, the results show that the feeding of the Technical Grade-containing pellets was found to significantly improve the reproductivity in each individual cow and to further significantly improve the reproductivity in the oestrus detection rate and pregnancy rate in each group. For all the parameters measured the No Observed Adverse Effect Levels (NOAELs) were greater than 20 mg/kg bw/day of CNSL. On the basis of this data Technical Grade at the tested dose does not evidently have any adverse toxic effects on the reproductivity of cattle. The data is valuable since it provides information on another mammalian group other than rodents.


 


Table 1        Effects of feeding Technical Grade on the reproductivity of cows (after Kaneda and Mochizuk (2016)


 


































































 


Endpoint



 


Mean results for the Technical Grade group (n = 9)


 



 


Mean results for the control group (n = 5)



 


Variation



Number of insemination times



2.0



2.6



-0.6



Non-pregnant days



111



137



-26



Non-pregnant days: 90 days of less (%)



33.3



20.0



13.3



Non-pregnant days: 120 days of more (%)



33.3



60.0



-26.7



Actual non-pregnant days



32.1



58.3



-26.2



Days of first insemination



78.9



78.7



0.2



Pregnancy rate at first service (%)



33.3



16.7



16.6



Pregnancy rate (%)



50.0



38.5



11.5



Oestrus detection rate (%)



79.1



68.9



10.2



 


 


 


Notes:


Number of insemination = times of artificial insemination


Non-pregnant days = last conception day - last calving day


Actual non-pregnant days = days from first to last insemination  


Days of first insemination = first insemination day after calving - last calving day


Pregnancy rate at first service = rate of conception achieved by first insemination


Pregnancy rate = [number of individuals with conception (group total)/total times of artificial insemination] x 100


Detection rate of oestrus (whole group) = [average number of insemination/((average actual days of non-pregnant/21)+1] x100


 


 


2.1.2 Absence of evidence of reproductive and developmental toxicity in studies on Distilled Grade


Currently an extensive, reliable and relevant data set is available on the potential reproductive and developmental toxicity of Distilled Grade to rats from a Prenatal Developmental Toxicity Study (OECD TG414) and a Combined Repeat Dose Toxicity Study with Reproduction/Development Toxicity Screening Study (OECD TG422). A Repeated Dose 90-day Toxicity Study (OECD TG408) is also available which provides reliable data on the potential effects of Distilled Grade on the growth and maturation of male and female rats.


.


Reliable and relevant data (Klimisch Code 1) are available from a Combined Repeat Dose Toxicity Study with Reproduction/Development Toxicity Screening in the Rat via Oral Gavage (OECD TG422, 1996 Version) in which male and initially unmated female Sprague-Dawley rats were administered Distilled Grade via oral gavage for up to 49 days. This older version of the test (rather than the current June 2016 version) assessed the effects of the test substance on the mating of the parental males and females, pregnancy in females and offspring birth and development, which corresponds to the pre-mating, mating and early post-mating (pregnancy and lactation) phases of the EOGRTS test.  At the start of the study the animals were approximately eight weeks old. The test substance was administered by oral gavage to three treatment groups at dose levels of 15, 150 and 1000 mg/kg body weight/day. A control group of ten males and ten females was dosed with the in-test vehicle arachis oil alone (but no test substance). Groups of ten male and ten female animals were treated at each dose level for fourteen days prior to the pairing of the sexes. On Day 14, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of 14 days. Following evidence of mating, the males were returned to their original cages (in groups of five) and females were transferred to individual cages. Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum (as opposed to PND 13 in the updated version)..


Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study. Haematology (in terms of haemoglobin, erythrocyte and haemocrit measurements) and blood chemistry (in terms of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, bilirubin, cholesterol and urea measurements) were also evaluated prior to mating and on termination on selected animals from each dose group. Following mating and subsequent gestation, offspring development was monitored up to Day 4 post partum. All surviving adult females and offspring were terminated on Day 5 post partum, followed by the termination of all surviving males. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was also performed.


An extensive range of reproductive parameters were measured in the study. This older version of the test did not measure a series of endocrine disruption related parameters, namely oestrous cyclicity and thyroid hormone measurement in the parental generation and, anogenital distance, nipple retention and thyroid hormone measurement in the offspring.


Overall, the repeat dose and reproductive toxicity screening test provided evidence that general systemic toxic effects (i.e. a reduction in body weight gain) and localised effects on non-glandular mucosa of the stomach (forestomach, squamous mucosa) were occurring at the highest exposure dose (1000 mg/kg bw/day) of Distilled Grade. The observed local effects on stomach morphology were probably due to the known irritancy of the test substance, which could have also caused secondary effects on body weight gain. In contrast, no adverse effects on reproductive and developmental endpoints were evident at any treatment dose:



  • mating performance

  • pre-coital interval

  • mating index

  • pregnancy index

  • gestation length

  • females with live offspring

  • parturition index,

  • number of litters,

  • number of offspring born

  • number of live offspring born

  • offspring sex ratio

  • live birth index

  • viability index

  • total litter weight post- partum

  • offspring bodyweight post-partum


 


Reliable and relevant data (Klimisch Code 1) are also available from a Prenatal Developmental Toxicity Study (OECD TG414) on Distilled Grade which assessed the effects of test substance exposure on offspring birth and development. The test started with the arrival of groups of pre-mated female Wistar rats. Untreated females were mated at the organism supplier and were at Day 0 or 1 post-coitum on arrival (Day 0 post-coitum is the day of successful mating). Twenty-two time-mated females (of approximately 10 to 14 weeks age) were assigned to a control group (no exposure to test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 21 post-coitum, inclusive, which corresponds to the female pregnancy and foetal in-utero development phases of the EOGRTS test. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study along with an extensive series of developmental endpoints in the foetuses. These included litter size, post-implantation loss, foetal sex ratio, foetal body weights, foetal anogenital distance and external, visceral and skeletal malformations and developmental variations in foetuses.


Animals in the high dose treatment (1000 mg/kg bw/day) group experienced severe toxicity (as body weight loss, decreased food consumption and adverse clinical signs). Four animals were sacrificed on Study Days 14 (2 animals), 16 (1 animal) and 17 (1 animal). To prevent further suffering, it was decided by the veterinarian at the test laboratory to sacrifice all the remaining high dose (1000 mg/kg/day) animals on Study Day 18 for ethical reasons. It was concluded that the effects observed at the highest dose were probably due to the known irritancy of the test substance which caused localised changes in stomach morphology and limited food consumption. No mortality was observed for the animals in the remaining groups during the study.


The test substance administered at dose levels of 100 mg/kg bw/day was not considered to cause maternal toxicity. No systemic toxic effects and non-adverse local effects on the surface of the forestomach were observed. At the 300 mg/kg bw/day dose level, the test substance was maternally toxic causing both adverse systemic toxic effects (on body weight gain). Body weight gain at 300 mg/kg bw/day was slightly lower from Day 12 post-coitum onwards, reaching statistical significance on Days 18 and 21 post-coitum only (0.88x and 0.83x of controls, respectively). Body weight gain, corrected for the weight of the uterus of the pregnant female, was statistically significantly lower compared with concurrent controls (on average 50% lower). Adverse local effects on the surface of the forestomach were also evident at 300 mg/kg bw/day which could also have caused secondary effects on body weight gain.


 


The data from the OECD TG414 study indicates that general systemic toxic effects (in terms of body weight gain) were occurring at the highest exposure dose (300 mg/kg bw/day) where animals survived to necropsy.


Due to the premature sacrifice of the dams treated at 1000 mg/kg bw/day, no data on foetal parameters were determined at this dose level. However, in the 1000 mg/kg bw/day group all except two females were gravid on the day of unscheduled necropsy. In the study no statistically significant treatment-related effects were observed at doses up to 300 mg/kg bw/day for:



  • Developmental endpoints by dose for litters with implants, namely:

  • number of corpora luteum

  • number of implantation site

  • number of live and dead foetuses

  • number of early and late resorptions

  • number of late resorptions

  • number of pre- and post-implantation losses


 



  • Developmental endpoints by dose for litters with live foetuses, namely:

  • number of live offspring

  • foetal sex ratio

  • foetal body weights

  • anogenital distance of foetuses

  • any of the developmental parameters investigated as part of the external, visceral and skeletal examinations for malformations and variations.


 


No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity, i.e. Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH),  in females were observed in the OECD TG414 study on Distilled Grade.


Taken together the data from the OECD TG422 and TG414 studies provides definitive evidence that the test substance at the doses tested does not adversely affect the reproductive processes of mating and pregnancy and does not affect offspring numbers born to exposed females and the early development of these offspring.


An assessment of test substance exposure on the growth and maturation of post-weaning rats through to adulthood is available from a reliable and relevant data (Klimisch Code 1) Repeated Dose 90-day Toxicity Study (OECD TG408) on Distilled Grade. In the test, groups of ten male and ten female (nulliparous and non-pregnant) Wistar rats (approximately 6 weeks of age[1]) were allocated to a control group (no test substance) and three test substance exposure groups (i.e. doses of 50, 150 and 500 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study. In the study the additional sperm parameters measured in the EOGRTS were not addressed and the vaginal smears collected were only assessed at necropsy to determine the stage of the oestrous cycle and allow correlation with the histopathology of ovaries.


Exposure to Distilled Grade did not result in adverse effects in rats at levels of 50 and 150 mg/kg bw/day. Histopathological examinations showed epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females. At 150 and 500 mg/kg bw/day this correlated with macroscopically irregular stomach surfaces. These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. Furthermore, a lower body weight gain was observed in males at 500 mg/kg bw/day which was also considered to be adverse. Other test substance-related changes in clinical signs, body weight, clinical pathology, organ weights and histopathology were considered non-adverse. This included an assessment of the absolute weights of a series of reproductive organs/tissues (testes, epididymides, prostate gland and seminal vesicle in males and ovary and uterus in females) which showed that there were no changes, relative to those in the controls, at any exposure dose. However, when the results were adjusted relative to the terminal body weights of the rats, small, but statistically significant, increases in the weight of the testis (20%) and the epididymis (18%), relative to body weight, were evident in males exposed to 500 mg/kg bw/day when compared to measurements made on the controls. Histopathological examination of the organs/tissues in males and females showed that none of the reproductive organs/tissues in males and females showed observable changes compared to those from the control animals indicating that prolonged exposure did not affect the male and female reproductive systems.


In the study, a marked increase of TSH was observed in males at 150 and 500 mg/kg bw/day and females at 500 mg/kg bw/day, though there was marked variability between the responses of the individuals in these groups which in most instances were within the 95%ile of the historical control data. This change correlated with diverse follicular cell hypertrophy in the thyroid of males, which were generally of limited severity and without degenerative changes. Under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy. Overall no adverse effects on endocrine-disruption related apical morphological and histological endpoints and indicators of thyroid hormonal activity in both sexes were observed in the OECD TG408 study on Distilled Grade.


In summary, the data from the OECD TG422, TG414 and TG408 studies show that general systemic toxic effects were only observed in the presence of local effects due to the known irritancy of the test substance. Observed adverse systemic effects included a decreased body weight (gain) correlated with reduced food consumption which is potentially secondary to the local effects observed on forestomach morphology.  It is accepted that not all the parameters relating to reproductive and developmental toxicity measured in the EOGRTS have been addressed in the OECD TG422, TG414 and TG408 studies.


 


Absence of evidence of reproductive and developmental toxicity in studies on Distillation Residue Grade


Currently reliable and relevant data is available on the potential reproductive and developmental toxicity of Distillation Residue Grade to rats from a Prenatal Developmental Toxicity Study (OECD TG414). A Repeated Dose 90-day Toxicity Study (OECD TG408) is also available which provides reliable data on the potential effects of Distillation Residue Grade on the growth and maturation of male and female rats.


Reliable and relevant data (Klimisch Code 1) are available from a Prenatal Developmental Toxicity Study (OECD TG414) on Distillation Residue Grade which assessed the effects of test substance exposure on offspring birth and development. The test started with the arrival of groups of pre-mated female Wistar rats. Untreated females were mated at the organism supplier and were at Day 0 or 1 post-coitum on arrival (Day 0 post-coitum is the day of successful mating). Twenty-two time-mated females (of approximately 10 to 14 weeks age) were assigned to a control group (no exposure to test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 21 post-coitum, inclusive, which corresponds to the female pregnancy and foetal in-utero development phases of the EOGRTS test. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study along with an extensive series of developmental endpoints in the foetuses. These included litter size, post-implantation loss, foetal sex ratio, foetal body weights, foetal anogenital distance and external, visceral and skeletal malformations and developmental variations in foetuses.


In the study no statistically significant treatment-related maternal effects were evident up to 1000 mg/kg bw/day in terms of:



  • changes in thyroid and uterus weight (in terms of weight or change relative to total body weight)

  • gross pathological abnormalities at necropsy in exposed animals including the uterus

  • microscopic observations in the thyroid gland


In the study no statistically significant treatment-related effects were also observed for:



  • Developmental endpoints by dose for litters with implants, namely:

  • number of corpora luteum

  • number of implantation site

  • number of live and dead foetuses

  • number of early and late resorptions

  • number of late resorptions

  • number of pre- and post-implantation losses


 



  • Developmental endpoints by dose for litters with live foetuses, namely:

  • number of live offspring

  • foetal sex ratio

  • foetal body weights

  • anogenital distance of foetuses

  • any of the developmental parameters investigated as part of the external, visceral and skeletal examinations for malformations and variations.


In summary, the data from the OECD TG414 study indicates that no maternal systemic toxicity was observed in the 100, 300 and 1000 mg/kg/day groups, as no relevant endpoints showed significant differences in responses compared to the control animals. No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity, i.e. T3, T4 and TSH levels, in females were observed in the OECD TG414 study on Distillation Residue Grade.  Based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for (Distillation Residue Grade) of 1000 mg/kg/day was established.


An assessment of test substance exposure on the growth and maturation of post-weaning rats through to adulthood are available from a reliable and relevant data (Klimisch Code 1) Repeated Dose 90-day Toxicity Study (OECD TG408) on Distillation Residue Grade. In the test, groups of ten male and ten female (nulliparous and non-pregnant) Wistar rats (approximately 6 weeks of age[2]) were allocated to a control group (no test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study. In the study the additional sperm parameters measured in the EOGRTS were not addressed and the vaginal smears collected were only assessed at necropsy to determine the stage of the oestrous cycle and allow correlation with the histopathology of ovaries.


  At end of treatment no changes in the weights of a large number of organs were evident including the male and female reproductive organs (i.e. epididymis, seminal vesicle gland and testis in males and the ovary and uterus in females). Higher liver weights were noted for males treated at 300 and 1000 mg/kg bw/day and females treated at 300 (relative to body weight only) and 1000 mg/kg bw/day (absolute and relative to body weight).  This increased liver weight correlated to hepatocellular hypertrophy and an increased incidence of multinucleated hepatocytes, which were seen microscopically in males and females at 1000 mg/kg bw/day and males at 300 mg/kg bw/day. Additionally, clinical biochemistry measurements revealed concurrent changes in liver enzymes. A >4-fold increase in alanine aminotransferase activity (ALAT) was noted in males and females treated at 1000 mg/kg bw/day, in combination with lesser increases in aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP) activity and total bilirubin concentration, which suggests hepatocellular damage.  Therefore, the changes in the liver at 1000 mg/kg bw/day are considered adverse.  The liver changes at 300 mg/kg bw/day were, at the severity observed, not considered to be adverse.


During microscopic examination, erosion/ulcers were noted in the stomach of males at 1000 mg/kg/day.  Due to the degenerative nature of this finding, the effect was considered to be adverse and were considered to be due to the known irritancy of the test substance.


 


Histopathological examination of a large number of organs/tissues in males and females showed that test substance-related microscopic findings after treatment were only noted in the liver (as described previously), mesenteric lymph node, spleen and thyroid glands se well as the stomach of males of males and females. In general these effects were observed at the highest dose (1000 mg/kg bw/day), but were typically of limited severity. With regard to the effects on the thyroid gland, males, starting at the mid-dose of 300 mg/kg bw/day, showed an increased incidence and severity (compared to concurrent control males) of diffuse, bilateral, follicular cell hypertrophy, up to mild degree in the highest dose of 1000 mg/kg bw/day. Given the largely minimal severity of the response (except for a mild degree of severity observed in some males at the highest dose), which was without any degenerative changes, these findings were not considered to be adverse. In females a low incidence and minimal severity of follicular cell hypertrophy was noted in treated animals.  The effect in females was also without any degenerative changes so the findings were not considered to be adverse. In the study, a marked increase of TSH was observed in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day. However, there was marked variability in the responses of individuals at the highest exposure groups and most values were within the 95%ile for the historical control data. There was no accompanying change in thyroid weight in either males or females. Under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy. No test-substance induced changes in T3 and T4 levels were recorded in males and females during the study. Overall, it is concluded that no adverse effects on endocrine-disruption related apical morphological and histological endpoints and indicators of thyroid hormonal activity in both sexes were observed in the OECD TG408 study on Distillation Residue Grade. Based on the results observed in the stomach and liver, the No Observed-Adverse-Effect Level (NOAEL) for local and systemic toxicity was considered to be 300 mg/kg bw/day.


In summary, reliable and relevant subacute and subchronic (OECD TG414 and TG408) studies have been conducted which provide detailed and extensive data on a range of reproductive and developmental endpoints. The OECD TG414 study indicates that exposure to the test substance Distillation Residue Grade resulted in no adverse effects on the reproductive organs and fertility of parental animals and the development of resulting offspring. In an OECD TG408 Repeated Dose 90-day Toxicity Study there were no observable changes on reproductive organs/tissues in males and females compared to those from the control animals at the highest test dose. It is accepted that not all the parameters relating to reproductive and developmental toxicity measured in the EOGRTS have been addressed in the OECD TG414 and TG408 studies.


 


Control of risks to human health in all exposure scenarios


 


The Risk Characterisation Exercise in the Chemical Safety Report (CSR) carried out for Technical Grade using the updated Derived No Effect Levels (DNELs) generated using the new interpolated data from Annex IX and X testing with the OECD TG408 and TG414 studies shows that in all cases the RCR values for human health (workers and consumers) assessment in all the identified exposure scenarios were less than 1 (indicating an acceptable level of risk as humans are not exposed to the test substance at levels above the relevant “safe” threshold(s)).  .


Technical Grade is classified as a Skin Sensitiser Cat 1A, Skin Irritant Cat. 2 and Eye Damage Cat 1. Given the classification of Technical Grade as a skin irritant and sensitiser and an eye irritant, all skin and mucous membranes with potential exposure should be protected with appropriate PPE. Therefore, in all the exposure scenarios appropriate risk management measures in the CSR are specified, namely:



  • Hand protection to EN374 standard as minimum and Eye protection to EN166 standard as minimum are employed for all operations.


 



  • Protective clothing to EN368 standard as minimum is used to restrict exposure to the workforce.


 


On this basis workers are expected to be protected adequately from exposure (according to controlled risk described in the CSR) and thus systemic effects after dermal exposure are expected to be minimal. For consumers it is assumed that the advice provided on personal protection during the use of products containing Distillation Residue Grade are followed.


In addition, the OECD TG414 and TG408 studies for the target substances Distilled and Distillation Residue Grades (and by interpolation Technical Grade) show that general systemic toxic effects only occur in the presence of local effects due to the known irritancy of the constituents of the test substance. Therefore, sustained dermal exposure which could potentially lead to systemic reproductive and developmental effects in workers and consumers is not to be expected without significant local effects which would restrict exposure.


Overall, it is not expected that provision of further information from the EOGRTS would change the perceived risk posed to the general population (consumers) and workers by exposure to Technical Grade via the exposure scenarios identified in the Chemical Safety Report.


 


 


3. Summary


The following points are considered relevant to the adaption of the Extended One Generation Reproductive Toxicity Study for Technical Grade:


1. There is a definitive body of evidence that the test substance does not adversely affect the reproductive processes of mating and pregnancy and does not affect the numbers born to exposed females and the early development of the offspring. The absence of evidence of reproductive and developmental toxicity in cattle due to Technical Grade is from a series of studies by Kaneda and Mochizuk (2016). For the other members of the category Distilled and Distillation Residue Grades a series of reliable and relevant subacute and subchronic (OECD TG422, TG414 and TG408) studies have been conducted which provide detailed and extensive data on a range of reproductive and developmental endpoints. These studies indicate that exposure to either Distilled or Distillation Residue Grade resulted in no adverse effects on the reproductive organs and fertility of parental animals and the development of resulting offspring.


 


2. Although the conduct of the EOGRTS test could potentially provide additional data on reproductive and developmental toxicity endpoints it would not improve the overall conclusions on the perceived risks posed to human health by exposure to the test substance via the identified exposure scenarios in the Chemical Safety Report. In addition, studies show that general systemic toxic effects only occur in the presence of local effects due to the known irritancy of the test substance. Therefore, sustained dermal exposure which could potentially lead to systemic effects in workers and consumers is not to be expected without significant local effects. This would limit the total dose to which individuals would be exposed.


 


3. Taking account of all these points together it is proposed to adapt the conduct of the Extended One-Generation Reproductive Toxicity Study on Technical Grade and adopt a weight of evidence approach. Given the currently available dataset addressing key reproduction and developmental toxicity endpoints conduct of the EOGRTS would be disproportionate and inappropriate for this Natural Complex Substance.


 


References


ECHA (2017) Guidance on Information Requirements and Chemical Safety Assessment. Chapter R.7a: Endpoint specific guidance (Version 6.0, July 2017). European Chemical Agency, Helsinki.


ECHA Board of Appeal (2015) Judgement Case Number A-005-20014, Summary of Decision of 23 September 2015 of the Board of Appeal of the European Chemicals Agency. Available at: https://echa.europa.eu/documents/10162/b224dd5a-03a8-40a6-9e42-42b3e56a8c74


Kaneda, K and Mochizuki, M (2016) Milk yield and/or milk quality, perinatal disease preventative or therapeutic agent, and reproductivity improving agent for ruminants.  Confidential Report.


Klimisch, H-J, Andreae, M and Tillmann, U (1997) A systematic approach for evaluating the quality of experimental toxicological and ecotoxicological data. Regulatory Toxicology and Pharmacology, 25, 1–5


Judgement of the General Court (2019) In Case T-755/17. Details available at: http://curia.europa.eu/juris/celex.jsf?celex=62017TJ0755&lang1=en&type=TXT&ancre


 


[1] In the OECD TG408 study dosing should begin as soon as possible after weaning and, in all cases, before the animals are nine weeks old


 


[2] In the OECD TG408 study dosing should begin as soon as possible after weaning and, in all cases, before the animals are nine weeks old


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Guideline:
other: Investigating reproductivity as well as changes in milk yield and/or milk quality and the incidence of perinatal disease
Principles of method if other than guideline:
In the study evaluating the effect of feeding Technical Grade on reproductivity adult female Holstein cattle were used. Individuals which had calved were divided at random into a test group and control group. Technical Grade was fed to the individuals in the test group in pellets at an amount of 100 g (10 g in terms of CNSL) per day for 5 days from the date of calving.
GLP compliance:
not specified
Remarks:
Satisfying the K2 requirement:“Studies or data (mostly not performed according to GLP)…or in which investigations are described that cannot be subsumed under a testing guideline,but which are nevertheless well-documented and scientifically acceptable”
Justification for study design:
not applicable
Species:
other: cattle
Details on species / strain selection:
adult female Holstein cattle were used
Sex:
female
Route of administration:
oral: feed
Details on exposure:
Technical Grade was fed to the individuals in the test group in pellets at an amount of 100 g (10 g in terms of CNSL) per day for 5 days from the date of calving. The test group which was fed Technical Grade consisted of 9 cows (2 cows having had two calves, 4 cows having had three calves, 1 cow having had four calves, 1 cow having had five calves and 1 cow having had six calves). Five cows (all of which had had three calves) were allocated to control group and were not fed Technical Grade containing-pellets. Although the feed was modified depending on the condition of the cows, it was designed so that 7 kg of timothy hay (which is high in fibre and energy content but low in protein content) and 2 to 3 kg of a concentrate feed were fed on average per day.
Details on mating procedure:
Insemination
Dose / conc.:
10 other: g/day
Remarks:
Technical grade was fed to the individuals in the test group in pellets at an amount of 100 g (10 g in terms of CNSL) per day for 5 days from the date of calving. This corresponds to a dose of 20 mg/kg bw/day based on a typical animal weight of 450 to 680 kg depending on age
No. of animals per sex per dose:
The test group which was fed Technical Grade consisted of 9 cows (2 cows having had two calves, 4 cows having had three calves, 1 cow having had four calves, 1 cow having had five calves and 1 cow having had six calves).
Control animals:
yes, plain diet
The test results from the reproductivity study are shown in Table 1 and indicate that the feeding of the Technical Grade-containing pellet was found to result in improvements in almost all the endpoints for the test group compared with control group. In particular, significant effects in terms of reducing the number of inseminations, decreasing the number of non-pregnant days and actual non-pregnant days and improving the pregnancy rate were measured. Further, the feeding of Technical grade was able to achieve a conception rate exceeding 50-60% and an oestrus detection rate exceeding 70% or more of target levels, respectively. No change was noted in the days of first insemination. Overall, the results show that the feeding of the Technical Grade-containing pellet was found to significantly improve the reproductivity in each individual cow and to further significantly improve the reproductivity in the oestrus detection rate and pregnancy rate in each group. On the basis of this data Technical Grade does not evidently have any adverse toxic effects on the reproduction of cattle.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Dose descriptor:
dose level:
Generation:
F1
Effect level:
ca. 10 other: g/day
Based on:
test mat.
Sex:
not specified
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
no

Effects of feeding Technical grade on the reproductivity of cows

 

 

Endpoint

 

Mean results for the Technical grade group (n = 9)

 

 

Mean results for the control group (n = 5)

 

Variation

Number of insemination times

2.0

2.6

-0.6

Non-pregnant days

111

137

-26

Non-pregnant days: 90 days of less (%)

33.3

20.0

13.3

Non-pregnant days: 120 days of more (%)

33.3

60.0

-26.7

Actual non-pregnant days

32.1

58.3

-26.2

Days of first insemination

78.9

78.7

0.2

Pregnancy rate at first service (%)

33.3

16.7

16.6

Pregnancy rate (%)

50.0

38.5

11.5

Oestrus detection rate (%)

79.1

68.9

10.2

 

Notes:

Number of insemination = times of artificial insemination

Non-pregnant days = last conception day - last calving day

Actual non-pregnant days = days from first to last insemination  

Days of first insemination = first insemination day after calving - last calving day

Pregnancy rate at first service = rate of conception achieved by first insemination

Pregnancy rate = [number of individuals with conception (group total)/total times of artificial insemination] x 100

Detection rate of oestrus (whole group) = [average number of insemination/((average actual days of non-pregnant/21)+1] x100

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The basis for the adoption of a category approach for the three grades of processed Cashew Nutshell Extract is the commonality of the constituents and functional groups in the three grades and the common modes of action for specific localised endpoints that are manifest in physico-chemical, environmental fate and toxicological properties that are similar or follow a regular pattern as a result of structural similarity. The ECHA Final Decisions on the Annex IX and X (mammalian toxicology, ecotoxicology and environmental fate) Testing Proposals for three grades confirmed the applicability of a category approach which involves reading data across from the source substances Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to the target substance Cashew Nutshell Extract, Decarboxylated (Technical Grade) by interpolation. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”. Further details on the justification for using the interpolation based read-across approach are given in the attached document “Report on the grouping and read-across rationale for the three grades of processed Cashew Nutshell Extract”. The interpolation approach has been applied to the Annex IX OECD TG408 and TG414 testing on Distilled and Distillation Residue Grades. For the Annex VII and VIII endpoints a programme of testing has been carried out to provide comparative information for the three grades and develop a data matrix to support the category and read-across approaches. This has involved generating reliable data for all Annex VII and VIII physico-chemical and environmental fate endpoints and certain mammalian toxicity properties. However, based on the discussions surrounding the Final Decisions it was not considered necessary to apply this approach retrospectively to address all identified data gaps at Annexes VII and VIII. The original data used for the registration of a single substance (Cashew Nutshell Liquid, CAS Number 8007-24-7) was largely based on data for Distilled Grade since this was considered to be the most (eco) toxicologically active form, given the higher content of low molecular weight constituents (such as cardanol) and the low content of polymeric species. Therefore, based on the use of the category approach it is considered appropriate to read-across from the existing short-term mammalian toxicity data for Distilled Grade to the other two grades.
2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
For the mammalian reproductive toxicity endpoint no test data is available for the registered substance, Technical Grade. However data is available for the compositionally similar substance Distilled Grade which is part of the category of the three grades of CNSL. In a subacute 28-day Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test Distilled Grade was administered to 5 Sprague-Dawley rats/sex/dose via oral gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day). Changes in the lungs, mesenteric lymph node occurred at the highest dose of 1000 mg/kg bw/day resulting in a NOAEL of 150 mg/kg bw/day for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive toxicity based on an absence of effects of the test substance on the relevant reproductive parameters. It is considered appropriate to read-across from the data for Distilled Grade to Technical Grade with the result that it has been estimated that Technical Grade does not result effects on mammalian reproductive toxicity parameters at an exposure dose of 1000 mg/kg bw/day.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant deaths during the study.

Increased salivation was detected prior to dosing and up to 5 hours after dosing for animals of either sex treated with 1000 mg/kg bw/day from Day 9 onwards. One female treated with 150 mg/kg bw/day developed clinical signs consistent with inappropriate dosing on Day 5 and was subsequently terminated. One female treated with 1000 mg/kg bw/day had given birth to a number of pups of which the majority were found dead. Several clinical signs were observed in this animal, and the animal and litter were terminated.

BODY WEIGHT AND WEIGHT GAIN
A slightly reduced bodyweight gain was observed for 1000 mg/kg bw/day males during the first two weeks of the study. Reduced bodyweight gain was also observed for 1000 mg/kg bw/day females during the later stages of the gestation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency were detected.

FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency were detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
Haematological assessments revealed elevated platelet counts in animals of either sex treated with 1000 mg/kg bw/day. Elevated haemoglobin, erythrocyte and haematocrit was also evident for males treated at 1000 mg/kg bw/day. No such effects were detected at 150 and 15 mg/kg bw/day.

CLINICAL CHEMISTRY
An increase in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were observed for 1000 mg/kg bw/day animals of either sex, together with elevated inorganic phosphorus, bilirubin and urea, and reduced cholesterol levels.

URINALYSIS
N/A

NEUROBEHAVIOUR
Open field arena observations revealed increased salivation for individual animals of either sex treated with 1000 mg/kg bw/day from Week 3.

ORGAN WEIGHTS
Females treated with 1000 mg/kg bw/day showed elevated liver weights.

GROSS PATHOLOGY
None

HISTOPATHOLOGY: NON-NEOPLASTIC
Groups of alveolar macrophages were seen with a higher incidence for females treated with 1000 mg/kg bw/day.
A higher incidence of sinus histiocytosis and/or foamy histiocytes was observed in relation to treatment with 1000 mg/kg bw/day.
Hyperkeratosis, frequently associated with acanthosis was seen in the forestomachs of animals of either sex treated with 1000 mg/kg bw/day. Focal ulceration of the forestomach epithelium was also seen in one high dose female.
Mucosal hypertrophy was seen in three males at the top dose group. A low incidence of mucosal hypertrophy was observed in females in all dose groups.

OTHER FINDINGS
MATING
No adverse effects on mating or fertility were observed.

OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: BAsed on systemic changes in the lungs, mesenteric lymph node, stomach and duodenum.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment effects occurred on reproduction or offspring development.
Reproductive effects observed:
not specified

A document which justifies the read–across from Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Grade”) and Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (“Distillation Residue Grade”) to Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated (“Technical Grade”) is attached in the Category object.

Conclusions:
A NOAEL of 150 mg/kg bw/day has been identified for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.
Executive summary:

In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).

 

Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose.The NOAEL is 150 mg/kg bw/day.

 

This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The basis for the adoption of a category approach for the three grades of processed Cashew Nutshell Extract is the commonality of the constituents and functional groups in the three grades and the common modes of action for specific localised endpoints that are manifest in physico-chemical, environmental fate and toxicological properties that are similar or follow a regular pattern as a result of structural similarity. The ECHA Final Decisions on the Annex IX and X (mammalian toxicology, ecotoxicology and environmental fate) Testing Proposals for three grades confirmed the applicability of a category approach which involves reading data across from the source substances Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to the target substance Cashew Nutshell Extract, Decarboxylated (Technical Grade) by interpolation. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”. Further details on the justification for using the interpolation based read-across approach are given in the attached document “Report on the grouping and read-across rationale for the three grades of processed Cashew Nutshell Extract”. The interpolation approach has been applied to the Annex IX OECD TG408 and TG414 testing on Distilled and Distillation Residue Grades. For the Annex VII and VIII endpoints a programme of testing has been carried out to provide comparative information for the three grades and develop a data matrix to support the category and read-across approaches. This has involved generating reliable data for all Annex VII and VIII physico-chemical and environmental fate endpoints and certain mammalian toxicity properties. However, based on the discussions surrounding the Final Decisions it was not considered necessary to apply this approach retrospectively to address all identified data gaps at Annexes VII and VIII. The original data used for the registration of a single substance (Cashew Nutshell Liquid, CAS Number 8007-24-7) was largely based on data for Distilled Grade since this was considered to be the most (eco) toxicologically active form, given the higher content of low molecular weight constituents (such as cardanol) and the low content of polymeric species. Therefore, based on the use of the category approach it is considered appropriate to read-across from the existing short-term mammalian toxicity data for Distilled Grade to the other two grades.
2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
For the mammalian reproductive toxicity endpoint no rodent-based test data is available for the registered substance, Technical Grade. However, data on the growth and maturation of post-weaning rats through to adulthood is also available for the compositionally similar substance Distilled Grade which is part of the category of the three grades of CNSL. In a reliable and relevant (Klimisch Code 1) sub-chronic OECD TG408 Repeated Dose 90-day Toxicity Study, Distilled Grade was administered to groups of ten male and ten female (nulliparous and non-pregnant) Wistar rats (approximately 6 weeks of age) allocated to a control group (no test substance) and three test substance exposure groups (i.e. doses of 50, 1500 and 500 mg/kg bw/day). The test substance and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study.
Exposure to Distilled Grade did not result in adverse effects in rats at levels of 50 and 150 mg/kg bw/day. Histopathological examinations showed epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females. These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. Furthermore, a lower body weight gain was observed in males at 500 mg/kg bw/day which was also considered to be adverse. Other test substance-related changes in clinical signs, body weight, clinical pathology, organ weights and histopathology were considered non-adverse. This included an assessment of the absolute weights of a series of reproductive organs/tissues (testes, epididymides, prostate gland and seminal vesicle in males and ovary and uterus in females) which showed that there were no changes, relative to those in the controls, at any exposure dose. However, when the results were adjusted relative to the terminal body weights of the rats, small, but statistically significant, increases in the weight of the testis (20%) and the epididymis (18%), relative to body weight, were evident in males exposed to 500 mg/kg bw/day when compared to measurements made on the controls. Histopathological examination of the organs/tissues in males and females showed that none of the reproductive organs/tissues in males and females showed observable changes compared to those from the control animals indicating that prolonged exposure did not affect the male and female reproductive systems.
In the study, a marked increase of TSH was observed in males at 150 and 500 mg/kg bw/day and females at 500 mg/kg bw/day, though there was marked variability between the responses of the individuals in these groups with most values being within the 95%ile limit for historical control data. This change correlated with diverse follicular cell hypertrophy in the thyroid of males, which were without degenerative changes. Under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy. Overall no adverse effects on endocrine-disruption related apical morphological and histological endpoints and indicators of thyroid hormonal activity in both sexes were observed in the OECD TG408 study on Distilled Grade.
It is considered appropriate to read-across from the data for Distilled Grade to Technical Grade as part of a weight of evidence approach for the EOGRTS information requirement.
Qualifier:
according to guideline
Guideline:
other: OECD TG 408
Species:
rat
Strain:
Wistar
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
An assessment of test substance exposure on the growth and maturation of post-weaning rats through to adulthood is available from a reliable and relevant data (Klimisch Code 1) Repeated Dose 90-day Toxicity Study (OECD TG408) on Distilled Grade. In the test, groups of ten male and ten female (nulliparous and non-pregnant) Wistar rats (approximately 6 weeks of age ) were allocated to a control group (no test substance) and three test substance exposure groups (i.e. doses of 50, 150 and 500 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy.
Exposure to Distilled Grade did not result in adverse effects in rats at levels of 50 and 150 mg/kg bw/day. Histopathological examinations showed epithelial hyperplasia of the non-glandular mucosa of the stomach (forestomach, squamous mucosa) at 50, 150 and 500 mg/kg bw/day in males and females with a dose-related increase in severity, up to marked in males and moderate in females. At 150 and 500 mg/kg bw/day this correlated with macroscopically irregular stomach surfaces. These findings at 500 mg/kg bw/day were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. Furthermore, a lower body weight gain was observed in males at 500 mg/kg bw/day which was also considered to be adverse. Other test substance-related changes in clinical signs, body weight, clinical pathology, organ weights and histopathology were considered non-adverse. This included an assessment of the absolute weights of a series of reproductive organs/tissues (testes, epididymides, prostate gland and seminal vesicle in males and ovary and uterus in females) which showed that there were no changes, relative to those in the controls, at any exposure dose. However, when the results were adjusted relative to the terminal body weights of the rats, small, but statistically significant, increases in the weight of the testis (20%) and the epididymis (18%), relative to body weight, were evident in males exposed to 500 mg/kg bw/day when compared to measurements made on the controls. Histopathological examination of the organs/tissues in males and females showed that none of the reproductive organs/tissues in males and females showed observable changes compared to those from the control animals indicating that prolonged exposure did not affect the male and female reproductive systems.
In the study, a marked increase of TSH was observed in males at 150 and 500 mg/kg bw/day and females at 500 mg/kg bw/day, though there was marked variability between the responses of the individuals in these groups resulting in biomodal distributions of TSH levels. This change correlated with diverse follicular cell hypertrophy in the thyroid of males, which were without degenerative changes. Although this change in TSH level was considered to be test substance-related, under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and thyroid follicular cell hypertrophy. Overall no adverse effects on endocrine-disruption related apical morphological and histological endpoints and indicators of thyroid hormonal activity in both sexes were observed in the OECD TG408 study on Distilled Grade.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The basis for the adoption of a category approach for the three grades of processed Cashew Nutshell Extract is the commonality of the constituents and functional groups in the three grades and the common modes of action for specific localised endpoints that are manifest in physico-chemical, environmental fate and toxicological properties that are similar or follow a regular pattern as a result of structural similarity. The ECHA Final Decisions on the Annex IX and X (mammalian toxicology, ecotoxicology and environmental fate) Testing Proposals for three grades confirmed the applicability of a category approach which involves reading data across from the source substances Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to the target substance Cashew Nutshell Extract, Decarboxylated (Technical Grade) by interpolation. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”. Further details on the justification for using the interpolation based read-across approach are given in the attached document “Report on the grouping and read-across rationale for the three grades of processed Cashew Nutshell Extract”. The interpolation approach has been applied to the Annex IX OECD TG408 and TG414 testing on Distilled and Distillation Residue Grades. For the Annex VII and VIII endpoints a programme of testing has been carried out to provide comparative information for the three grades and develop a data matrix to support the category and read-across approaches. This has involved generating reliable data for all Annex VII and VIII physico-chemical and environmental fate endpoints and certain mammalian toxicity properties. However, based on the discussions surrounding the Final Decisions it was not considered necessary to apply this approach retrospectively to address all identified data gaps at Annexes VII and VIII. The original data used for the registration of a single substance (Cashew Nutshell Liquid, CAS Number 8007-24-7) was largely based on data for Distilled Grade since this was considered to be the most (eco) toxicologically active form, given the higher content of low molecular weight constituents (such as cardanol) and the low content of polymeric species. Therefore, based on the use of the category approach it is considered appropriate to read-across from the existing short-term mammalian toxicity data for Distilled Grade to the other two grades.
2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
For the mammalian reproductive toxicity endpoint no rodent-based test data is available for the registered substance, Technical Grade. Data on the growth and maturation of post-weaning rats through to adulthood is also available for the compositionally similar substance Distillation Residue Grade which is part of the category of the three grades of CNSL. In a reliable and relevant (Klimisch Code 1) sub-chronic OECD TG408 Repeated Dose 90-day Toxicity Study, Distillation Residue Grade was administered to groups of ten male and ten female (nulliparous and non-pregnant) Wistar rats (approximately 6 weeks of age) allocated to a control group (no test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study.
In the study there were no observable changes on reproductive organs/tissues in males and females compared to those from the control animals at the highest test dose. Adverse findings were noted in the livers of males and females (in terms of weights, histopathology and enzyme function) at 1000 mg/kg bw/day. Erosion/ulcers were also noted in the stomach of males at 1000 mg/kg bw/day. Due to the degenerative nature of this finding, the effect was considered to be adverse and was considered to be due to the known irritancy of the test substance. It is concluded that no adverse effects on endocrine-disruption related apical morphological and histological endpoints and indicators of thyroid hormonal activity (i.e. T3, T4 and TSH levels) in both sexes were observed in the OECD TG408 study on Distillation Residue Grade. A NOAEL of 1000 mg/kg bw/day has been identified for systemic toxicity based on these results.
It is considered appropriate to read-across from the data for Distillation Residue Grade to Technical Grade as part of a weight of evidence approach for the EOGRTS information requirement.
Qualifier:
according to guideline
Guideline:
other: OECD TG408
Species:
rat
Strain:
Wistar
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The basis for the adoption of a category approach for the three grades of processed Cashew Nutshell Extract is the commonality of the constituents and functional groups in the three grades and the common modes of action for specific localised endpoints that are manifest in physico-chemical, environmental fate and toxicological properties that are similar or follow a regular pattern as a result of structural similarity. The ECHA Final Decisions on the Annex IX and X (mammalian toxicology, ecotoxicology and environmental fate) Testing Proposals for three grades confirmed the applicability of a category approach which involves reading data across from the source substances Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to the target substance Cashew Nutshell Extract, Decarboxylated (Technical Grade) by interpolation. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”. Further details on the justification for using the interpolation based read-across approach are given in the attached document “Report on the grouping and read-across rationale for the three grades of processed Cashew Nutshell Extract”. The interpolation approach has been applied to the Annex IX OECD TG408 and TG414 testing on Distilled and Distillation Residue Grades. For the Annex VII and VIII endpoints a programme of testing has been carried out to provide comparative information for the three grades and develop a data matrix to support the category and read-across approaches. This has involved generating reliable data for all Annex VII and VIII physico-chemical and environmental fate endpoints and certain mammalian toxicity properties. However, based on the discussions surrounding the Final Decisions it was not considered necessary to apply this approach retrospectively to address all identified data gaps at Annexes VII and VIII. The original data used for the registration of a single substance (Cashew Nutshell Liquid, CAS Number 8007-24-7) was largely based on data for Distilled Grade since this was considered to be the most (eco) toxicologically active form, given the higher content of low molecular weight constituents (such as cardanol) and the low content of polymeric species. Therefore, based on the use of the category approach it is considered appropriate to read-across from the existing short-term mammalian toxicity data for Distilled Grade to the other two grades.
2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
For the mammalian reproductive toxicity endpoint no rodent-based test data is available for the registered substance, Technical Grade. However, data on offspring birth and development are also available for the compositionally similar substance Distilled Grade which is part of the category of the three grades of CNSL. In a reliable and relevant (Klimisch Code 1) OECD TG414 Prenatal Developmental Toxicity Study, Distilled Grade was administered to twenty-two time-mated females (of approximately 10 to 14 weeks age) assigned to a control group (no exposure to test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 21 post-coitum, inclusive. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study along with an extensive series of developmental endpoints in the foetuses.
In the study animals in the high dose treatment (1000 mg/kg bw/day) group experienced severe toxicity (as body weight loss, decreased food consumption and adverse clinical signs) over Days 14 to 17. To prevent further suffering, it was decided by the veterinarian at the test laboratory to sacrifice all the remaining high dose (1000 mg/kg/day) animals on Study Day 18 for ethical reasons. It was concluded that the effects observed at the highest dose were probably due to the known irritancy of the test substance which caused localised changes in stomach morphology and limited food consumption. No mortality was observed for the animals in the remaining groups during the study.
The test substance administered at dose levels of 100 mg/kg bw/day was not considered to cause maternal toxicity. No systemic toxic effects and non-adverse local effects on the surface of the forestomach were observed. At the 300 mg/kg bw/day dose level, the test substance was maternally toxic causing both adverse systemic toxic effects (on body weight gain). Body weight gain at 300 mg/kg bw/day was slightly lower from Day 12 post-coitum onwards, reaching statistical significance on Days 18 and 21 post-coitum only (0.88x and 0.83x of controls, respectively). Body weight gain, corrected for the weight of the uterus of the pregnant female, was statistically significantly lower compared with concurrent controls (on average 50% lower). Adverse local effects on the surface of the forestomach were also evident at 300 mg/kg bw/day which could also have caused secondary effects on body weight gain.
No toxicologically significant changes were noted in any of the developmental parameters after treatment up to 300 mg/kg bw/day. Due to the premature sacrifice of the dams treated at 1000 mg/kg bw/day, no data on foetal parameters were determined at this dose level. However, in the 1000 mg/kg bw/day group all except two females were gravid on the day of unscheduled necropsy.
The data from the OECD TG414 study indicates that general systemic toxic effects (in terms of body weight gain) were occurring at the highest exposure dose (300 mg/kg bw/day) where animals survived to necropsy whilst potentially endocrine-mediated effects on reproductive and developmental endpoints were not evident at any treatment dose (100 and 300 mg/kg bw/day). No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity (i.e. T3, T4 and TSH levels) in females were observed in the OECD TG414 study on Distilled Grade. It is considered appropriate to read-across from the data for Distilled Grade to Technical Grade as part of a weight of evidence approach for the EOGRTS information requirement.
Qualifier:
according to guideline
Guideline:
other: OECD TG 414
Species:
rat
Strain:
Wistar
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: maternal toxicity
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Conclusions:
Reliable and relevant data (Klimisch Code 1) are also available from a Prenatal Developmental Toxicity Study (OECD TG414) on Distilled Grade which assessed the effects of test substance exposure on offspring birth and development. The test started with the arrival of groups of pre-mated female Wistar rats. Untreated females were mated at the organism supplier and were at Day 0 or 1 post-coitum on arrival (Day 0 post-coitum is the day of successful mating). Twenty-two time-mated females (of approximately 10 to 14 weeks age) were assigned to a control group (no exposure to test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle (propylene glycol) were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 21 post-coitum, inclusive, which corresponds to the female pregnancy and foetal in-utero development phases of the EOGRTS test.
effects on body weight gain.
No toxicologically significant changes were noted in any of the developmental parameters after treatment up to 300 mg/kg bw/day. Due to the premature sacrifice of the dams treated at 1000 mg/kg bw/day, no data on foetal parameters were determined at this dose level. However, in the 1000 mg/kg bw/day group all except two females were gravid on the day of unscheduled necropsy.
The data from the OECD TG414 study indicates that general systemic toxic effects (in terms of body weight gain) were occurring at the highest exposure dose (300 mg/kg bw/day) where animals survived to necropsy whilst potentially endocrine-mediated effects on reproductive and developmental endpoints were not evident at any treatment dose (100 and 300 mg/kg bw/day). No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity (i.e. T3, T4 and TSH levels) in females were observed in the OECD TG414 study on Distilled Grade.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The basis for the adoption of a category approach for the three grades of processed Cashew Nutshell Extract is the commonality of the constituents and functional groups in the three grades and the common modes of action for specific localised endpoints that are manifest in physico-chemical, environmental fate and toxicological properties that are similar or follow a regular pattern as a result of structural similarity. The ECHA Final Decisions on the Annex IX and X (mammalian toxicology, ecotoxicology and environmental fate) Testing Proposals for three grades confirmed the applicability of a category approach which involves reading data across from the source substances Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to the target substance Cashew Nutshell Extract, Decarboxylated (Technical Grade) by interpolation. In this context interpolation is “the estimation of a value for a member of the group using measured values from other members on both sides of that member within the defined group spectrum”. Further details on the justification for using the interpolation based read-across approach are given in the attached document “Report on the grouping and read-across rationale for the three grades of processed Cashew Nutshell Extract”. The interpolation approach has been applied to the Annex IX OECD TG408 and TG414 testing on Distilled and Distillation Residue Grades. For the Annex VII and VIII endpoints a programme of testing has been carried out to provide comparative information for the three grades and develop a data matrix to support the category and read-across approaches. This has involved generating reliable data for all Annex VII and VIII physico-chemical and environmental fate endpoints and certain mammalian toxicity properties. However, based on the discussions surrounding the Final Decisions it was not considered necessary to apply this approach retrospectively to address all identified data gaps at Annexes VII and VIII. The original data used for the registration of a single substance (Cashew Nutshell Liquid, CAS Number 8007-24-7) was largely based on data for Distilled Grade since this was considered to be the most (eco) toxicologically active form, given the higher content of low molecular weight constituents (such as cardanol) and the low content of polymeric species. Therefore, based on the use of the category approach it is considered appropriate to read-across from the existing short-term mammalian toxicity data for Distilled Grade to the other two grades.
2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
For the mammalian reproductive toxicity endpoint limited no rodent-based test data is available for the registered substance, Technical Grade. Data on offspring birth and development are also available for the compositionally similar substance Distillation Residue Grade which is part of the category of the three grades of CNSL. In a reliable and relevant (Klimisch Code 1) OECD TG414 Prenatal Developmental Toxicity Study, Distillation Residue Grade was administered to twenty-two time-mated females (of approximately 10 to 14 weeks age) assigned to a control group (no exposure to test substance) and three test substance exposure groups (i.e. doses of 100, 300 and 1000 mg/kg bw/day). The test substance and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 21 post-coitum, inclusive. Clinical signs, functional observations, body weight development and food and water consumption were monitored during the study along with an extensive series of developmental endpoints in the foetuses.
In the study there were no statistically significant treatment-related effects on the thyroid gland in females (in terms of weight or change relative to total body weight) at doses up to 1000 mg/kg bw/day. No statistically significant treatment-related effects were observed for a number of reproductive endpoints related to corpora luteum, implantation sites, viable foetuses, dead foetuses, early resorptions, late resorptions, pre-implantation loss and post-implantation loss. No test substance-related changes were noted in any of the developmental parameters investigated as part of the external, visceral and skeletal examinations for malformations and variations. The data from the OECD TG414 study indicates that no maternal systemic toxicity was observed in the 100, 300 and 1000 mg/kg/day groups, as no relevant endpoints showed significant differences in responses compared to the control animals. No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity (i.e. T3, T4 and TSH levels) in females were observed in the OECD TG414 study. Based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) of 1000 mg/kg bw/day was established.
It is considered appropriate to read-across from the data for Distillation Residue Grade to Technical Grade as part of a weight of evidence approach for the EOGRTS information requirement.
Qualifier:
according to guideline
Guideline:
other: OECD TG414
Species:
rat
Strain:
Wistar
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: maternal toxicity
Remarks on result:
not measured/tested
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For the assessment of the toxicity to reproduction the read across substances are Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade). A document which justifies the read–across from Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated (Technical Grade) is attached.


 


Short description of key information:


In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).


Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose. The NOAEL is 150 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.


This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.


In valid (reliable without restrictions, Klimisch Code 1) OECD TG414 Prenatal developmental toxicity studies on the source substances Distilled and Distillation Residue Grades groups of time-mated Wistar Han female rats were exposed to doses of 100, 300 and 1000 mg/kg bw/day by once daily oral gavage for 14 days from Day 6 to 20 post-coitum.


 


In the study on Distilled Grade severe toxicity of the test substance was observed in females treated at 1000 mg/kg bw/day and this complete high dose group was euthanized on animal welfare grounds in extremis on Days 12-18 post-coitum.  In these animals, slight to severe body weight loss (up to 20%) and/or strongly reduced body weight gain, strongly reduced food consumption (up to 50% lower than controls), severe macroscopic alterations of the stomach wall (irregular surface) and/or severe clinical signs. At the mid-dose 300 mg/kg bw/day treatment mild to moderate test substance-related effects were evident. Microscopy revealed ulceration/erosion, hyperplasia with keratosis and sub-epithelial inflammatory cell infiltrates. The forestomach findings are considered a local test substance-related effect, most likely caused by the known irritating properties of cardol and, to a lesser extent, cardanol constituents of the test substance. At this dose level body weight gain was slightly lower from Day 12 post-coitum onwards, reaching statistical significance on Days 18 and 20 post-coitum only (0.88x and 0.83x of controls, respectively). 


 


In the corresponding study on Distillation Residue Grade no maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups, as no relevant endpoints showed significant differences in responses compared to the control animals.


 


No test substance-related microscopic observations in the thyroid glands of females were observed in animals surviving to necropsy in both studies as the recorded responses were within the range of normal background pathology encountered in rats of this age and sex for both grades. In both studies no test substance induced changes in triiodothyronine (T3) or thyroxine (T4) were evident in adult females surviving to necropsy (300 and 1000 mg/kg bw/day for Distilled and Distillation Residue Grades respectively). In both studies increases in thyroid stimulating hormone (TSH), showing considerable inter-group variability, but which are largely consistent with the 95%ile limit for historical control data of the test laboratory, were evident in adult females surviving to necropsy. Given that the possible adversity of these effects could not be assessed within this type of study the finding was not taken into account when determining the maternal NOAEL.


 


In both studies no toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, fetal anogenital distance, external, as well as visceral and skeletal malformations and developmental variations) were evident in animals surviving to necropsy.


 


The data from the oral gavage OECD TG414 study on Distilled Grade is consistent with the data from the OECD TG408 studies and indicates that there is localised epithelial hyperplasia of the non-glandular mucosa (forestomach, squamous mucosa) in the stomach at higher exposure doses. The stomach findings were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. This response is considered to be a result of the known irritancy of the test substance due to the presence of the constituents cardol, and to a lesser extent, cardanol. No comparable effects were observed in the OECD TG414 study on Distillation Residue Grade. In the study on Distillation Residue Grade gross lesions (dark red discoloration) were noted in the mesenteric lymph nodes for 3/22 females at 1000 mg/kg bw/day. However, no test substance-related microscopic findings were observed. No comparable effects were observed in the study on Distilled Grade.


 


These Prenatal developmental toxicity studies are acceptable and satisfy the guideline requirement for an OECD TG414 study in rats.


In valid (reliable without restrictions, Klimisch Code 1) OECD TG414 Prenatal developmental toxicity studies on the source substances Distilled and Distillation Residue Grades groups of time-mated Wistar Han female rats were exposed to doses of 100, 300 and 1000 mg/kg bw/day by once daily oral gavage for 14 days from Day 6 to 20 post-coitum.


 


 In the study on Distilled Grade severe toxicity of the test substance was observed in females treated at 1000 mg/kg bw/day and this complete high dose group was euthanized on animal welfare grounds in extremis on Days 12-18 post-coitum.  In these animals, slight to severe body weight loss (up to 20%) and/or strongly reduced body weight gain, strongly reduced food consumption (up to 50% lower than controls), severe macroscopic alterations of the stomach wall (irregular surface) and/or severe clinical signs. At the mid-dose 300 mg/kg bw/day treatment mild to moderate test substance-related effects were evident. Microscopy revealed ulceration/erosion, hyperplasia with keratosis and sub-epithelial inflammatory cell infiltrates. The forestomach findings are considered a local test substance-related effect, most likely caused by the known irritating properties of cardol and, to a lesser extent, cardanol constituents of the test substance. At this dose level body weight gain was slightly lower from Day 12 post-coitum onwards, reaching statistical significance on Days 18 and 20 post-coitum only (0.88x and 0.83x of controls, respectively). In the corresponding study on Distillation Residue Grade no maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups, as no relevant endpoints showed significant differences in responses compared to the control animals.


 


In both studies no toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, foetal body weights, foetal anogenital distance, external, as well as visceral and skeletal malformations and developmental variations) were evident in animals surviving to necropsy.


 


The data from the oral gavage OECD TG414 study on Distilled Grade is consistent with the data from the OECD TG408 studies and indicates that there is localised epithelial hyperplasia of the non-glandular mucosa (forestomach, squamous mucosa) in the stomach at higher exposure doses. The stomach findings were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. This response is considered to be a result of the known irritancy of the test substance due to the presence of the constituents cardol, and to a lesser extent, cardanol. No comparable effects were observed in the OECD TG414 study on Distillation Residue Grade. In the study on Distillation Residue Grade gross lesions (dark red discoloration) were noted in the mesenteric lymph nodes for 3/22 females at 1000 mg/kg bw/day. However, no test substance-related microscopic findings were observed. No comparable effects were observed in the study on Distilled Grade.


 


These Prenatal developmental toxicity studies are acceptable and satisfy the guideline requirement for an OECD TG414 study in rats.


 


 

Effects on developmental toxicity

Description of key information

In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).


Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose. The NOAEL is 150 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.


This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.


In valid (reliable without restrictions, Klimisch Code 1) OECD TG414 Prenatal developmental toxicity studies on the source substances Distilled and Distillation Residue Grades groups of time-mated Wistar Han female rats were exposed to doses of 100, 300 and 1000 mg/kg bw/day by once daily oral gavage for 14 days from Day 6 to 20 post-coitum.


 


 In the study on Distilled Grade severe toxicity of the test substance was observed in females treated at 1000 mg/kg bw/day and this complete high dose group was euthanized on animal welfare grounds in extremis on Days 12-18 post-coitum.  In these animals, slight to severe body weight loss (up to 20%) and/or strongly reduced body weight gain, strongly reduced food consumption (up to 50% lower than controls), severe macroscopic alterations of the stomach wall (irregular surface) and/or severe clinical signs. At the mid-dose 300 mg/kg bw/day treatment mild to moderate test substance-related effects were evident. Microscopy revealed ulceration/erosion, hyperplasia with keratosis and sub-epithelial inflammatory cell infiltrates. The forestomach findings are considered a local test substance-related effect, most likely caused by the known irritating properties of cardol and, to a lesser extent, cardanol constituents of the test substance. At this dose level body weight gain was slightly lower from Day 12 post-coitum onwards, reaching statistical significance on Days 18 and 20 post-coitum only (0.88x and 0.83x of controls, respectively). In the corresponding study on Distillation Residue Grade no maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups, as no relevant endpoints showed significant differences in responses compared to the control animals.


 


In both studies no toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, foetal body weights, foetal anogenital distance, external, as well as visceral and skeletal malformations and developmental variations) were evident in animals surviving to necropsy.


 


The data from the oral gavage OECD TG414 study on Distilled Grade is consistent with the data from the OECD TG408 studies and indicates that there is localised epithelial hyperplasia of the non-glandular mucosa (forestomach, squamous mucosa) in the stomach at higher exposure doses. The stomach findings were interpreted to be adverse due to the overall severity and concurrent presence of ulceration in some animals. This response is considered to be a result of the known irritancy of the test substance due to the presence of the constituents cardol, and to a lesser extent, cardanol. No comparable effects were observed in the OECD TG414 study on Distillation Residue Grade. In the study on Distillation Residue Grade gross lesions (dark red discoloration) were noted in the mesenteric lymph nodes for 3/22 females at 1000 mg/kg bw/day. However, no test substance-related microscopic findings were observed. No comparable effects were observed in the study on Distilled Grade.


 


These Prenatal developmental toxicity studies are acceptable and satisfy the guideline requirement for an OECD TG414 study in rats.


 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

For the assessment of developmental toxicity the read across substances are Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade).  A document which justifies the read–across from Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) and Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) to Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated (Technical Grade) is attached.

Toxicity to reproduction: other studies

Additional information

No adverse effects on reproduction or development were observed in a 28-day reproductive screening study. A similar conclusion is expected for Cashew Nutshell Extract, Decarboxylated (Technical Grade) based on the read-across justification.


 


A document which justifies the read–across from Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) to Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated (Technical Grade) is attached.

Justification for classification or non-classification

No adverse effects on reproduction or development were observed in a 28-day OECD 422 Combined repeat dose/reproductive screening study on Distilled Grade and OECD 414 Prenatal developmental toxicity studies on Distilled and Distillation Residue Grades and therefore classification is not considered appropriate. A similar conclusion is expected for Cashew Nutshell Extract, Decarboxylated (Technical Grade) based on the read-across justification.

Additional information