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Key study repeated dose toxicity oral: 90 day rat oral (OECD TG 408, GLP) NOEL = 5 mg/kg bw/d

Key value for chemical safety assessment

Additional information

Repeated dose toxicity: rodents

In the key study, a 90 day rat oral toxicity study (following OECD 408 with a few deviations), six test groups each consisting of 14 animals per sex were dosed with lysmeral (analytical purity 97.8%) via gavage with 2, 5, 25, and 50 mg/kg body weight/day (five days a week) (Givaudan 1986A). For the high dose group a satellite group of 14 animals per sex was included for a post-treatment period of 4 weeks. As an adverse clinical sign, alopecia was observed in the females of the high dose group. Organ specific toxicity included the liver, as seen by elevated absolute (24% - 45% and 57% - 69% increase in males and females respectively) and relative (21% - 45% and 59% - 75% increase in males and females respectively) liver weights starting at 25 mg/kg bw/day. A histopathologic correlate (hepatic lipid droplet content) at 50 mg/kg was observed in both genders. Furthermore, a significant decrease in plasma cholinesterase activity ranging from 30% to 70% of respective controls and lower plasma cholesterol levels ranging from 40% to 70% of respective controls at 25 and 50 mg/kg bw/day in both genders was detected.

 

A slight but significant increase in aspartate aminotransferase activity was observed in males of the high dose group, whereas other liver enzymes such as alanine aminotransferase activities were not influenced. Effects on clinical chemistry were reversible in the recovery group. In addition, in female rats treated with 25 and 50 mg/kg bw/day, elevated absolute (16% - 30% increase) and relative (18% - 36% increase) weights of adrenal glands and hypertrophy of the zona fasciculata were observed. Findings in both, adrenals and liver were shown to be reversible in the recovery group.

 

In parallel, test substance related testicular toxicity such as spermatoceles in the epididymides and testicular atrophy was observed at 50 mg/kg bw/day (see Attachment 1). Disturbances of spermatogenesis and spermiogenesis, testicular increases in Sertoli cell-only tubules and increased surface density in Leydig cells were described along with a decreased density of spermatozoa, nucleated cells and spermatoceles in the epididymides of the high dose animals. In the 4 week recovery group, the same testicular pathology was observed to a lesser extent. In the lower dose groups (2-25 mg/kg bw/d) either no or low incidences of pathological findings in testes and epididymides comparable to the findings in control group animals were observed. Therefore, a NOAEL for testicular toxicity effects is set at 25 mg/kg bw/day.

A NOEL has been set at 5 mg/kg bw/d based on the decreased plasma cholinesterase activity, and a NOAEL has been set at 25 mg/kg bw/d based on the effects on testes and additional systemic toxicity.

Oral administration of lysmeral (analytical purity 99.1%) and lysmerylic acid to rats (50 mg/kg bw/day by gavage) for 1, 2, 3, 4 or 14 days was performed in a study with main focus on male reproductive organs (BASF SE 2006A). This study aimed for the comparison of lysmeral and lysmerylic acid in terms of potency, time dependency of adverse testicular and spermatotoxic effects and species specificity based on an analogous study performed in mice ((BASF SE 2006B). In rats, slight to severe testicular atrophy with an incidence of 2/5 animals for lysmeral and 3/5 animals for lysmerylic acid after a single application, and in all animals after longer application periods was observed (BASF SE 2006A). Generally, testicular effects were described as diffuse tubular testicular degeneration, fine vacuolar change of pachytene spermatocytes up to apoptotic cell death. Furthermore, sperm parameter examined i.e. sperm motility, spermatid count in testes, cauda epididymal sperm count, and sperm morphology were affected solely after an application period of 14 days, as seen for two test substances. Although not statistically significant, body weight gains were found to be decreased by 25% and 20% below controls after application of lysmeral and lysmerylic acid for 14 days.

 

For comparison, oral administration of lysmeral and lysmerylic acid via gavage in mice (50 mg/kg bw/day) for 1, 2, 3, 4 or 14 days led to a reduction in the ratio of normal to abnormal sperm in animals exposed only for 3 and 4 days (BASF SE 2006B). However, other treatment periods did not influence this parameter and other sperm parameters, i.e. sperm motility, spermatid count in testes or cauda epididymidis. Single administration (1 day) of lysmerylic acid led to a significant reduction in the ratio of normal to abnormal sperm and reduced total sperm numbers in cauda epididymidis in the group exposed for 2 days was observed. All other examined sperm parameters were not influenced for all treatment periods. For both substances, macroscopic and microscopic evaluation of the testes revealed no pathological changes in all groups observed. A statistically non-significant decrease in body weight gains (33% below controls) was found after application of lysmerylic acid after 14 days. Since the changes observed in single sperm parameters were inconsistent, did not follow a kinetic and were not verified histologically in testes, a substance related origin is unlikely. In line, a further study supports the absence of testicular toxicity in mice, as presented further below.

As supportive evidence, several oral gavage studies for 5 consecutive days in rats at doses ranging from 25 to 400 mg/kg bw/day confirmed clinical signs of toxicity, body weight loss and macroscopic changes in the liver starting from 50 mg/kg bw/day lysmeral. At same dose levels, changes in seminiferous epithelium with degenerated/reduced numbers of germ cells, were found, whereas decreased testes and kidney weights and decreased sizes of prostate & seminal vesicles became evident at higher dose levels (Givaudan 1986B, Givaudan 1991A, Newberne 1990).

However, oral administration of 100 mg/kg bw/day lysmeral for five consecutive days in male mice or guinea pigs showed neither any general adverse systemic effects nor adverse effects on the male reproductive organs (Givaudan 1983; Newberne 1990).Five male albino SPF mice and 5 male Himalayan spotted SPF guinea-pigs (each from Institute of Biological and Medical Research, Füllinsdorf, Switzerland) were orally administered with 100 mg of lysmeral suspended in rape oil per kg body weight per day. Furthermore, 5 mice and 5 guinea-pigs were dosed in an identical manner with the vehicle. All animals were treated once daily, for 5 consecutive days.

At commencement of treatment, individual body weights were 43 – 61 g for mice and 604 – 676 g for guinea pigs. Animals were acclimatized for 6 days before treatment, were individually caged (temperature 19 – 23° C, mean relative humidity 45 – 65 %, artificial light for 12 hours) and were allowed free access to food and tap water. After the 5th treatment day, all animals were kept in individual metabolism cages for 24 hours to collect urine. Mortality, general symptoms, and body weight development were recorded once daily. After a gross necropsy, testes of all animals were weighed and fixed in mixture of Bouin and embedded in Paraplast Plus. Testes were sectioned at a nominal thickness of 5 µm and stained with haematoxilin and eosin. Testes and epididymides of all animals were microscopically examined. The condition of the seminiferous tubules of all animals was semiquantitatively evaluated. One hundred cross-sectioned seminiferous tubules were examined per testis cross section. During the inspection, each testis section was meanderingly moved under the microscope and every two (mice) or every three or five (guinea-pigs) cross-sectioned seminiferous tubules were graded. Graduation of the seminiferoμs tubules:

0) normal cellularity of the epithelium

1) normal cellularity of the epithelium, however with some to many degenerated cells or detritus in the lumen of the seminiferous tubule

2) many degenerated cells in the epithelium and disorganization of the epithelial structure

3) severe destruction of the epithelium

All mice and all guinea-pigs survived the test period. Symptoms of incompatibility were not seen. The body weight development was normal. Necropsy findings were observed in the heart, liver, and lung of 1 control mouse as well as in the lung of 1 treated mouse. Changes seen in the guinea-pigs were fine concrements in the milky contents of the urinary bladder (5 control guinea-pigs, 2 treated guinea-pigs) and white spots or regions in the liver (4 control guinea-pigs, 3 treated guinea-pigs). All changes were not considered to be related to treatment because they were incidental and distributed among control and treated animals.

No changes were observed in testes and epididymides of all animals. Absolute and relative testes weights of control and treated animals were comparable. There was no difference between germinal epithelium of control animals and that of treated animals. In mice treated with lysmeral, 79.6%, 19.6%, 0.7%, 0.1% of the tubuli seminferi were graded 0, 1, 2 and 3 respectively (versus 77.1%, 22.1%, 0.4%, 0.4% for the grades 0, 1, 2 and 3 in control animals, respectively). In line with these results, in guinea pigs treated with lysmeral, 86.2%, 13.5%, 0.3%, 0.0% of the tubuli seminferi were graded 0, 1, 2 and 3 respectively (versus 87.8%, 12.2%, 0.0%, 0.0% for the grades 0, 1, 2 and 3 in control animals, respectively).

Dermal administration of lysmeral (analytical purity 99.1%) to rats 6 hours per day for 5 days (250, 500 1000, 2000 mg/kg bw/day) caused very slight decrease in body weights by 2% and marked testicular atrophy at the high dose only (Givaudan 1991A). Seminiferous tubules with disorganization of the epithelial structure, decrease of the number of germ cells, increase of the number of degenerating germ cells (inclusive giant cells) were observed in combination with immature/degenerating germ cells in epididymides and the occurrence of spermatocele. No clinical signs and substance related necropsy findings were observed. No further observations were performed in this study to assess adverse effects other than testicular toxicity.

 

Repeated dose toxicity: non-rodents

In a pilot study, lysmeral (analytical purity 95%) was administered to two male beagle dogs by oral administration via gelatine capsules in subsequently increasing doses (47 -564 mg/kg bw/day) for 9 weeks (Givaudan 1990A). As general adverse effects, occasional vomiting in both animals, diarrhoea in one animal and body weight reduction together with an increase in clinico-chemical parameters (glutamate dehydrogenase, alanine aminotransferase) was found. Histological examinations revealed multifocal inflammation in the liver of the two animals. In parallel, these dogs showed mild atrophy in seminiferous tubules (necrosis of germ cells, multinucleated giant cells in tubular lumen).

Further studies in beagle dogs were performed, i.e. administration of 4.4, 22.3 or 44.6 mg/kg bw/day lysmeral (analytical purity 97.6%) to each 3 male/female dogs (Givaudan 1990B) or 200 mg/kg bw/day to 3 female dogs for 90 days in gelatine capsules (Givaudan 1990C). In the former study occasional diarrhoea at 22.3 or 44.6 mg/kg bw/day and vomiting at the high dose group was observed but no other alterations and no findings from the latter study were attributable to treatment. In male animals, no alterations on reproductive organs were observed.

Based on the indications of adverse testicular effects observed in the two dogs of the pilot study (Givaudan 1990A), a testicular toxicity screening study in beagle dogs at comparable dose levels was performed for further confirmation. This study intended to clarify, whether testicular toxicity after oral administration of lysmeral occurs in a non-rodent species. In this study, lysmeral (analytical purity 99.1%) was administered to groups of 4 purebred male Beagle dogs via gelatine capsules at dose levels of 0, 40, 200 and 1000/500 mg/kg bw/day for 2 weeks (reduction of dose levels in the high dose group due to vomitus and diarrhoea) (BASF SE 2008A). Besides clinical/hematological examinations, urinalyses and a gross-pathological assessment, specific histopathological investigation on reproductive organs and liver was performed.

Systemic effects, such as a retardation in body weight gains and body weight loss in distinct animals together with decreases in food efficiency were observed in combination with vomitus and soft faeces/diarrhea in all animals of the mid and high dose group. Furthermore, significant absolute and relative liver weight increases between 30-40% above control values, and centrilobular hypertrophy of hepatocytes were observed in the mid and high dose groups.

Distinct clinical parameters were altered, i.e. prolongation in activated partial thromboplastin time, increases in serum magnesium, potassium and inorganic phosphate levels in mid/high dose animals and decreased glucose levels in high dose animals. Decreases in aspartate aminotransferase and alanine aminotransferase were found in mid/high dose animals.

A massive diffuse degeneration of seminiferous tubules combined with a hyperplasia of Leydig cells in the testes and an aspermia and epithelial vacuolation in the epididymides was found in one dog of the mid dose, which showed also a decrease in relative testis weights. Furthermore, a reduced size of testes and epididymides was observed in this animal. A second animal in the mid dose group showed a slight, one-sided and focal degeneration of seminiferous tubules, which was observed in historical control data as well and might therefore be considered as spontaneous in nature. In contrast, no such adverse testicular effects were observed in animals of the low and high dose group. Decreases in prostate sizes were observed in low dose and high dose group animals. However, due to the lack of histopathological findings and the absence of a dose response relationship, these effects are not considered to be substance related.

To further clarify the findings of the study above, a follow-up study, involving a higher animal number per dose group and additional andrological/ spermatological examinations prior and during the test substance administration period was performed. Lysmeral was administered to groups of 10 male purebred Beagle dogs via gelatine capsules at concentrations of 0 and 200 mg/kg body weight/day for 2 weeks (BASF SE 2008B).

Mean body weight loss (-0.2 kg compared to 0.1 kg in controls after day 14) due to mainly 2 of 10 animals with a massive body weight loss were observed, together with a slightly reduced food consumption (up to 25% below controls) starting from day 3 onwards. In line, a negative value for food efficiency was found. Vomitus in 7 of 10 animals and diarrhoea in 4 of 10 animals was observed as further parameters for systemic toxicity. Significant increases in absolute and relative liver weights (14% and 17% above controls, respectively) with centrilobular hypertrophy of hepatocytes became evident in the dosed animals.

Furthermore, distinct clinical parameters were statistically significantly altered (values refer to respective mean control levels after day 14), i.e. increases in alanine aminotransferase by 80% and aspartate aminotransferase activities by 310%, prolongation in activated partial thromboplastin time by 10%, decrease in serum triglyceride levels by 35% compared to control. Decreases in red blood cell counts and haemoglobin by 5%, and hematocrit values by 10% together with a decrease in reticulocyte counts by 60% indicate an anemic situation after test substance application. Increases in serum urea by 45%, creatinine by 25%, calcium by 5% and magnesium levels by 20% indicate adverse effects on the kidneys, however, no kidney weight changes were observed.

Decreases in absolute and relative testes weights by approx. 25% along with a slight to severe degeneration of seminiferous tubules in 9 of 10 animals were observed. Unilateral decrease in testicular length or width was found in 6 of 10 animals.

Furthermore, effects on spermatological parameters, i.e. decrease of progressively motile spermatozoa and/or morphological alterations were found in 9 of 10 dogs after treatment when compared to the values of the respective animals before treatment.

Morphological sperm alterations consisted mainly of mid-piece anomalies (cytoplasmatic droplets) and less frequently in sperm neck anomalies (paraxial tail attachment, cytoplasmatic droplets). Prostate weights were slightly decreased and respective minimal to moderate multifocal atrophies were found in 3 of 10 animals.

 

Considering the findings from the available studies in dogs, a NOAEL is set at 44.6 mg/kg bw/day based on the testicular toxicity observed.

A screening study on the male reproductive function in rabbits was performed in order to clarify, whether testicular toxicity or spermatotoxic effects after oral administration of lysmeral occurs in a further non-rodent species.

In this screening study, rabbits were treated via gavage for 15 days at doses of 30, 100 and 300 mg/kg bw/day lysmeral (analytical purity 99.1%; BASF SE 2008C). No test substance related findings on clinical observations, body weights and food consumption were observed in all dosing groups. Neither testes nor cauda epididymis weights were affected. A moderate diffuse degeneration of the seminiferous tubules combined with a moderate oligospermia and a moderate mixed inflammation in the epididymides was observed in 1/5 animals of the low dose group. The inflammation found in the epididymides is assumed to be causative for the degenerative changes in the testis. In the mid dose group, a reduced testes and epididymides size with severe diffuse degeneration of seminiferous tubules in the examined left testis and a severe atrophy plus aspermia in the left epididymides was observed in 1/5 animals. However, sperm evaluation did not reveal any treatment related effect in this or any other treated animal. Based on the absence of a dose response relationship, the isolated occurrence in one single animal and the absence of adverse effects on spermatological parameters in the respective animal, a treatment related origin of the observed findings seem unlikely.

In a study on primates, using a limited number of animals, oral administration of 100 mg/kg bw/day lysmeral for 5 days did not lead to any general adverse effects or testicular toxicity (Newberne 1990; Givaudan 1984G). In this study using 2 male rhesus monkeys (Macaca mulatta), clinical signs of toxicity and mortality was monitored and animals were weighed at test day 1 and 6. Animals were sacrificed by perfusion with glutaraldehyde and subjected to a complete necropsy. All organs and tissues were examined grossly and testes and epididymides were examined by histopathology.

No endpoints related to liver were investigated. No incompatibility reactions were seen in clinical observations during the in-life period and body weights were not significantly affected. In histological examinations, only small foci in one epididymis of one animal and small hollow spaces in the epithelium of one epididymis of the other animal was observed. The testes of both animals were found to be free of lesions. Seminiferous tubules with orderly arrangement of intact stages of spermatogenesis was predominantly found whereas decreased numbers of spermatozoa per tubule to less than 10 was a minor finding. Unlike effects observed in rats, no tubules with complete loss of cells, sertoli-cell-only-syndrome or with slight/severe reduction and destruction of the germinal epithelium was observed in both testes of the two animals. The findings in one of the epididymis of each animal does not represent a test substance related effect, since other male reproductive tissues were not affected. Overall, general and testicular toxicity was not observed under the conditions of this study in primates.

Other relevant information

In vitro data using primary rat hepatocytes indicate inhibition of hepatic lipogenesis and gluconeogenesis by lysmeral metabolites (McCune et al. 1982; see Chapter “Toxicity to reproduction). TBBA has been shown to inhibit fatty acid synthesis and glucose synthesis. An inhibition of fatty acid synthesis by 50% required 5-10 µM TBBA. Addition of glycine, a pivotal substrate for hippurate (TBHA) formation, had no effect on TBBA inhibition of lipogenesis. Furthermore, TBBA treatment decreased CoA, acetyl-CoA and citrate levels and addition of octanoate protected against the inhibitory effect of TBBA on lipogenesis. In a recent in vitro study in rat hepatocytes, TBBA and lysmeral were found to rapidly and dose dependently transform to TBBA-CoA conjugates and an accumulation to stable levels occurs (Givaudan 2017; see Chapter “Toxicity to reproduction).

As established by these in vitro studies, liver toxicity observed for lysmeral is likely to be caused by disruption of CoA-dependent metabolic processes triggered by the metabolite TBBA.The formation of TBBA-CoA might either be directly toxic or result in the inhibition of CoA dependent processes. Xenobiotic-acyl-S-CoA thioesters have been described to covalently modify proteins (Darnell et al. 2015; Lassila et al 2015), interfere with endogenous lipid metabolism (Darnell et al. 2013) or deplete the CoA pool. Since the CoA pool is important in a large number of catabolic and anabolic biochemical reactions, but is rather small and cannot be increased quickly, a disruption of a permanent turnover of acyl-CoAs might impair many biochemical pathways (Brass 2002).

The disruption of CoA dependent processes has been confirmed in vivo based on metabolome analyses of lysmeral and structurally related substances (BASF 2017; 99C0208/05004; 99C0179/97075; 99C0032/07002; 30S0875/08053). In these studies, Wistar rats were treated for 28 days via gavage with lysmeral (15 and 45 mg/kg bw/d; two independent experiments). For comparison, the related alcohol lysmerol (10 and 50 mg/kg bw/d) or meta-lysmeral (150 and 450 mg/kg bw/d) were administered to Wistar rats accordingly. For each dose group five male and five female animals were used, while the control group consisted of 10 untreated males and 10 untreated females. For the metabolome analysis of lysmeral and lysmerol treated animals, blood samples were taken from retro-orbital venous plexus after 7, 14 and 28 days, respectively. Meta-lysmeral treated animals were sampled after 28 days only. Plasma metabolome was examined using GC/MS and LC/MS-MS techniques. For all these metabolites, changes in plasma were calculated as the ratio of the mean of metabolite levels in individual rats in a treatment group relative to mean of metabolite levels in rats in a matched control group (time point, dose level, sex). For the identification of biologically relevant changes in the metabolome, common significant changes (p = 0.05) for male animals (treated with 45 mg/kg BW/d lysmeral) have been identified in plasma derived from two independent rat studies. Mean changes of metabolite levels were considered relevant, if statistically significant and changed into the same direction in 4 out of 6 timepoints investigated. The dose chosen is associated with lysmeral induced liver and testicular toxicity, as seen in other repeated dose and reproductive toxicity studies.


In comparison to these changes, the metabolome ratios of the respective metabolites for lysmerol and meta-lysmeral treated male rats have been added (for further information on the methodology refer to Kamp et al., 2012 and van Ravenzwaay et al., 2015). The metabolites showing common significant changes are listed in Attachment 2.

By comparing the ratios of the 204 different metabolites assessed, a vast majority of metabolites that are commonly changed between the two independent studies represent lipids, fatty acids and fatty acid related metabolites. Plasma levels of all these metabolites are decreased when compared to respective untreated animal levels. These decreases are generally seen after 7, 14 and 28 days exposure. When compared with the metabolite changes induced by lysmerol treated rats, which is known to lead to comparable testicular toxicity as lysmeral treatment, a very comparable pattern and a predominant decrease of complex lipids and fatty acid class associated metabolite levels were observed. In contrast, meta-lysmeral treatment has been shown to lead to no testicular toxicity phenotype and does provide a divergent metabolite pattern especially for complex lipids such as sphingolipids, ceramides and phosphatidylcholines.

 

  1. Brass, E. P. (2002); Pivalate-Generating Prodrugs and Carnitine Homeostasis in Man. Pharmacol. Rev. 54, 589-598.
  2. Darnell et al. (2013); Metabolism of xenobiotic carboxylic acids: focus on coenzyme A conjugation, reactivity, and interference with lipid metabolism. Chem. Res. Toxicol. 26, 1139-1155.
  3. Darnell et al. (2015); Significantly different covalent binding of oxidative metabolites, acyl glucuronides, and S-acyl CoA conjugates formed from xenobiotic carboxylic acids in human liver microsomes. Chem. Res. Toxicol. 28, pp. 886-896.
  4. Kamp H. (2012); Application of in vivo metabolomics to preclinical/toxicological studies: case study on phenytoin-induced systemic toxicity. Bioanalysis. Sept;4(18): 2291-2301.
  5. Lassila et al. (2015); Toxicity of carboxylic acid-containing drugs: the role of acyl migration and CoA conjugation investigated. Chem. Res. Toxicol. 28, 2292-2303.
  6. Van Ravenzwaay B. et al (2015); The development of a database for metabolomics - looking back on ten years of experience. Int J Biotechnol 14:47–68. doi: 10.1504/IJBT.2015.074801.

Justification for classification or non-classification

The present data on repeated dose toxicity do not fulfill the criteria laid down in 67/548/EEC and 1272/2008/EEC, and therefore, a non-classification is warranted.