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Effects on fertility

Description of key information

Key informatiom: Extended one-generation study (OECD TG 443): Systemic NOAEL >= 300 mg/kg bw. Fertility NOAEL >= 300 mg/kg bw.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
A DRF for the extended one generation study is present similar to the Reproduction/developmental toxicity screening test (similar to OECD TG 421) and an extended one-generation reproduction toxicity study are available. In addition, in two oral gavage studies of 28- and 90-day no fertility effects are seen at the highest dose tested of 500 and 1000 mg/kg bw, respectively.
Effect on fertility: via dermal route
Quality of whole database:
The dermal studies are insufficient for deriving information on systemic toxicity and fertility. Below a summary of the dermal studies is included in the overall summary of all repeated dose studies.
Additional information

Several studies are available in which effects on fertility and/or developmental toxicity was assessed. The key information from the EOGRT (OECD TG 443) is presented first. The information from the DRF for the EOGRT (somewhat similar to OECD TG 421)is then presented and thereafter the information from the repeated dose toxicity studies.

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 consumption of 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.

Haematology investigations 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 chemistry investigations 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 investigation of 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 investigation of 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 investigation of 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: Relative liver weights 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. Relative kidney weights 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.

Effects 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.

Repeated dose Reproscreen study DRF for the OECD TG 443 somewhat similar to OECD TG 421

This 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.

Heamatology: 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.

Reproductive toxicity in the repeated dose toxicity 90 days in rat - oral (OECD TG 408)

The safety of the test substance OTNE was examined in a subchronic (13 week) oral toxicity study in Wistar rats, according to OECD TG 408. OTNE was administered by daily oral gavage as a dilution in corn oil at levels of 0 (vehicle control), 30, 120 and 500 mg/kg body weight/day to groups of 10 rats/sex during 13 weeks. No relevant findings were seen on fertility and there were no histopathological lesions in reproductive organs. The increase of the relative testes weight at the mid dose only is not toxicologically relevant (absolute is more important than relative weight for this organ). Some dilatation of the uterus was reported, however as it this was also observed in the control group it was not considered an effect related to exposure to OTNE. Overall no effects on fertility were observed in the animals exposed to OTNE. The NOAEL for fertility is >=500 mg/kg bw,

Reproductive toxicity in the repeated dose toxicity 90 days in mouse and rat - dermal with oral exposure - NTP studies

The study was performed to assess the dermal repeated dose systemic toxicity as a result of dermal exposure to OTNE in the rat and mouse. The study was performed comparable to OECD TG 411, with some deviations (Dermal application without occlusion (not justified); partial oral exposure through grooming is expected, the top dose tested in mice was above the recommended dose)). Solutions of OTNE in ethanol were applied to the skin of rat and mice for 5 days per week for 3 months. There were 10 rodents in each dose group. In rat and mice, doses for which reproductive effects were evaluated were 25% and 50% OTNE in ethanol and 100% OTNE. These doses correspond approximately to 125 to 500 mg/kg bw/day in rat 500 to 2000 mg/kg bw/day in mice. During the course of this study, samples were collected for estrous cycle characterization. At the end of the study, samples were collected for reproductive tissue evaluations. No effects were seen in rat. Exposure via dermal application in mice showed some minor but significant fewer sperm and lower sperm motility in the 2000 mg/kg bw/day male group. In females at 2000 mg/kg bw the estrus cycle was significantly extended at this dose with one day. The effects in mice are all observed at the high dose of 2000 mg/kg bw in which a very high increase of relative liver weight was seen (up to 90%). At 1000 mg/kg bw no such adverse effects on fertility are seen in male and female mice.

Reproductive toxicity in repeated dose toxicity 28 days in rat - oral (OECD TG 407)

This study was performed to assess the systemic toxicity of OTNE to the rat according to OECD TG 407. OTNE was administered by oral gavage, once daily, to three groups of 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 bw/day. After the 28-day exposure period, no effects on fertility were observed in the animals exposed to OTNE, therefore the NOAEL is set at >= 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

Rat: Prenatal development toxicity study (OECD TG 414): maternal NOAEL 240 mg/kg bw/day, developmental NOAEL >=480 mg/kg bw/day

Rabbit: Prenatal developmental toxicity study (OECD TG 414): maternal toxicity NOAEL is 200 mg/kg bw, developmental NOAEL >=500 mg/kg bw.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The quality of the database is high because 2 OECD TG 414 in rat and rabbits are available beside developmental toxicity in a DRF for the EOGRT test (OECD TG 443) and the actual EOGRT test (OECD TG 443). In addition, a study in which the transfer across the placenta and into milk of rats during and after pregnancy following repeated oral administration was studied. These studies adequately fulfill the REACH requirements.
Additional information

Rat developmental toxicity study (OECD TG 414)

A developmental toxicity study was performed in accordance with the US FDA Guideline on detection of toxicity to reproduction for medicinal products, which is equivalent to OECD TG 414. The study was performed under GLP conditions. Groups of pregnant rats (25/dose group) were gavaged with 0 (control), 96, 240, or 480 mg/kg bw/day on gestation days 7-17 at dosage volumes of 0.5, 0.1, 0.25, or 0.5 ml/kg bw, respectively. Viability, abnormal clinical signs, body weights, abortions, premature deliveries, and feed consumption were recorded. Mating performance was assessed. On gestation day 21, surviving rats were euthanized by carbon dioxide asphyxiation and Caesarean sections were performed to remove the foetuses. Rats underwent gross necropsy. Foetuses were assessed for litter parameters and gross external, soft tissue, or skeletal malformations. Maternal animals showed reduced body weights and persistent clinical signs (e.g., increased salivation and urine-stained abdominal fur) at the highest dose. There were no treatment-related effects on developmental parameters at any dose, although reduction (not statistically significant) in foetal body weight was noted at the highest dose. Based on these results, the maternal NOAEL was 240 mg/kg bw/day and the developmental NOAEL was >=480 mg/kg bw/day.

Dose range finding study for the rat developmental toxicity study

Introduction and Method: For the DRF for the OECD TG 414, forty presumed pregnant Crl:C~SD)IGSBR VAFlPlus@ rats were randomly assigned to five dosage groups, eight rats per group. The test article, OTNE, or the control article, reverse osmosis membrane process deionized water (R.O. water), were administered orally via gavage once daily to these naturally-bred female rats on days 7 through 17 of presumed gestation Doses were 0, 240. 480. 960 and 1920 mg/kg bw. The dosage volumes were 2.0. 0.25, 0.5, 1.0 and 2.0 mI/kg bw and were adjusted daily on the basis of the individual body weights recorded immediately before dosage administration. Checks for viability were made twice daily. Clinical observations were recorded daily before dosage and 60 ± 10 minutes after administration. These observations were also recorded once daily during the post dosage period. Body weights were recorded on DO 0 and daily during the dosage and post dosage periods. Feed consumption values were recorded on DOs 0, 7, 10, 12, 15, 18 and 21. All surviving rats were sacrificed on DG 21 and examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Foetuses were weighed and examined for gross external alterations and sex.

Results: One rat in the 1920 mg/kg bw dosage group was moribund sacrificed on DO 16. All other rats survived until scheduled sacrifice. Test-article related clinical observations included chromorhinonhea, excess salivation and a dried red or red perioral substance in all treated groups; a dried red or red substance on the fur in the 240, 960 and 1920 mg/kg bw dosage groups; urine-stained abdominal fur and a dried red or red perivaginal substance in the 960 and 1920 mg/kg bw. dosage groups and amber coloured urine in the 1920 mg/kg bw dosage group. Body weight changes in the 240 and 480 mg/kg bw dosage groups tended to be slightly reduced as compared to the control group value during the first three days of dosage administration. Body weight gains were reduced in the 960 and 1920 mg/kg bw dosage groups-for the entire dosage period, the entire gestation period after the initiation of dosage administration and for the entire gestation period. Body weights were generally comparable among the dosage groups throughout the study. Absolute and relative feed consumption values were reduced for the 960 and 1920 mg/kg bw dosage groups. Absolute and relative feed consumption values were reduced in these groups for the entire dosage period, the entire gestation period after the initiation of dosage administration and for the entire gestation period. The values were comparable to the control group values for the entire post dosage period. No Caesarean-sectioning or litter parameters were affected by dosages of the test article as high as 1920 mg/kg/day. Conclusion: Based on the results of this study, dosages of 0 (control article), 240, 480 and 960mg/kg bw are recommended for the developmental toxicity study in rats. The 240 mg/kg bw dosage is expected to be a NOAEL for both maternal and embryo/foetal toxicity, and the 960 mg/kg bw is expected to produce maternal toxicity and little or no developmental toxicity.

Final dosing: For the final study the 480 mg/kg bw was used as the highest dose.

Rabbit developmental toxicity study (OECD TG 414)

In this rabbit developmental toxicity test according to OECD 414 (in compliance with GLP) the effects of the test substance was tested on pregnant New Zealand White rabbits and the development of the embryo and fetus following daily oral administration by gavage during gestation days (GD) 6-28.

Method:The dose levels were set at 0, 75, 200 and 500 mg/kg bw based on repeated dose toxicity studies with rats and DRF with rabbits. In the DRF rabbits no body weight effects were seen but the increase in relative liver weight in rat up to 50% at 500 mg/kg bw (oral gavage 90 -day study) was included in the decision not to overload the metabolic capacity too much for rabbits because also for rabbits the glucuronic pathway is considered key for excretion of the substance. This is because for both species the ketone and after reduction to the alcohol will be the key site of conjugation. The dose levels are set on a tolerable liver weight increase of ca 25% . Eighty-eight time-mated rabbits arrived at the test facility and were allocated to one control group and three groups receiving the test substance by oral gavage. A dose volume of 2 mL/kg body weight was applied in all groups and corn oil was used as vehicle. Control animals received the vehicle only. In-life parameters included signs of morbidity and mortality, body weight and food consumption. On gestation day 29 the dams were sacrificed and examined macroscopically. Fetuses, placentas, reproductive organs and kidneys and livers were weighed. The fetuses were macroscopically examined and processed for visceral and skeletal examinations.

Results, Maternal toxicity:

Two animals died before the scheduled necropsy on gestation day 29, these deaths were not treatment-related. Indications of maternal toxicity were noted in the high dose group. Mean body weight of these animals was lower throughout the study compared to the control and other treatment groups, reaching a difference of ~5% on day 29. This is mainly ascribed to a significantly lower food consumption (~32% compared to the control) and the resulting body weight loss during the first three days of dosing (day 6 to 9). This growth retardations reflects the lower body weights throughout the study. The overall food consumption in the high dose group was reduced with ~17% compared to the control group. The significant increase in the relative liver weight in the high dose group (16%) may be adaptive (induced to metabolise the substance) but is conservatively used to help setting the NOAEL. The body weight effects and reduced food consumption and the increased relative liver weights are used to derive the NOAEL of 200 mg/kg bw.

Results, Developmental toxicity:

No effects of treatment were observed on pre-implantation and post-implantation loss. The number of live fetuses per dam was not affected by the treatment. The ossification status of the vertebrae and sternebrae did not indicate a treatment-related delayed development of the fetuses. Incomplete ossification of the forelimb and hindlimb was observed in all groups, as is expected in normal fetal development. Skeletal malformations of the vertebra and fused ribs occurred incidental and were randomly distributed in all groups, indicating no treatment related effect. This is supported by the absence of a treatment-related effect on variations in the shape vertebra. Malformation and variations in the shape of sternebrae were more common than skeletal malformation of the vertebra and ribs, but lacked a treatment-related trend. This results in a NOAEL of >=500 mg/kg bw for developmental toxicity.

Conclusions

Based on the indications of maternal toxicity in the highest dose group, i.e. increased relative liver weight and decreased food consumption and body weight change, the NOAEL for maternal toxicity in rabbits is 200 mg/kg bw/day. Based on the absence of effects on implantation sites , i.e. resorptions, live/dead fetuses, and fetal development, i.e. malformations, variations, teratomas, the NOAEL for developmental toxicity in rabbits is ≥ 500 mg/kg bw/day.

Justification for classification or non-classification

Based on the results, which indicate no fertility and developmental toxicity effects of OTNE, the substance does not need to be classified as toxic to reproduction according to EU CLP Regulation (EC 1272/2008 and its amendments).