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Administrative data

Description of key information

Key study: Repeated dose toxicity via oral gavage in rat (OECD TG 408): NOAEL 120 mg/kg bw/day.

(other available repeated dose toxicity studies are summarised in the 'Additional information' section below. The OECD TG 443 is fully presented in the fertility section (IUCLID 7.8)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Four repeated dose toxicity studies are available, conducted according to OECD TG 408, 443, 407, and 421 and all are performed in compliance with GLP.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Quality of whole database:
The dermal NTP studies were not used for assessment for the dermal route becau e oral grooming resulted in uncertainties on exposure.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Quality of whole database:
The dermal NTP studies were not used for assessment for the dermal route becau e oral grooming resulted in uncertainties on exposure.

Additional information

Summary of the repeated dose toxicity studies

Four oral repeated dose toxicity studies are available: 90-day gavage study in rats according to OECD TG 408 (Triskelion, 2017), extended one-generation reproductive toxicity in rats according to OECD TG 443 (Covance 2020), 28-day gavage study in rats according to OECD TG 407 (Huntingdon, 1997), DRF for EOGRT (OECD TG 443), screening for reproductive / developmental toxicity in rats similar to OECD TG 421 (Triskelion 2019). A 14 -day juvenile study was also performed (Covance, 2019) to make sure that juveniles were not more sensitve than adults or pups (data are summarised in a paragraph below). In addition, two dermal 90-day studies (similar to OECD TG 411) in rat and mice are carried out by NTP (2007/2016). In these two studies, the application sites were not covered and due to grooming the dermal exposure and local and systemic effects is confounded because high oral exposure has occurred. Therefore, the local and systemic long-term dermal effects cannot be evaluated according the standard OECD 411 criteria.

The key systemic effects seen in all 6 studies are summarized in the table. The four oral studies have similar NOAELs: 120-300 mg/kg bw and LOAELs: 500-1000 mg/kg bw. Effects on body weight are observed at dose levels of 500 mg/kg bw and higher. The other effects on haematology, spleen, liver, and kidney are not observed at dose levels below 300 mg/kg bw. In general, when determined, no neurotoxic, immunotoxic or fertility effects are observed. The slightly sperm effects and slightly longer oestrus cycle in mice are at very high doses of 2000 mg/kg bw (twice the limit dose of 1000 mg/kg bw) and are accompanied with 89% increase in liver weights at this dose.

The overall NOAEL is 120 mg/kg bw based on the 90-day oral gavage toxicity study on which bases the systemic DNELs via all routes will be derived. This study is considered key as is has the lowest NOAEL. Furthermore, more parameters were investigated compared to the OECD 421 and 443 studies such as water consumption, ophthalmoscopic examination, urinalysis, and behaviour. Finally, in this study accumulation of hyaline droplets in tubules of the outer cortex of the kidneys accompanied by an increased number of basophilic tubules, and/or dilated tubules with eosinophilic casts consisting of granular debris was identified by immunohistochemical staining being indicative for alpha-2u globulin hydrocarbon nephropathy. Summary of the repeated dose toxicity studies is presented in the Table below..

Table: Summary of results for systemic toxicity of all repeated dose toxicity studies in order of fulfilling the repeated dose toxicity endpoint.

Systemic toxicity

OECD 408

OECD 443

OECD 407

OECD 421

OECD 411 (1)

OECD 411 (1)

Exposure duration

90 days

120 days

28 days

38 days

90 days

90 days

Species

Rat

Rat

Rat

Rat

Rat

Mice

LOAEL (mg/kg bw)

500

>300

1000

500

500 (1)

500 (1)

NOAEL (mg/kg bw)

120

300

150

120

250 (1)

250 (1)

Neurotoxicity and immunotoxic effects

No effects

NA

No effects

NA

No effects

No effects

Reproductive and/or Endocrine organs

No effects

No effects

No effects

No effects

No effects

No effects

Lowest Adverse Effects (mg/kg bw)

 

 

 

 

 

 

Body weight and body weight gain effect > 10%

500

No effects

1000

No effects

500

1000

Haematology

500

No effects

No effects

500

No effects

500

Spleen

500

No effects

No effects

500

No effects

No effects

Liver: Increase in weight and hypertrophy

500

No effects

1000

500

500

2000

Kidney: Increase in weight and a2u-hydrocarbon nephropathy

500

No effects

1000

500

No effects

No effects

Male fertility: sperm

No effects

No effects

NA

NA

No effects

2000

Female fertility: Oestrus cycle

No effects

No effects

NA

NA

No effects

2000

(1) The dermal NTP studies were not used for NOAEL/LOAEL derivation because dermal doses with no occlustion were confounded due to oral grooming and oral exposure occurred.

 90-day oral gavage repeated dose study (OECD TG 408, Triskelion, 2017)

The key study is a sub-chronic (13-week) repeated dose toxicity study performed according to OECD TG 408 and GLP principles, rated Klimisch 1. Wistar Han IGS rats (Crl:WI(Han)) were administered daily by oral gavage at dose levels of 30, 120 and 500 mg/kg bw/day. A control group treated with vehicle (corn oil) was included. In each dose group 10 male and 10 female animals were included. Analytical verification of dose and stability of the substance showed that the rats received the intended concentrations. All parameters are measured in accordance with the current guideline.

Clinical signs and body weights: There was no mortality. Salivation was noted just prior to or after dosing, mainly in the high dose group. There were no other treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. Ophthalmoscopic examination did not reveal any treatment related ocular changes. There were no relevant changes in growth parameters. Food and water intake were increased in the high-dose group in both sexes.

Haematology: Haematology parameters were studied in all rats at necropsy. In the high-dose group haemoglobin concentration was decreased in both sexes, and red blood cell count and packed cell volume were decreased in males at maximum with 12%. Thrombocytes were increased in males, and prothrombin time was reduced in females of this group. Total white blood cell counts were increased in males of the high-dose group. The red blood cell parameters decrease combined with the spleen effects (see below) the effects were considered adverse at the high dose group.

Clinical chemistry was conducted in all rats at necropsy. An increase cholesterol and phospholipids concentrations in females of the high-dose group was seen. In combination with the high liver weight increase and hypertrophy this is expected to be due to high metabolic demand. At the high dose this is considered adverse.

Urinalysis, conducted in all rats in week 13 of the study, did not reveal relevant changes in renal concentrating ability or in semi-quantitative (dipstick) urinary measurements. Microscopic examination of the urinary sediment showed increases in epithelial cells, amorphous material and casts in males of the high-dose group. Casts were also noted in two males of the mid-dose group. These effects can be related to alpha2u-hydrocarbon nephropathy.

Organs: Spleen: the relative weight of the spleen (37%) was increased in males of the high-dose group. Macroscopic examination at necropsy revealed no treatment-related changes. The changes in the spleen were characterized by increased extramedullary erythropoiesis in 8/10 high-dose males, in 3/10 mid-dose females and in 5/10 high-dose females. Extramedullary erythropoiesis in the spleen may be regarded as a physiological mechanism to meet an increased demand for the production of red blood cells. However, a reason for the increased demand could not be established; the bone marrow did not show abnormalities indicating that the normal production of red blood cells was impaired. Further, no signs of an increased red blood cell turnover were found (such as haemorrhages, elevated reticulocytes or increased accumulation of iron pigment in the spleen). Taking into account that the extramedullary erythropoiesis noted in a few females in the mid-dose group was not accompanied by functional disturbances in red blood cell system, this finding in the mid-dose group is not considered adverse. Liver: The relative weight of the liver was increased in males and females of the mid- and high dose groups (12-15% in the mid-dose group and 50% in the high-dose group). The microscopic changes in the liver were characterized by centrilobular hepatocellular hypertrophy (enlarged cells as well as enlarged nuclei) and hepatocellular vacuolation. The incidence of centrilobular hepatocellular hypertrophy was 3/10, 6/10 and 9/10 in the low-, mid- and high-dose males, respectively; and 8/10 and 9/10 in the mid- and high-dose females, respectively. In addition, 10/10 mid-dose and 10/10 high-dose females showed a minimal increased incidence of macro-vesicular hepatocellular vacuolation. This minimal vacuolation was seen in all groups in the males with a lower incidence. The incidence of hepatocellular hypertrophy in the liver showed a clear dose-effect relationship in males of all dose groups and in females of the mid- and high dose group. The elevated blood lipids, increase in relative liver weight in combination with hepatocellular hypertrophy. These effects may be considered a reaction to an increased metabolic demand. Liver weight increase and hepatocellular hypertrophy in the absence of clinical pathology or histologic alterations indicative of overt hepatocellular damage may be considered an adaptive non-adverse effect (ESTP Expert Workshops 2012, 2016). This may be concluded for the high dose too but in view of the high relative liver weight increase (and accompanying effects) the mid-dose is considered non-adverse for liver effects. Kidney: The relative weight of the kidneys was increased in males of the mid- and high-dose groups (13% and 36%, respectively) and in females of the high-dose group (13%). Microscopy revealed alpha2u-urinary microglobulins (accumulation of hyaline droplets in tubules of the outer cortex of the kidneys accompanied by an increased number of basophilic tubules, and/or dilated tubules with eosinophilic casts consisting of granular debris) and identified by immunohistochemical staining.

Other effects: There were no neurotoxicity, immunological toxic and fertility toxicity findings in this study. There were also no neoplastic findings in this study.

Conclusion

In conclusion, a NOAEL of 120 mg/kg bw/day could be derived based on the effects in the spleen and liver which were accompanied by changes in haematology or clinical biochemistry in the high dose group only. There were no neurotoxicity, immunological toxic and fertility toxicity findings in this study. There were also no neoplastic findings in this study.

The extended one generation toxicity study according to OECD TG 443

Introduction:

The test substance was investigated, in a study performed according to OECD TG 443, on systemic and reproductive toxicity when administered continuously by oral gavage to Han Wistar rats. In the F0 generation, 24 Han Wistar rats per sex received OTNE at dose levels of 30, 100 or 300 mg/kg/day. The dose was based on all repeated dose toxicity studies showing increase relative liver weights with hypertrophy at 500 mg/kg bw of ca. 50% indicative for overloading the metabolic pathway. The NOAEL for this effect was set at 120 mg/kg bw and therefore 300 mg/kg bw was anticipated to show some liver toxicity, which could be well tolerated. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation (resulting in exposure up ca 120 days. In the F1 generation, 40 rats per sex were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and as the F0 generation resulting in approximately 70 exposure days for the F1A cohort and 77 exposure days for the F1B cohort. A similarly constituted Control group received the vehicle, corn oil.

Measured parameters:

For the F0 generation data were recorded on clinical condition, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (haematology, blood chemistry and thyroid-related hormones), sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 and 20 of age. Blood samples were collected from selected offspring on Day 4 and 22 of age for biomarker investigations.

For F1 generation - Cohort A, data were recorded on clinical condition, body weight, food consumption, sexual maturation, vaginal opening and estrous cycles. Clinical pathology (haematology, blood chemistry, thyroid hormones and urinalysis), sperm assessment, ovarian follicle counts, organ weight, macroscopic pathology, full microscopic pathology and spleen cell immunophenotyping investigations were performed.

For F1 generation - Cohort B, data was recorded on clinical condition, body weight, food consumption, estrous cycles, sexual maturation, organ weight, and a targeted set of macroscopic pathology investigations were performed.

Results – systemic toxicity:

F0 adults (and F1 offspring up to weaning)

Mortality: There were two deaths throughout the duration of study; Control male No. 12 and Female No. 267 that received 100 mg/kg/day were euthanized prematurely on animal welfare grounds during the 10-week pre-pairing treatment period and on Day 1 of lactation respectively. Female No. 267 had shown signs comprising decreased activity, irregular breathing and was abnormally cold to touch, macropathological examination revealed abnormal contents of the gastrointestinal tract and thickened uterus horns. When compared to the animals killed at scheduled termination, these findings were atypical and therefore the death of female No. 267 was not considered related to treatment.

Clinical signs: There were no clinical signs considered to be related to treatment in F0 males or in the F0 females which reared their litters to weaning.

Body weight gain of males and females before pairing and of females during gestation and lactation was unaffected by treatment.

Food consumptionof both sexes before pairing, males after pairing and females during gestation and lactation was generally similar to that of the Controls. From Days 14 to 21 of lactation, food intake was marginally high for females that received 100 or 300 mg/kg/day.

Haematologyinvestigations revealed slightly low haematocrit and haemoglobin concentrations for males that received 300 mg/kg/day and an increase in mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and mean corpuscular volume for females that received 300 mg/kg/day. For females that received 30, 100 or 300 mg/kg/day, a slight increase in haemoglobin levels was evident. All these effects were <6% and considered incidental and not adverse.

Blood chemistryinvestigations revealed marginally low alkaline phosphatase levels for males that received 100 or 300 mg/kg/day and females that received 30, 100 or 300 mg/kg/day (< 20%). Low aspartate aminotransferase and phosphate levels were evident for males that received 300 mg/kg/day. Gamma-glutamyl transpeptidase increased from zero to 1. Total protein and albumin levels increased minimally for males that received 300 mg/kg/day and protein levels at this dose for females but <=6%. Bilirubin and bile acid levels were low and for females that received 30, 100 or 300 mg/kg/day. For females, cholesterol was slightly increased at the 300 mg/kg/day (+16%). The toxicological relevance decreases of alkaline phosphate levels, low aspartate transferase and bile acid levels is unclear, because increase is considered to be related to liver effects. Others liver related parameters such as, increase in gamma-glutamyl transferase and some increase in cholesterol are considered to be due to increased liver function.

Macropathology: There were no test item related macropathology findings in any of the organs.

Organ weights and Histopathology:Liver: Relative Liver weights were dose dependently statistically significantly high among males and females that received 100 or 300 mg/kg/day (9.5 and 29.5%, respectively). For females only, relative liver weights were dose dependently increased up to 26.5%. Treatment-related changes in the liver were present in both males and females of F0 generation. In some males and approximately half of the females treated with OTNE at 300 mg/kg/day, the hepatic changes were consistent with minimal centrilobular or general hepatocellular hypertrophy (males only). Some changes in blood chemistry values such as the increase in gamma glutamyl peptide transferase, increase in total protein, increase in cholesterol, increase in liver weight and slight liver hypertrophy is anticipated to be related to an adaptive increase of liver function and not considered adverse.Thyroid: Relative thyroid weights were also increased in males but less so in females. No histopathological findings were seen and therefore their increases were not considered adverse.Spleen: The higher mean weights of the spleen and in F0 males that received 300 mg/kg/day were not supported by any microscopic pathology changes and thus biological significance and any relationship to treatment is not evident.Kidneys: Treatment-related changes in the kidneys were present in males only. Increased amounts of hyaline droplets within the renal proximal tubular epithelium were considered to be treatment related in kidneys of males treated with OTNE at 300 mg/kg/day and at 100 mg/kg/day examined: 15/25 and 23/24 animals, respectively. Additionally, higher incidence of minimal focal to multifocal, tubular basophilia was observed in the kidneys of males treated with OTNE at 300 mg/kg/day: 11/24 animals. Hyaline droplets and the increase in tubular basophilia are considered to be related to alpha-2u-globulin hydrocarbon nephropathy, an adaptive response, male sex specific and not considered adverse for humans; but no immune staining was done.

Results Reproductive toxicity (F0)

Fertility P0: Irregular, extended, and acyclic estrous cycles were observed in both control and treatment groups. The cause was thought to be related due to a light cycle error in the animal facility room and therefore these abnormalities were not considered related to treatment. There was no effect of treatment on pre-coital interval, fertility, gestation length and gestation index. Sperm motility, counts and morphology were unaffected by treatment.

Developmental toxicity: At 30, 100 or 300 mg/kg/day, there was no effect of treatment on post implantation survival, the mean number of offspring born and the number of live offspring on Day 1. Offspring survival after birth was unaffected by treatment and there was no clear effect on offspring sex ratio. There was no effect of treatment on offspring ano-genital distance, nipple counts in males, organ weights or macropathological findings. Male and female reproductive organs were not affected. There was no effect of treatment on serum T4 levels in adults or offspring, and no conclusive effect on TSH levels.

Results: Selected F1 offspring - Cohorts 1A and 1B

Clinical signs: Treatment of the F1A or F1B males and females was generally well tolerated and there were no test-item related changes in clinical condition observed from weaning on Day 21 of age up until scheduled termination at approximately Week 13 or 14 of age.

Body weight: At 30, 100 or 300 mg/kg/day, body weight gain of both sexes between Days 21 to 25 of age was generally similar to that of the Controls. For males that received 300 mg/kg/day, group mean body weight gain from Day 1 to 57 after the formal commencement of the F1 generation was marginally low when compared to the controls (<=-5%). Food consumption was not affected.

Haematology investigationof the F1A cohort did not reveal any treatment related changes. Two significant finding in females were not considered toxicologically relevant: Hemoglobulin concentrations decrease of 5% and platelet count increase of 17% in absence of other related findings.

Blood chemistry investigationof the F1A cohort revealed low alanine amino transferase and glucose levels for males that received 300 mg/kg/day. For males and females that received 30, 100 or 300 mg/kg/day, bile acids were low when compared to the controls. Low alkaline phosphatase values were evident in females that received 300 mg/kg/day. Calcium was minimally but significantly increased (+3%). For females that received 100 or 300 mg/kg/day, cholesterol was high (+17 and +30%, respectively) when compared to the controls. Except for calcium most parameters are liver related.

Urinalysis investigationof the F1A cohort revealed low pH in males and females that received 300 mg/kg/day: from ca 7 to 6.1. For males that received 100 or 300 mg/kg/day, total protein and protein was high when compared to the controls 92 and 275%, respectively. Additionally, total sodium levels for males that received 300 mg/kg/day was low and for males that received 30 or 100 mg/kg/day, Females that received 300 mg/kg/day, urine volume, total protein and total sodium was low whereas, protein, urine potassium and urine chloride was high when compared to the controls. Urine sodium levels were high. These effects are considered minor and not considered adverse in absence of related finding in kidney.

Macropathology: There were no test item related macropathology findings on all organs.

Organ weight: Relativeliverweights were high for cohort 1A males and females that received 30, 100 or 300 mg/kg/day (<=28% for the high dose). For cohort 1A and 1B males that received 300 mg/kg/day and cohort 1A and 1B females that received 300 mg/kg/day.Thyroid and parathyroidweights were high in F1A and 1B males when compared to the controls (<=37%); in females it was also increased but not significantly (<11%), which are anticipated to be related to increase in liver function. RelativeKidneyweights were statistically significantly high among cohort 1A males and females that received 300 mg/kg/day (<=16%) and slightly high for males that received 100 mg/kg/day (+7%).

Histopathology, Liver: Treatment-related changes in the liver were present in both males and females of the F1 generation. In some males and approximately half of the females treated with OTNE at 300 mg/kg/day, minimal centrilobular hypertrophy was apparent with one male showing minimal general hypertrophy. These are similar to what is seen in the F0 generation.

Microscopic pathology changes related to treatment with OTNE were seen in the kidneys and the liver but not in any other organs. Increases in the weight of some organs in some cohorts e.g. thyroid, prostate and oviduct were therefore not considered adverse.Kidney: Treatment-related changes in the kidneys were present in males only. In males of the F1 generation, increased amounts of hyaline droplets within the renal proximal tubular epithelium was considered to be treatment related in kidneys of males treated with OTNE at 300 and 100 mg/kg/day. Additionally, higher incidence of minimal to slight, focal to multifocal, tubular basophilia was observed in the kidneys of males treated with OTNE at 100 or 300 mg/kg/day (number of animals). These effects are considered to be related to alpha-2u-globulin hydrocarbon nephropathy.

Reproductive toxicity (F1A and F1B): Reproductive parameters for the F1 group, there was no adverse effect of treatment on sperm motility, counts or morphology. At 300 mg/kg/day there was a slight increase in cauda epididymal weight and sperm concentration, which is not considered toxicological relevant. Age at sexual maturation, time between vaginal opening and first estrous and oestrus cycles and ovarian follicle counts were unaffected by treatment. For cohort 1A females that received 100 or 300 mg/kg/day and cohort 1B females that received 300 mg/kg/day, ovaries and oviduct weights were high when compared to the controls just <=11%. No histopathological findings were seen and therefore biological significance or any relationship to treatment is uncertain. Prostate weights of cohort 1B males that received 300 mg/kg/day was high when compared to the controls (+19%). No histopathological findings were seen.

Conclusion: Considering systemic toxicity for F0 and F1 animals, minor effects were seen on haematology and blood chemistry of which the latter can be related to increased liver function. In the liver the increased relative liver weights and increased minimal hypertrophy is considered adaptive. Relative kidney weights and histopathological changes in the kidneys of F0 and F1 Cohort 1A males, and changes in the liver of F0 and F1 Cohort 1A males and females were not considered adverse within the context of this study and for males these were related to alpha 2u-globulin hydrocarbon nephropathy. The NOAEL for systemic toxicity in the F0 and F1 A adult animals was concluded to be the high dose of 300 mg/kg/day. Based on the results and absence of effects obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 animals was the high dose of 300 mg/kg/day for males and females.

28-day oral gavage repeated dose study (OECD TG 407, Huntingdon, 1995)

A 28-day oral repeated dose toxicity study via gavage in rats in accordance with OECD TG 407, Klimisch 1, is available.

Method: OTNE was administered by oral gavage, once daily, to three groups of Sprague-Dawley rats for a minimum of twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in corn oil at concentrations of 0.3, 3.0 or 20% w/v and was administered at a dosage volume of 5 ml/kg/day. Control animals received the vehicle (corn oil) alone at the same dose volume (5 ml/kg/day). All rats of Groups 2 and 3 (15 and 150 mg/kg/day respectively) and five males and five females from each of Groups 1 and 4 (Control and 1000 mg/kg/day respectively) were killed following the four week treatment period. The remaining animals (five males and five females from Groups 1 and 4) were retained for a two-week recovery period. Bodyweights, food and water consumption and clinical observations were recorded during the study. Blood and urine samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically; specified tissues were then prepared for histopathological examination.

Results

Clinical signs and body weight: There were no treatment-related mortalities. Evidence of poor grooming was noted for high dosage group rats during the treatment period. Lower than control bodyweight gains were recorded over the four-week treatment period for male rats treated at 1000 or 150 mg/kg/day. Higher than control water consumption was noted for male and female rats of the high dosage group: during Week 3. There were no other differences from control for other parameters measured, including bodyweight, food and water consumption, and urinalysis that were considered to be related to treatment.

Haematology: No effects observed.

Clinical chemistry: Some liver related parameters were affected at the high dose. Cholesterol levels were higher than control for male and female rats treated at 1000 mg/kg/day. Additionally, higher than control gamma-glutamyltransferase levels were recorded for some male and female rats of this high dosage group and glutamic pyruvic transaminase (GPT) levels were lower than control for males from the high dosage group. Following the two-week recovery period, higher cholesterol levels for females and lower GPT levels for males of the high dosage group were recorded.

Organ:Liver: Relative liver weights were higher than control for male and female rats treated at 1000 mg/kg/day, resulting in enlarged livers. At microscopy centrilobular hepatocyte enlargement in the liver of male and female rats was seen at the high dose. The report considered the liver effects observed at the highest dose level to be adaptive in nature (as they were not observed in the recovery animals and therefore were reversible) and probably related to the metabolism of the test material. However, a review by the RIFM Expert Panel concluded that these effects could not be ignored and need to be considered for the NOAEL.Kidney: In the kidneys of male rats eosinophilic inclusions in the cortical tubules were observed. The kidney finding was also present in male rats of the intermediate dosage group. At the end of the recovery period, the kidney finding was present, but to a lesser extent, for male rats from the high dosage group. The kidney finding is characteristic for the alpha2u globulin hydrocarbon nephropathy syndrome, which is specific to male rats, and is therefore not considered predictive of a similar effect in man.

Other effects: Yellow/brown stained fur was noted among high dosage group male and female rat. There were no neurotoxicological, immunological or fertility findings in this study.

Conclusion

The key target organ is the liver showing high increased liver weight accompanied by some liver clinical chemistry parameters and increase in centrilobular hepatocyte enlargement. Though these effects can be considered adaptive they were considered for deriving the NOAEL, resulting in a NOAEL of 150 mg/kg bw. 

Screening for reproductive / developmental toxicity study (DRF for OECD TG 443 (somewhat similar to OECD TG 421, Triskelion, 2019); See IUCLID Chapter 7.8.1

A reproduction/developmental toxicity screening test, performed similar to OECD TG 421 and following GLP, served as a dose-range finding study for an extended one generation reproduction toxicity study.

Method: The test substance was administered daily by oral gavage as a dilution in corn oil at levels of 0 (vehicle control), 30, 120 and 500 mg/kg body weight/day. Male animals were dosed for 38 days after which half of the number of the male animals were sacrificed on day 38 and the other male animals were kept untreated up to sacrifice on day 74. Female animals were dosed for 4 weeks pre-mating, during mating, gestation and up to lactation day 13 and were sacrificed on postnatal day 14-16. Pups were sacrificed on postnatal day 13.

Results:Mortality: There was no mortality.

Clinical signs: Salivation was noted just prior to and after dosing mainly in the high-dose animals and all animals of the high-dose group glided with their ventral part of the head and neck over the cage bottom just after dosing. There were no other treatment-related clinical signs.

Body weight: There were no relevant changes in body weight and body weight changes.

Food consumption was increased in the high-dose group, mainly in the female animals.

Hematology: At the end of the treatment period, in high-dose male animals, the concentration of haemoglobin, the mean corpuscular volume, the mean corpuscular haemoglobin and the mean corpuscular haemoglobin concentration were decreased and the number of thrombocytes was increased. After the recovery period, no effects on the red blood cell parameters were observed in male animals. In female animals, no effects on red blood cell and coagulation parameters were noted. At the end of the treatment period, the number of neutrophils was increased in the blood of high-dose male animals. No effects were observed on white blood cell parameters after the recovery period. In female animals, no effects on any of the white blood cell parameters was observed.

Biochemistry: At the end of the treatment period, in high-dose male animals, the concentration of total protein was increased. After the recovery period, no effects on clinical chemistry parameters were noted. In female animals of the high-dose group, the activity of gamma glutamyl transferase was increased. No effects of T4 hormone was observed in male and female adult animals and in pups on postnatal day 13.

Organ weight: Liver: At the end of the treatment period, the relative weight of the liver was increased in males of the mid- and high-dose groups. The difference from controls was 12% in the mid-dose group and 52% in the high-dose group. After the recovery period, no effect was observed in the mid-dose group whereas the difference in relative liver weight with controls in the high-dose group was only 8%. - In female animals, the relative liver weight was dose-related increased in all groups (7, 11 and 29% in the low-, mid- and high-dose groups, respectively). Kidney: At the end of the treatment period, the relative weight of the kidneys of male animals of the high-dose group was increased (+22%). No effect was observed after the recovery period. Spleen: At the end of the treatment period, the relative weight of the spleen of male animals of the high-dose group was increased (+25%). No effect was observed after the recovery period.

Reproduction: No treatment-related effects were observed on reproductive performance and on any of the early postnatal developmental parameters.

Conclusion: In male animals, all observed effects on clinical signs, haematology- and clinical chemistry parameters and on organ weights appeared to be reversible after a 37 days recovery period. Although not examined in this study, most probably, the same will be the case for comparable effects observed in female animals. Many of the effects observed in this study were consistent with the effects observed in in the sub-chronic (13 week) oral gavage study and in the present study it was shown that the effects appeared to be reversible to treatment. The increase in absolute- and relative liver weights of at least 29% as was observed in male and female animals of the high-dose group is considered as an adverse effect of treatment. Based on the results of this study the NOAEL for general toxicity is 120 mg/kg bw/day and the NOAEL for fertility and early postnatal developmental toxicity is >= 500 mg/kg bw/day.

DRF for OECD TG 443 – Juvenile tolerable study

Introduction: The purpose of this study was to assess the influence of daily oral gavage administration of OTNE for two weeks to immature rats (from Day 21 to 34 days of age). The study assessed the effects on the general condition, maturation and development of the rats, in order to aid identification of suitable dose levels for investigation in an extended one generation (OECD 443) study.

Method: Three groups, each comprising six male and six female Wister rats, received OTNE at doses of 15, 60 or 300 mg/kg/day. A similarly constituted control group received

the vehicle, corn oil, at the same volume dose as treated groups. During the study, clinical condition, body weight, food consumption, organ weight and macropathology investigations were undertaken.

Results:There wereno premature deaths and no test-item related changes in clinical condition, body weight gain or food intake. On Days 8, 9, 11 and 12 of treatment, signs associated with dose administration comprised salivation in several males and females receiving 300 mg/kg/day. In addition, on Days 13 and 14 of treatment, chin rubbing was also evident in several males and females. At scheduled termination on Day 35 of age, mean absolute and adjusted liver weights were slightly high for males and females at 300 mg/kg/day only. Macroscopic examination revealed no test-item related abnormalities.

Conclusion:Based on the results of this preliminary juvenile toxicity study, the only effect of treatment was that at 300 mg/kg/day. Slightly increased liver weights recorded in males and females receiving 300 mg/kg/day was considered not to be adverse. Based on the degree of liver weight increase seen in males and females receiving 300 mg/kg/day, it is advised that the high dose level in the forthcoming main OECD TG 443 study should not exceed 300 mg/kg/day.

90 -day dermal (with oral exposure) repeated dose study (OECD TG 411, rat, NTP, 2016)

In the rat study, OTNE was tested in a sub-chronic dermal toxicity study according to OECD Guideline 411. The study is rated Klimisch 2, because the substance was administered through dermal application without coverage resulting in oral exposure (due to grooming), which is not in line with the required coverage according to the guideline and a real systemic dermal NOAEL cannot be derived.

Method: Male and female F344/NTac rats were administered OTNE dermally for 3 months. Groups of 10 male and 10 female rats received no treatment (untreated control) or dermal application of OTNE in 95% aqueous ethanol at concentrations of 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, or 100% (neat) 5 days per week for 3 months (31.25, 62.5, 123, 250 and 500 mg/kg bw). Animals in the 100% OTNE groups were compared to untreated controls, and the remaining dose groups were compared to the vehicle controls. Formulations were administered at a volume of 0.5 mL/kg body weight, on approx. 10% of body surface area (25 cm2).

Results:

Local dermal effects: For the local irritant effects the dose in concentration will be used because concentration is a better indicator for irritancy than the dose. In male and female rats administered 12.5 to 100% OTNE, the incidences of minimal to mild hyperplasia and hyperkeratosis (except in 12.5% OTNE males) at the site of application were significantly greater than those in the vehicle control groups. The dermal exposure and effects are confounded for the following reasons: 1) grooming by the animals decreased the actual dermal dose; 2) dermal exposure time is decreased and; 3) the scratch, rubbing and licking of the exposed site may have enhanced the dermal penetration. In view of these confounders a dose descriptor for local effects via the dermal exposure route is deemed inappropriate.

Clinical signs and body weight: All rats survived to the end of the study. There were no biologically significant clinical or behavioural effects. Final mean body weights and body weight gains of dosed male and female rats were similar to those of the respective control groups with the exception of the 500 mg/kg bw/day OTNE male rats, which had a statistically significant but minor decrease in mean body weight gain (-6%) compared to the untreated control.

Clinical chemistry: Alanine aminotransferase and Alkaline phosphatase are significantly decreased becoming dose depending from 125 mg/kg bw onwards (starting with a decrease of 25%) in all dosed groups of males and in 125 to 500 mg/kg bw/day OTNE females. In general, the toxicological relevance of a decrease of these enzymes is generally doubtful but it is consistently seen.

Organ effects:

Liver: Relative liver weights at 250 and 500 mg/kg bw/day OTNE were significantly increased (> 10-31%) compared to the control. At 250 mg/kg bw this increase is considered adaptive at 500 mg/kg bw these may be considered adverse. Kidney: The relative kidney weights of the 500 mg/kg bw/day OTNE male and female rats were significantly increased by 10 and 8%, respectively. These increased are not considered adverse. Other effects: There were no neurotoxic, immunological toxic or fertility effects seen. There were no biologically significant neoplastic findings found.

Conclusion

A local long-term repeated dose NOAEL could not be established because the concentration to which the animals are exposed doubtful due to oral grooming. A systemic dermal repeated dose NOAEL cannot be established because beside dermal exposure the oral exposure route is a major route e.g. the relative liver weights in the oral and dermal studies is similar increased (ca 50% at 500 mg/kg bw) in the oral 90-day IFF study as in this NTP study. Only a combined systemic dermal/oral NOAEL can be established. This can be set at 250 mg/kg bw considering the relative liver weight increase of < 30% as an adaptive effect in absence of other liver related findings of toxicological relevance.

 

90 -day dermal repeated dose study (OECD TG 411, mice, NTP, 2016)

In the mouse study, OTNE was tested in a sub-chronic dermal toxicity study according to OECD Guideline 411. The study is rated Klimisch 2, because the substance was administered through dermal application without coverage resulting in oral exposure and a real systemic dermal NOAEL cannot be derived.

Method

Solutions of OTNE in ethanol were applied to the skin of mice 5 days per week for 3 months, without occlusion. Ten mice were tested in each dose group. Doses were 6.25%, 12.5%, 25% and 50% OTNE in ethanol and 100% OTNE, on approximately 10% of body surface area (6.6 cm²). These doses correspond approximately to 125, 250, 500, 1000 and 2000 mg/kg bw. One group received ethanol alone and served as the control group for the dose groups that were exposed to 125 to 1000 mg/kg bw/day OTNE in ethanol. Another control group received no administration of ethanol and served as the control group for the dose group exposed to 2000 mg/kg bw/day OTNE, which had no ethanol. During the course of this study, samples were collected for clinical chemistry and oestrous cycle characterization. At the end of the study, samples were collected for reproductive tissue evaluations, genetic toxicology studies, and >40 tissues were collected from each animal for histopathology diagnosis.

Results

Local dermal effects: In all exposure groups >=6.25%, there was an increase in the incidence of skin lesions (hyperkeratosis, hyperplasia, chronic active inflammation, fibrosis, epidermal inflammation, and hair follicle hyperplasia) and therefore a NOAEL cannot be set for local dermal repeated exposure. The dermal exposure is confounded for the following reasons: 1) grooming by the animals decreased the actual dermal dose; 2) dermal exposure time is decreased and; 3) the scratch, rubbing and licking of the exposed site may have enhanced the dermal penetration. In view of these confounders a dose descriptor for local effects via the dermal exposure route is deemed inappropriate.

Clinical signs and body weight: All animals survived to the end of the studies. There were no biologically significant clinical (except for skin irritation) or behavioural findings related to OTNE administration. Small but significant decreases in body weights were observed in male mice from 500 mg/kg bw onwards remaining below 10% up to 1000 mg/kg bw but a decrease of 14% was seen at 2000 mg/kg bw.

Haematology: The haematological effects concerned decreased erythrocyte count and haematocrit, lowered haemoglobin concentrations and an increase in platelets. These effects were found significant at dose levels of 1000 and 2000 mg/kg bw/day in male and females.

Organ: Heart: Absolute and relative heart weights were increased approximately 14% in the 1000 and 2000 mg/kg bw/day females. The relative heart weights of 500 and 1000 mg/kg bw/day males were also increased, but this may be due to the decreased male body weights. Thymus: The absolute thymus weights of males and females as well as the relative thymus weight of females were decreased by approximately 22% in 2000 mg/kg bw/day groups compared to untreated controls without macro or microscopic findings. Liver: A dose-dependent increased relative liver weight in males and females was found. At 500, 1000 and 2000 mg/kg bw this increase was ca > 30%, 50 and 89%, respectively. Also liver hypertrophy was seen in males. Reproductive organs-Fertility: No findings in males were seen up to and including 1000 mg/kg bw. At 2000 mg/kg bw (twice the limit dose) in males fewer caudal sperm and caudal sperm/mg, with lower motility (11%) was seen. These findings were not associated with any histopathologic changes in the testes or epididymis. No findings were seen in females up to and including 1000 mg/kg bw. Female mice administered 2000 mg/kg bw/day exhibited an increase in cycle length of approximately 1 day and displayed an increased probability of extended oestrus as compared to the untreated controls. In view of minimal effects seen and absence of macro- and microscopic effect in both male and female reproductive organs, these effects are not considered adverse.

Other effects: There were no neurotoxic or immunologic findings. There were no biologically significant neoplastic findings related to the substance.

Conclusion on the sub-chronic dermal tests

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals were exposed are doubtful due to oral grooming. A systemic dermal repeated dose NOAELcannot be established because beside dermal exposure the oral exposure route may be the major route. Therefore only a systemic dermal/oral NOAEL can be derived. This NOAEL is 500 mg/kg bw considering the relative liver weight increase as an adaptive effect in absence of other liver findings.

Justification for classification or non-classification

Based on all available oral sub-chronic repeated dose toxicity data, the NOAEL relevant for human hazard assessment is determined to be 120 mg/kg bw/day from the key 90 -day oral repeated dose toxcicity study. The dose and the effects seen result in absence of classification and labelling for this endpoint according to EU CLP (EC 1272/2008 and its amendments).