Registration Dossier

Administrative data

Description of key information

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Feb 2017 to 17 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21st September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Directive 2001/59/EC, Official Journal of the European Communities, L225, 21.8.2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han IGS (Crl:WI(Han))
Details on species / strain selection:
The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. This rat strain was used because it is routinely used at the test facility for this type of studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6-7 weeks old at the start of the treatment period.
- Weight at study initiation: The body weights at initiation of treatment were within ± 20% of the mean weight for each sex, and ranged from 143- 171 g (mean 155 g) for males and from 113- 134 g (mean 123 g) for females.
- Housing: The animals were kept in macrolon cages with wood shavings (Lignocel) as bedding material, and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex. On the day of FOB testing and motor activity assessment, the animals were temporarily kept singly in macrolon cages. During urine collection, animals were kept singly in stainless-steel metabolism cages.
- Diet: From their arrival until the end of the study (unless precluded by the collection of concentrated urine in week 13, or the collection of blood from overnight fasted rats prior to scheduled necropsy), the rats received a powdered, cereal-based rodent diet (VRF1 (FG))from a commercial supplier (SDS Special Diets Services, Witham, England) batch 2771. The diets were provided ad libitum in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. During the study, the food in the cans was replaced by fresh portions from the freezer weekly and filled up as needed.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 Feb 2017 To: 16 (males) and 17 (females) May 2017
Route of administration:
oral: gavage
Details on route of administration:
The oral route was used because this is a possible route of human exposure and the key route for assessing the toxicity of the substance as such.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials (one vial per group per day) in a refrigerator. The vehicle for dosing the controls wassimilarly be stored. All gavage liquids were continuously stirred on a magnetic stirrer during the dosing procedure, to ascertain the homogeneity of the test substance in the vehicle.

VEHICLE
- Concentration in vehicle: 0, 6, 24 and 100 mg/mL, corresponding to dose levels of 0, 30, 120 and 500 mg/kg bw/ day respectively.
- Amount of vehicle (if gavage): 5 mL/kg bw/ day, the dose volumes were adjusted weekly to the latest recorded body weights for each individual rat.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the test substance in gavage liquid were conducted using a Gas Chromatography (GC) method, based on the method provided by the sponsor. The test substance was used as the reference standard for the preparation of calibration samples.

METHOD VALIDATION
Before analysis of study samples, the analytical method was validated by analyzing three spiked samples per dose level, to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996.
- Recovery: the mean recovery of the test substance from gavage liquid should be between 85% and 115% at each of the dose levels of the study.
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
- Specificity: signals should be corrected in case the signal obtained for blank samples was ≥ 5% of the signal obtained for low-dose samples..

ANALYSIS OF GAVAGE LIQUIDS
Preparation of validation samples:
Validation samples with nominal concentrations of 0, 6, 24 and 100 mg test substance per ml gavage liquid were prepared by addition of approximately 0, 12, 48 and 200 mg test substance to 2 ml gavage liquid (control, low-dose, mid-dose and high-dose, respectively).

Validation samples were analyzed in triplicate and study samples were analyzed in duplicate.

Homogeneity:
The homogeneity of the test substance was assessed in the batch of gavage liquids, prepared for the study. Three samples of each test gavage liquid, taken at different locations in the gavage liquid container, and 1 sample of the control gavage liquid were analyzed in duplicate. For each concentration level, a one-way analysis of variance (Anova) was performed using the sample location (1-3) as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the three locations in the gavage liquid container). The test substance was considered to be homogeneously distributed in the gavage liquid if p ≥ 0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the three locations was ≤ 5%.

Stability:
The stability of the test substance in gavage liquid was assessed in the batch of gavage
liquids, prepared for the study (4 hours animal room and 1 week at 2-10 °C). One sample of each test gavage liquid and one sample of the control gavage liquid were analyzed in duplicate.
For each concentration level, a one-way analysis of variance (Anova) was performed using time as grouping factor. An associated F-value with probability p < 0.01 was considered to be significant (i.e. the mean concentrations differ significantly before and after storage). The test substance was considered to be stable in gavage liquid if p ≥ 0.01 and/or if the mean concentration after storage was within 90-110% of the mean concentration at t = 0.

Content:
The content of the test substance was determined in the batches of gavage liquids, prepared for the study five data spread evenly within the study period.
The content of the test substance in gavage liquid was considered to be “close to intended” if the mean measured concentration was between 90 and 110% of the intended concentration.

Duration of treatment / exposure:
13 consecutive weeks (7 days/week)
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The doses were intended to provide information on the sub-chronic oral toxicity of the test substance and to establish a no-observed-adverse-effect level (NOAEL).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment (including post-dosing signs) were recorded.

DETAILED CLINICAL OBSERVATIONS AND NEUROBEHAVIOURAL EXAMINATION:
In addition to the above daily general clinical observations, detailed clinical examinations (in an arena outside the home cage) were performed on all rats prior to the first exposure and then once weekly throughout the study. Behavioral endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats at the end of the study (in week 12 or 13 for females and in week 13 for males). Motor activity tests data recorded on DVD were removed from the study dossier after submission of the final report.

BODY WEIGHT:
The body weight of each animal was recorded once during the acclimatization period (day -1), at initiation of treatment (day 0), and once per week thereafter. The animals were weighed after fasting prior to scheduled necropsy in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION
Food consumption was measured per cage by weighing the feeders. The consumption was measured over weekly periods of for all animals in the cage. The results were expressed in g per animal per day.

WATER CONSUMPTION
Water consumption was measured per cage, by weighing the drinking bottles daily, during 5- day periods in weeks 1, 6 and 11. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic observations were made prior to the start of treatment (on day -5) in all rats, and in the last week of the treatment period (on day 83 and 85 for males and females, respectively) in all rats of the control group and the high-dose group. Because no treatment related ocular changes were observed in the high-dose group, eye examination was not extended to the animals of the intermediate-dose groups at the end of the study. Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulfate.

HAEMATOLOGY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). EDTA or citrate (for prothrombin time) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out: hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBC), reticulocytes, total white blood cells (WBC), differential white blood cells (Lymphocytes, neutrophils, eosinophils, basophils and monocytes), prothrombin time and thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY:
At necropsy, blood samples were taken from the abdominal aorta of all rats whilst under CO2/O2 anesthesia. The rats were fasted overnight before necropsy (water was freely available). The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were discarded after analysis. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, (fasting) glucose, bilirubin (total), cholesterol (total), triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate (PO4).

URINALYSIS:
In week 13 (day 84-85 and 85-86 for males and females, respectively), all rats were deprived of water for 24 hours and of food during the last 16 hours of this period. During the last 16 hours of deprivation, the rats were individually kept in metabolism cages and urine was collected. Urine samples were discarded after analysis. The following determinations were carried out in individual samples: volume and specific gravity (to investigate the concentrating ability of the kidneys), appearance, pH, glucose, microscopy of the urinary sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, worm eggs, sperm cells), occult blood, ketones, protein, bilirubin and urobilinogen.
Sacrifice and pathology:
GROSS PATHOLOGY:
Early in week 14, after overnight fasting (water was freely available), the animals were killed on two successive working days (day 91 and 92 for males and females, respectively) in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO2/O2 anesthesia and then examined grossly for pathological changes.
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated on the basis of the terminal body weight of the animals: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (with coagulating glands), spleen, testes, thymus, uterus.

HISTOPATHOLOGY:
The tissues to be examined microscopically were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin.
Histopathological examination (by light microscopy) was performed on all tissues and organs listed below, of all animals of the control group (1) and the high-dose group (4) with the exception that gross lesions, liver and spleen from all animals (groups 1-4) were examined. The kidneys were examined in all male rats of all dose groups. Additionally, renal α2- microglobulin was assessed by immunohistochemistry. For that purpose, sections of the kidneys of all male animals of all dose groups were processed for immunohistochemical staining of α2-microglobulin using an α2-microglobulin antibody (Thermofisher PA5-47788).

The following organs and tissues were examined: adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum, cerebellum), cecum, colon, duodenum, epididymides, esophagus, eyes, GALT (gut associated lymphoid tissue, including Peyer's patches), heart, ileum, jejunum, kidneys, liver, lungs, mammary gland (females), mesenteric lymph nodes, nerve-peripheral (sciatic), ovaries, oviducts (=fallopian tubes), pancreas, parathyroid, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles+ coagulating glands, skeletal muscle (thigh), skin (flank), spinal cord (retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar)), spleen, sternum with bone marrow, stomach (non glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix), vagina, all gross lesions.
Statistics:
Please refer to 'other information on materials and methods incl. tables'
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted mainly in the high-dose group and occasionally also in the lower dose groups. This finding is ascribed to the dosing of the rats by oral gavage. Post dosing signs comprised salivation just prior to and/or after dosing and, at a few occasions, sliding with the ventral parts of the head and neck over the cage bottom after dosing. These findings were noted mainly in the high-dose group and occurred only incidentally in the other groups. There were no other treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
There was no mortality in any group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were statistically significantly decreased in males of the low- and mid-dose group at several stages. The differences with the controls were only slight (between 3-8%) and there was no dose-response relationship. Body weights were statistically significantly increased in females of the high-dose group and (at two occasions) in the mid-dose group. The differences with the controls were very slight (less than 5%). There were no marked differences in body weight gain between test groups and controls. Occasionally, statistically significant differences in body weight gain were noted, but these were not consistent. In conclusion, there were no relevant changes in growth parameters.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was increased in the high-dose group in both sexes. The differences with the controls were statistically significant at most of the weekly stages. Incidentally, a statistically significant difference with controls also occurred in the low- or middose group, but these findings were not consistent and the overall food intake was not affected in these groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake was increased in the high-dose group in both sexes. In the first week of the study, the differences with the controls were only slight (overall 8%) and occurred in highdose males only. At the subsequent stages, these differences aggravated (overall ≥ 41% for males and ≥ 24% for females) and were often statistically significant. In the other groups water intake was not affected; an incidental statistically significant decrease in water intake in low-dose males on day 5 of the study was considered a chance finding.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination did not reveal any treatment-related changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group, statistically significant decreases were noted in red blood cell count, hemoglobin concentration and packed cell volume in males, and in hemoglobin concentration in females. Thrombocytes were increased in males of the high-dose group. Prothrombin time was reduced in females of the high-dose group. An incidental increase in MCHC was noted in males of the low-dose group.
Total white blood cell count was statistically significantly increased in males of the high-dose group. The absolute counts of lymphocytes, neutrophils and basophils were also increased but there were no relevant changes in the percentage distribution.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol and phospholipids were statistically significantly increased in females of the highdose group. The values obtained in this group were outside the range of historical control data.
The following changes were statistically significant but were not considered to be of toxicological significance:
- Ca concentration was increased in the high-dose group in both sexes. Ca concentration was also increased in low-dose males but there was no dose response relationship. The values in the various groups were within the range of historical control data and therefore the fluctuations in calcium level were not considered to be adverse.
- ASAT activity was decreased in females of the high-dose group. However, an increase rather than a decrease in this variable may represent a toxic effect .
- Creatinine concentration was decreased in females of the high-dose group.
- An incidental decrease in Albumin/globulin ratio was noted in mid-dose males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The renal concentration test showed a statistically significantly increased urinary volume in males of the high-dose group. The specific gravity was not significantly affected. This finding was probably related to the higher water intake in the high-dose group. Semi-quantitative (dipstick) urinary measurements showed a slight though statistically significant decrease in urinary pH in males of the mid- and high-dose group. Protein and ketones were decreased in males of the high-dose group. These findings are not considered to be of toxicological significance. Urinary appearance (color and clarity) is presented in individual animals only. There were no clear differences in urinary appearance among the groups. Microscopic examination of the urinary sediment showed a statistically significant increase in epithelial cells, amorphous material and casts in males of the high-dose group. The casts were noted in six males of this group and also in two males of the mid-dose group. In females of the high-dose group, urinary crystals were increased, but there was considerable variation in the severity of this finding in individual rats in the various groups. Urinary crystals are considered of limited, if any, toxicological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the substance in rats.
Immunological findings:
no effects observed
Description (incidence and severity):
In general, the results of this study did not indicate any immunologica effects (in or decrease of white blood cells)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant differences with the controls were noted:
- The relative weight of the liver showed a dose-related increase in males and females of the mid- and high-dose groups. The differences with the controls were 12-15% in the mid-dose group and more than 50% in the high-dose group. The absolute liver weights were increased in high-dose males and in mid- and high-dose females.
- 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%). The absolute kidney weights were increased in high-dose males and in mid- and high-dose females.
- The relative weight of the spleen was increased (37%) in males of the high-dose group. The absolute weight of the spleen was also increased in males of this group.
- The relative weight of the heart was increased (11%) in males of the high-dose group.

The following changes were statistically significant but were not considered to be of toxicological significance:
- The absolute weight of the heart was decreased in males of the mid-dose group. This finding was not reflected in significant changes in the relative weight of this organ and is therefore considered a chance finding.
- The relative weight of the testes was increased in mid-dose males. This finding is ascribed to the lower terminal body weights in this group. During growth retardation, rats tend to maintain their (absolute) weight of the testes, and there is a well-known inverse correlation between terminal body weight and relative testes weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy no treatment related macroscopic changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation revealed treatment-related microscopic changes in the kidneys, the liver and the spleen.

- The microscopic changes in the kidneys comprised intra- and extracellular accumulation of hyaline droplets in tubules of the outer cortex of the kidneys of 10/10 high-dose males, in all cases accompanied by an increased number of basophilic tubules. In seven of these males the kidneys also showed dilated tubules with eosinophilic casts consisting of granular debris. The characteristics of the findings in the kidneys, along with the observation that they occurred in males only, were highly suggestive for accumulation of alpha 2 urinary microglobulins (alpha 2u). To further specify these changes the kidneys were immunohistochemically stained with a polyclonal antibody against alpha 2u. Evaluation of the immunohistochemically stained kidneys revealed that the structures identified as hyaline droplets and granular casts in the HE stained slides showed a positive reaction (in Table 15 indicated as ‘ihc (immunohistochemically) positive for alpha-2u’). Microscopy of the kidneys of the low- and mid-dose males revealed alpha 2u-positive hyaline droplet accumulation in 9/1 low-dose males and in 10/10 mid-dose males. The severity of these findings was generally highest in the high-dose group and lower in the low- and mid-dose groups.
- 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 hepatocellular hypertrophy was 3/10, 6/10 and 9/10 in the low-, mid- and highdose males, respectively and 8/10 and 9/10 in the mid- and high-dose females, respectively. The incidence of macro-vesicular hepatocellular vacuolation was increased in 10/10 mid-dose and 10/10 high-dose females.
- The microscopic changes in the spleen were characterized by increase of extramedullary erythropoiesis in 8/10 high-dose males, in 3/10 mid-dose females and in 5/10 high-dose females.

The other organs and tissues did not reveal treatment-related histopathological changes. The histopathological changes observed were about equally distributed between the high-dose group and the controls or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Analysis of the dosing dilutions

- The substance was homogeneously distributed in the carrier at each test concentration.

- The substance was stable in the carrier under the experimental conditions (storage in the animal room for 4 hours or in the refrigerator (2-10°C for 7 days).

- The content of the substance in the test dilutions was close to intended in all five batches analysed, except for the low-dose level prepared on 13 March 2016 (the difference with intended was -16%). This finding was, however, attributed to the relatively high intercept of the calibration curve obtained during this analytical run.

- On the basis of the above it is concluded that the rats received the intended concentrations.

Conclusions:
Oral (gavage) administration of the substance, for a period of 13 weeks to Wister Han IGS (Crl:WI(Han)) rats revealed a NOAEL of 120 mg/kg bw/day based on toxicological significant effects on liver and spleen in the high dose group.
Executive summary:

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 test in vehicle 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.

Clinical chemistry was conducted in all rats at necropsy. An increase cholesterol and phospholipids concentrations in females of the high-dose group was seen.

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.

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 alpha 2urinary 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 and immunological toxic 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
For repeated dose toxicity a subchronic oral study is available, conducted according to OECD TG 408 in compliance with GLP. The resulting information is adequate for the endpoint and prevails the result of the 28-day repeated dose toxicity study (K1) as the dose descriptor is worst-case and duration of this study is longer.
System:
other: Liver weight increase and liver hypertrophy also some effects were seen on haematopoieses (decrease in red blood cell parameters, increase in extracellular haematopoieses in the spleen)
Organ:
liver
spleen

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Dermal application without covering. Oral exposure due to grooming has occurred.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
The dermal applied dose is not covered, grooming and oral exposure has occurred.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: F344/NTac
Details on species / strain selection:
For many years, the NTP used the inbred F344/N rat for its toxicity and carcinogenicity studies. However, this model has exhibited sporadic seizures, idiopathic chylothorax, and consistently high rates of mononuclear cell leukemia and testicular neoplasia. Therefore the F344 rat from the Taconic commercial colony (F344/NTac) was chosen as a more fecund rat model that could be used in both reproductive and carcinogenesis studies for comparative purposes.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female F344/NTac rats were obtained from the commercial colony at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the rats were 5 to 6 weeks old
- Weight at study initiation: 82-84 g (female) 83-86 g (male)
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 (males) or 13 (females) days

DETAILS OF FOOD AND WATER QUALITY:
Diet Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1.668° C)
- Humidity (%): 50% ± 15%
- Air changes (per hr): ≥ 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day, 12 hours/night

IN-LIFE DATES:
Date of First Dose: December 12 (males) or December 13 (females), 2006
Necropsy Date: March 13 (males) or March 14 (females), 2007


Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: approximately 10% of body surface area (corresponds to approximately 25 cm2)
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL/kg body weight (rats)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50% or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used

CALCULATION OF LOCAL EXPOSURE: average body weight (kg) * exposure concentration (µg/kg bw/day) / surface area (25 cm2)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed three times by the study laboratory using GC/FID by analysis. All 30 of the dose formulations analyzed for rats were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; 28 of 30 were within 10% of the target concentrations.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Dose / conc.:
500 mg/kg bw/day
Remarks:
100% OTNE
Dose / conc.:
250 mg/kg bw/day
Remarks:
50% OTNE
Dose / conc.:
125 mg/kg bw/day
Remarks:
25% OTNE
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
12.5% OTNE
Dose / conc.:
31.25 mg/kg bw/day
Remarks:
6.25% OTNE
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Probably based on the information of the 28-day oral gavage study.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily, histopathology at end of study period

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: hemoglobin; erythrocyte, reticulocyte, and platelet counts; erythrocyte distribution of width; mean platelet volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Animals fasted: No
- How many animals:10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- gross observation for evidence of disease, the health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program
HISTOPATHOLOGY: Yes
- Histopathology: adrenal gland, bone (femur) with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, oral cavity and larynx, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (site of application and control sites), spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
- Sperm Motility and Vaginal Cytology: At the end of the studies, sperm samples were collected from male rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 16 consecutive days prior to the end of the studies from female rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for vaginal cytology evaluations.
- Liver Cytochrome P450 Activity: Liver samples were collected from five male and five female special study rats per dose group on day 23 and from five male and five female core study rats per dose group at the end of the studies. Livers were analyzed for total microsomal protein content and CYP2E1 activity.
Statistics:
- The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance between dosed and vehicle control groups and between the 100% OTNE and untreated control groups.
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epididymal spermatozoal data, (skewed distributions) were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus, were constructed based on a Markov chain model proposed by Girard and Sager (1987). Equality of transition matrices among dose groups and between the control group and each dosed group was tested using chi-square statistics.
- A t-test was used to determine significant differences in organ and body weight data, and Wilcoxon’s (1945) rank sum test was used for hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epidymal spermatozoal data.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no biologically significant clinical findings related to OTNE administration
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Suppurative inflammation of the dermis in 1/10 Male rats treated with 100% OTNE, and 1/10 Female rats treated with 100% OTNE.
Other skin effects are reported under histopathological findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gains of dosed (except 100% OTNE) males and females were similar to those of the respective control groups. The mean body weight gain of 100% OTNE males was significantly less than that of the untreated control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in the hematology parameters attributable to the dermal administration of OTNE
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alkaline phosphatase activities were decreased in various male and female dosed groups at all time points (only average value, incidence not given): days 3 (100% (500 mg/kg bw OTNE) and 23 (50% (250 mg/kg bw) and 100% OTNE) and week 13 (all dose groups). Decreases were consistently observed in 100% OTNE males and females at all time points compared to the untreated control groups.
At the end of the study (week 13), alanine aminotransferase activities were decreased in 25% (125 mg/kg bw) OTNE or greater females and in all dosed groups of males compared to their respective control groups (only average value, incidence not given). The significance of these decreases is not known but may be related to an alteration in liver metabolism. Other changes in clinical chemistry results were not considered toxicologically or biologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the twice-daily observations, the behavior of the animals did not indicate any neurotoxic potential of the substance.
Immunological findings:
not examined
Description (incidence and severity):
Not specifically examined, however the obtained results did not indicate any immunotoxic potential of the substance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Only average value provided, incidence is not given:
Increased absolute (males 100% OTNE , females 50% and 100% OTNE) and relative (50% and 100% OTNE males and females) liver weights
Increased relative kidney weight (males and females 100% OTNE)
The concentrations 25, 50 and 100%, relates to 120, 250 and 500 mg/kg bw.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
Not specifically examined, however the behavior of the animals during the study was normal and did not indicate any neurotoxic potential of the substance. Furthermore, no histopathological abnormalities were observed in the brain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin (site of application):
- hyperkeratosis (mild to minimal):
1/10 in the 6.25% (31.25 mg/kg bw/day) group;
3/10 in the 12.5% (62.5 mg/kg bw/day) group;
4/10 in the 25% (125 mg/kg bw/day) group;
5/10 in the 50% (250 mg/kg bw/day) group;
8/10 in the 100% (500 mg/kg bw/day) group;

- hyperplasia (mild to minimal)
2/10 in the 31.25 mg/kg bw/day group;
4/10 in the 62.5 mg/kg bw/day group;
5/10 in the 125 mg/kg bw/day group;
8/10 in the 250 mg/kg bw/day group;
10/10 in the 500 mg/kg bw/day group;

Other treatment-related histopathologic, toxicologically relevant, changes were not observed in the organs of male or female rats
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Male rats did not display any OTNE-related changes in testis and epididymis weights or sperm parameters. Female rats administered OTNE did not display any toxicologically significant effects on cycle length, or estrous cyclicity.
Dose descriptor:
other: NOAEC
Remarks:
Local
Effect level:
6.25 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dermal effects are confounded by grooming
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic effects are confounded by grooming
Critical effects observed:
yes
Lowest effective dose / conc.:
12.5 other: %
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The effects reported in this study need to be evaluated in light of the used route of exposure. The study was intended as a sub-chronic dermal study; however, the animals were dermally exposed to OTNE in ethanol without occlusion of the treated site. Oral absorption (voluntarily or involuntarily), is expected as a result of oral grooming by the animals. In the present study, the grooming could have a contributed to an unknown extent to the systemic exposure and toxicity. Furthermore, also the local effects observed as a result of dermal exposure are confounded, a , as grooming by the animals can have influenced the actual dermal dose, dermal exposure time and could have given te opportunity for the animals to scratch, rub or lick the exposed site. As a result of these methodological limitations, the derivation of a dose descriptor for systemic or local effects due to dermal exposure is deemed inappropriate.

Conclusions:
As a result of methodological limitations of this dermal repeated dose toxicity study (confounding of exposure route due to oral grooming), the derivation of a dose descriptor for systemic or local effects is deemed inappropriate by the assessor. Nevertheless, the syetemic (hepatic) effects at the high doses (>=500 mg/kg bw) support the findings in the two oral repeated dose toxicity studies.
Executive summary:

Introduction: 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.

Systemic effects

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 and immunological effects seen. There were no biologically significant neoplastic findings found.

Conclusion

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals are exposed doubtful due to oral grooming

A systemic dermal repeated dose NOAELcannot 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.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Dermal application without covering and oral exposure has occurrentified.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
The site of dermal application was not covered and therefore oral exposure due to grooming has occurred.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female B6C3F1/N mice were obtained from the NTP colony at Taconic Farms, Inc
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the mice were 6 to 7 weeks old
- Weight at study initiation: Male 22.6-23.7 g, Female 18.4-19.1 g
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum, Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly
- Water (e.g. ad libitum): ad libitum, Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)
- Acclimation period: 14 (males) or 15 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1.668° C)
- Humidity (%): 50 ± 15
- Air changes (per hr): ≥ 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Date of First Dose: December 14 (males) or December 15 (females), 2006
Necropsy Date: March 15 (males) or March 16 (females), 2007

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: approximately 10% of body surface area (corresponds to approximately 6.6 sq cm)
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg body weight (mice)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50%, or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration (if solution): 100%, 50%, 75%, 87,5%, 93,75% Ethanol
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used

CALCULATION OF LOCAL EXPOSURE: average body weight (kg) * exposure concentration (µg/kg) / surface area(25 sq cm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed three times by the study laboratory using GC/FID by analysis. All 30 of the dose formulations analyzed were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; 26 of 30 were within 10% of the target concentrations.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
6.25% OTNE
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
12.5% OTNE
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
25% OTNE
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
50% OTNE
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
100% OTNE
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily, histopathology at end of study period

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: hemoglobin; erythrocyte, reticulocyte, and platelet counts; erythrocyte distribution of width; mean platelet volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Animals fasted: No
- How many animals:10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- gross observation for evidence of disease, the health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program
HISTOPATHOLOGY: Yes
- Histopathology: adrenal gland, bone (femur) with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, oral cavity and larynx, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (site of application and control sites), spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
- Sperm Motility and Vaginal Cytology: At the end of the studies, sperm samples were collected from male mice in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 16 consecutive days prior to the end of the studies from female mice in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for vaginal cytology evaluations.
- Liver Cytochrome P450 Activity: Liver samples were collected from five male and five female core study mice per dose group at the end of the studies. Livers were analyzed for total microsomal protein content and CYP2E1 activity.
Statistics:
- The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance between dosed and vehicle control groups and between the 100% OTNE and untreated control groups.
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epididymal spermatozoal data, (skewed distributions) were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus, were constructed based on a Markov chain model proposed by Girard and Sager (1987). Equality of transition matrices among dose groups and between the control group and each dosed group was tested using chi-square statistics.
- A t-test was used to determine significant differences in organ and body weight data, and Wilcoxon’s (1945) rank sum test was used for hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epidymal spermatozoal data.
Clinical signs:
no effects observed
Description (incidence and severity):
No other clinical findings other than dermal iritation, specified below.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Clinically, irritation was observed in the skin (site of application) of four males and two females administered 50% OTNE and in all males and females administered 100% OTNE. In addition, lesions considered to be ulcers were observed at the site of application in five males and two females administered 100% OTNE; these lesions were observed between day 43 in males and day 57 in females until the end of the study. Other skin effects are reported under histopathological findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights of 125, 500, and 1000 mg/kg bw/day OTNE males and mean body weight gains of 125 and 500mg/kg bw/day OTNE males were significantly less than those of the vehicle control group; those of 2000 mg/kg bw/day OTNE males were significantly less than those of the untreated controls
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to the vehicle (dosed groups) or untreated (2000 mg/kg bw/day OTNE groups) controls, significant decreases (≤ 14%) in the erythrocyte counts, hemoglobin concentrations, and hematocrit values occurred in the 500mg/kg bw/day OTNE or greater males and 100% OTNE females. Erythrocyte distribution widths, coefficients of variation of erythrocyte volumes (i.e., increased values indicate increased variation in sizes), were significantly increased in the 500mg/kg bw/day OTNE or greater males and in all dosed groups of females compared to their respective control groups. The parameters of erythrocyte count, hemoglobin concentration, and hematocrit value are quantitative estimates of the circulating erythroid mass. The changes in these parameters, paired with the changes in the red cell distribution width, were consistent with an alteration in erythropoiesis.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No other effects other than hematological findings
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the twice-daily observations, the behavior of the animals did not indicate any neurotoxic potential of the substance.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver
Absolute liver weights: all female and all male dosed groups (except 6.25% OTNE males) were increased (up to 92%) compared to their respective controls.
Relative liver weights: all male and female dosed groups were increased (up to 89%) compared to their respective controls.

Heart
Absolute and relative heart weights were increased approximately 14% in the 50% and 100% OTNE females
Relative heart weights of 25% and 100% OTNE males were also increased, but this may be due to the decreased body weights noted in the males.

Thymus
Absolute thymus weights of males and females as well as the relative thymus weight of females were decreased by approximately 22% in the 100% OTNE groups compared to untreated controls

Testis
In males, the absolute testis weight was decreased by 10% in the 50% OTNE group but not in the high dose and therefore not considered relevant.

Lung
In females, the relative lung weight of the 100% OTNE group was decreased by 21% compared to untreated controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- At the site of application, the incidences of minimal to moderate hyperplasia and chronic active inflammation were significantly increased in all dosed groups of males and females.
- In the liver, the incidence of centrilobular hypertrophy in 100% OTNE males was significantly greater than that in the untreated controls. There were no hepatic lesions in female mice. Centrilobular hypertrophy was of mild severity; affected hepatocytes were larger than the surrounding hepatocytes and had increased amounts of lightly eosinophilic to granular cytoplasm and loss of cytoplasmic glycogen.

Minimal to mild hyperkeratosis
Males: 250 mg/kg bw/day (5/10), 500 mg/kg bw/day (5/10), 1000 mg/kg bw/day (8/10), 2000 mg/kg bw/day (10/10)
Females: (5/10) for 125 and 250 mg/kg bw/day, (6/10) for 1000 mg/kg bw/day, (10/10) for 500 mg/kg bw/day, 2000 mg/kg bw/day

Fibrosis
Males: 125 mg/kg bw/day (4/10), 500 mg/kg bw/day (8/10), 1000 mg/kg bw/day (8/10), 2000 mg/kg bw/day (10/10)
Females : 250 mg/kg bw/day (10/10), 500 mg/kg bw/day (10/10), 1000 mg/kg bw/day (4/10), 2000 mg/kg bw/day (9/10)

Epidermis suppurative inflammation
Males: 2000 mg/kg bw/day (5/10)
Females: 500 mg/kg bw/day (4/10), 1000 mg/kg bw/day (4/10), 2000 mg/kg bw/day (7/10)

Hair follicle hyperplasia
Males: 1000 mg/kg bw/day (7/10), 2000 mg/kg bw/day (8/10)
Females: 500 mg/kg bw/day (4/10), 1000 mg/kg bw/day (3/10), 2000 mg/kg bw/day (10/10)

In the skin adjacent to the site of application, the incidences of hyperplasia, hyperkeratosis, chronic active inflammation, fibrosis, and epidermis suppurative inflammation were significantly increased, however these lesions were considered secondary to the spread of the test material from the site of application rather than a systemic effect.

Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reproductive effects
- Male mice administered 2000 mg/kg bw/day OTNE displayed fewer caudal sperm and caudal sperm/mg; these findings were not associated with any histopathologic changes in the testes or epididymides
- Male mice in the 2000 mg/kg bw/day OTNE group also exhibited lower sperm motility (~11%)

- Female mice administered 2000 mg/kg bw/day OTNE exhibited an increase in cycle length of approximately 1 day and displayed an increased probability of extended estrus as compared to the untreated controls.
- The increased probability of extended estrus was also observed in the 500 mg/kg bw/day and 1000 mg/kg bw/day OTNE groups as compared to the vehicle controls.
- When displayed as days in any given stage and presented as a percentage, the 500 mg/kg bw/day, 1000 mg/kg bw/day, and 2000 mg/kg bw/day OTNE groups exhibited more time in estrus than the respective control groups.
Dose descriptor:
other: NOAEC
Remarks:
Local
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dermal local effects are confounded by grooming
Dose descriptor:
NOAEL
Remarks:
Systemic
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic effects are confounded by grooming
Critical effects observed:
yes
Lowest effective dose / conc.:
6.25 other: %
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The effects reported in this study need to be evaluated in light of the used route of exposure. The study was intended as a sub-chronic dermal study; however, the animals were dermally exposed to OTNE in ethanol without occlusion of the treated site. Oral absorption (voluntarily or involuntarily), is expected as a result of oral grooming by the animals. In the present study, the grooming could have a contributed to an unknown extent to the systemic exposure and toxicity. Furthermore, also the local effects observed as a result of dermal exposure may have been confounded, a , as grooming by the animals can have influenced the actual dermal dose, dermal exposure time and could have given te opportunity for the animals to scratch, rub or lick the exposed site. As a result of these methodological limitations, the derivation of a dose descriptor for systemic or local effects due to dermal exposure is deemed inappropriate.

Conclusions:
As a result of methodological limitations of this dermal repeated dose toxicity study (confounding of exposure route due to oral grooming), the derivation of a dose descriptor for systemic or local effects after dermal exposure is deemed inappropriate. However, the systemic (liver) and local dermal (skin irritant) effects reported in this study are considered to support the respective findings in the oral repeated dose toxicity, and the skin Irritation endpoint.
Executive summary:

Introduction

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.

Systemic effects:

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 finding.

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, the moderate to severe systemic effects seen at this 2000 mg/kg bw, 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

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals are exposed 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 is NOAEL is 500 mg/kg bw considering the relative liver weight increase as an adaptive effect in absence of other liver findings.

Endpoint conclusion
Quality of whole database:
The information is not used for deriving a NOAEL because the dermal sites were not covered and grooming occurred. The dermal systemic effects can be contributed to oral exposure are therefore this information is considered supporting information.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Dermal application without covering. Oral exposure due to grooming has occurred.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
The dermal applied dose is not covered, grooming and oral exposure has occurred.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: F344/NTac
Details on species / strain selection:
For many years, the NTP used the inbred F344/N rat for its toxicity and carcinogenicity studies. However, this model has exhibited sporadic seizures, idiopathic chylothorax, and consistently high rates of mononuclear cell leukemia and testicular neoplasia. Therefore the F344 rat from the Taconic commercial colony (F344/NTac) was chosen as a more fecund rat model that could be used in both reproductive and carcinogenesis studies for comparative purposes.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female F344/NTac rats were obtained from the commercial colony at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the rats were 5 to 6 weeks old
- Weight at study initiation: 82-84 g (female) 83-86 g (male)
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 (males) or 13 (females) days

DETAILS OF FOOD AND WATER QUALITY:
Diet Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
Water Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1.668° C)
- Humidity (%): 50% ± 15%
- Air changes (per hr): ≥ 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day, 12 hours/night

IN-LIFE DATES:
Date of First Dose: December 12 (males) or December 13 (females), 2006
Necropsy Date: March 13 (males) or March 14 (females), 2007


Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: approximately 10% of body surface area (corresponds to approximately 25 cm2)
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL/kg body weight (rats)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50% or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used

CALCULATION OF LOCAL EXPOSURE: average body weight (kg) * exposure concentration (µg/kg bw/day) / surface area (25 cm2)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed three times by the study laboratory using GC/FID by analysis. All 30 of the dose formulations analyzed for rats were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; 28 of 30 were within 10% of the target concentrations.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Dose / conc.:
500 mg/kg bw/day
Remarks:
100% OTNE
Dose / conc.:
250 mg/kg bw/day
Remarks:
50% OTNE
Dose / conc.:
125 mg/kg bw/day
Remarks:
25% OTNE
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
12.5% OTNE
Dose / conc.:
31.25 mg/kg bw/day
Remarks:
6.25% OTNE
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Probably based on the information of the 28-day oral gavage study.
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily, histopathology at end of study period

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: hemoglobin; erythrocyte, reticulocyte, and platelet counts; erythrocyte distribution of width; mean platelet volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Animals fasted: No
- How many animals:10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- gross observation for evidence of disease, the health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program
HISTOPATHOLOGY: Yes
- Histopathology: adrenal gland, bone (femur) with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, oral cavity and larynx, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (site of application and control sites), spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
- Sperm Motility and Vaginal Cytology: At the end of the studies, sperm samples were collected from male rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 16 consecutive days prior to the end of the studies from female rats in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for vaginal cytology evaluations.
- Liver Cytochrome P450 Activity: Liver samples were collected from five male and five female special study rats per dose group on day 23 and from five male and five female core study rats per dose group at the end of the studies. Livers were analyzed for total microsomal protein content and CYP2E1 activity.
Statistics:
- The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance between dosed and vehicle control groups and between the 100% OTNE and untreated control groups.
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epididymal spermatozoal data, (skewed distributions) were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus, were constructed based on a Markov chain model proposed by Girard and Sager (1987). Equality of transition matrices among dose groups and between the control group and each dosed group was tested using chi-square statistics.
- A t-test was used to determine significant differences in organ and body weight data, and Wilcoxon’s (1945) rank sum test was used for hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epidymal spermatozoal data.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no biologically significant clinical findings related to OTNE administration
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Suppurative inflammation of the dermis in 1/10 Male rats treated with 100% OTNE, and 1/10 Female rats treated with 100% OTNE.
Other skin effects are reported under histopathological findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gains of dosed (except 100% OTNE) males and females were similar to those of the respective control groups. The mean body weight gain of 100% OTNE males was significantly less than that of the untreated control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no changes in the hematology parameters attributable to the dermal administration of OTNE
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alkaline phosphatase activities were decreased in various male and female dosed groups at all time points (only average value, incidence not given): days 3 (100% (500 mg/kg bw OTNE) and 23 (50% (250 mg/kg bw) and 100% OTNE) and week 13 (all dose groups). Decreases were consistently observed in 100% OTNE males and females at all time points compared to the untreated control groups.
At the end of the study (week 13), alanine aminotransferase activities were decreased in 25% (125 mg/kg bw) OTNE or greater females and in all dosed groups of males compared to their respective control groups (only average value, incidence not given). The significance of these decreases is not known but may be related to an alteration in liver metabolism. Other changes in clinical chemistry results were not considered toxicologically or biologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the twice-daily observations, the behavior of the animals did not indicate any neurotoxic potential of the substance.
Immunological findings:
not examined
Description (incidence and severity):
Not specifically examined, however the obtained results did not indicate any immunotoxic potential of the substance.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Only average value provided, incidence is not given:
Increased absolute (males 100% OTNE , females 50% and 100% OTNE) and relative (50% and 100% OTNE males and females) liver weights
Increased relative kidney weight (males and females 100% OTNE)
The concentrations 25, 50 and 100%, relates to 120, 250 and 500 mg/kg bw.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
Not specifically examined, however the behavior of the animals during the study was normal and did not indicate any neurotoxic potential of the substance. Furthermore, no histopathological abnormalities were observed in the brain.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin (site of application):
- hyperkeratosis (mild to minimal):
1/10 in the 6.25% (31.25 mg/kg bw/day) group;
3/10 in the 12.5% (62.5 mg/kg bw/day) group;
4/10 in the 25% (125 mg/kg bw/day) group;
5/10 in the 50% (250 mg/kg bw/day) group;
8/10 in the 100% (500 mg/kg bw/day) group;

- hyperplasia (mild to minimal)
2/10 in the 31.25 mg/kg bw/day group;
4/10 in the 62.5 mg/kg bw/day group;
5/10 in the 125 mg/kg bw/day group;
8/10 in the 250 mg/kg bw/day group;
10/10 in the 500 mg/kg bw/day group;

Other treatment-related histopathologic, toxicologically relevant, changes were not observed in the organs of male or female rats
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Male rats did not display any OTNE-related changes in testis and epididymis weights or sperm parameters. Female rats administered OTNE did not display any toxicologically significant effects on cycle length, or estrous cyclicity.
Dose descriptor:
other: NOAEC
Remarks:
Local
Effect level:
6.25 other: %
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dermal effects are confounded by grooming
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic effects are confounded by grooming
Critical effects observed:
yes
Lowest effective dose / conc.:
12.5 other: %
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The effects reported in this study need to be evaluated in light of the used route of exposure. The study was intended as a sub-chronic dermal study; however, the animals were dermally exposed to OTNE in ethanol without occlusion of the treated site. Oral absorption (voluntarily or involuntarily), is expected as a result of oral grooming by the animals. In the present study, the grooming could have a contributed to an unknown extent to the systemic exposure and toxicity. Furthermore, also the local effects observed as a result of dermal exposure are confounded, a , as grooming by the animals can have influenced the actual dermal dose, dermal exposure time and could have given te opportunity for the animals to scratch, rub or lick the exposed site. As a result of these methodological limitations, the derivation of a dose descriptor for systemic or local effects due to dermal exposure is deemed inappropriate.

Conclusions:
As a result of methodological limitations of this dermal repeated dose toxicity study (confounding of exposure route due to oral grooming), the derivation of a dose descriptor for systemic or local effects is deemed inappropriate by the assessor. Nevertheless, the syetemic (hepatic) effects at the high doses (>=500 mg/kg bw) support the findings in the two oral repeated dose toxicity studies.
Executive summary:

Introduction: 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.

Systemic effects

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 and immunological effects seen. There were no biologically significant neoplastic findings found.

Conclusion

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals are exposed doubtful due to oral grooming

A systemic dermal repeated dose NOAELcannot 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.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Dermal application without covering and oral exposure has occurrentified.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
The site of dermal application was not covered and therefore oral exposure due to grooming has occurred.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female B6C3F1/N mice were obtained from the NTP colony at Taconic Farms, Inc
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the mice were 6 to 7 weeks old
- Weight at study initiation: Male 22.6-23.7 g, Female 18.4-19.1 g
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum, Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly
- Water (e.g. ad libitum): ad libitum, Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)
- Acclimation period: 14 (males) or 15 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1.668° C)
- Humidity (%): 50 ± 15
- Air changes (per hr): ≥ 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Date of First Dose: December 14 (males) or December 15 (females), 2006
Necropsy Date: March 15 (males) or March 16 (females), 2007

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: approximately 10% of body surface area (corresponds to approximately 6.6 sq cm)
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg body weight (mice)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50%, or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration (if solution): 100%, 50%, 75%, 87,5%, 93,75% Ethanol
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used

CALCULATION OF LOCAL EXPOSURE: average body weight (kg) * exposure concentration (µg/kg) / surface area(25 sq cm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed three times by the study laboratory using GC/FID by analysis. All 30 of the dose formulations analyzed were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; 26 of 30 were within 10% of the target concentrations.
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
6.25% OTNE
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
12.5% OTNE
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
25% OTNE
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
50% OTNE
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
100% OTNE
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: twice daily, histopathology at end of study period

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed and clinical findings were recorded initially, on day 8, weekly thereafter, and at the end of the studies.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: hemoglobin; erythrocyte, reticulocyte, and platelet counts; erythrocyte distribution of width; mean platelet volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from the retroorbital plexus of special study rats on days 3 and 23 and from core study rats at the end of the study for hematology and clinical chemistry. Blood was collected from the retroorbital sinus of core study mice at the end of the study for hematology.
- Animals fasted: No
- How many animals:10 male and 10 female special study rats, and all core animals at the end of the study
- Parameters: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- gross observation for evidence of disease, the health of the animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program
HISTOPATHOLOGY: Yes
- Histopathology: adrenal gland, bone (femur) with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung (with mainstem bronchus), lymph nodes (mandibular and mesenteric), mammary gland, nose, oral cavity and larynx, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin (site of application and control sites), spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus.
Other examinations:
- Sperm Motility and Vaginal Cytology: At the end of the studies, sperm samples were collected from male mice in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per gram testis and per testis, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 16 consecutive days prior to the end of the studies from female mice in the untreated control, vehicle control, 25%, 50%, and 100% OTNE groups for vaginal cytology evaluations.
- Liver Cytochrome P450 Activity: Liver samples were collected from five male and five female core study mice per dose group at the end of the studies. Livers were analyzed for total microsomal protein content and CYP2E1 activity.
Statistics:
- The Fisher exact test (Gart et al., 1979), a procedure based on the overall proportion of affected animals, was used to determine significance between dosed and vehicle control groups and between the 100% OTNE and untreated control groups.
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epididymal spermatozoal data, (skewed distributions) were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
- Jonckheere’s test (Jonckheere, 1954) to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
- Extreme values identified by the outlier test of Dixon and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.
- Proportions of regular cycling females in each dosed group were compared to the vehicle control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus, were constructed based on a Markov chain model proposed by Girard and Sager (1987). Equality of transition matrices among dose groups and between the control group and each dosed group was tested using chi-square statistics.
- A t-test was used to determine significant differences in organ and body weight data, and Wilcoxon’s (1945) rank sum test was used for hematology, clinical chemistry, total microsomal protein, cytochrome P450 activity, spermatid, and epidymal spermatozoal data.
Clinical signs:
no effects observed
Description (incidence and severity):
No other clinical findings other than dermal iritation, specified below.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Clinically, irritation was observed in the skin (site of application) of four males and two females administered 50% OTNE and in all males and females administered 100% OTNE. In addition, lesions considered to be ulcers were observed at the site of application in five males and two females administered 100% OTNE; these lesions were observed between day 43 in males and day 57 in females until the end of the study. Other skin effects are reported under histopathological findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The final mean body weights of 125, 500, and 1000 mg/kg bw/day OTNE males and mean body weight gains of 125 and 500mg/kg bw/day OTNE males were significantly less than those of the vehicle control group; those of 2000 mg/kg bw/day OTNE males were significantly less than those of the untreated controls
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to the vehicle (dosed groups) or untreated (2000 mg/kg bw/day OTNE groups) controls, significant decreases (≤ 14%) in the erythrocyte counts, hemoglobin concentrations, and hematocrit values occurred in the 500mg/kg bw/day OTNE or greater males and 100% OTNE females. Erythrocyte distribution widths, coefficients of variation of erythrocyte volumes (i.e., increased values indicate increased variation in sizes), were significantly increased in the 500mg/kg bw/day OTNE or greater males and in all dosed groups of females compared to their respective control groups. The parameters of erythrocyte count, hemoglobin concentration, and hematocrit value are quantitative estimates of the circulating erythroid mass. The changes in these parameters, paired with the changes in the red cell distribution width, were consistent with an alteration in erythropoiesis.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No other effects other than hematological findings
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
During the twice-daily observations, the behavior of the animals did not indicate any neurotoxic potential of the substance.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver
Absolute liver weights: all female and all male dosed groups (except 6.25% OTNE males) were increased (up to 92%) compared to their respective controls.
Relative liver weights: all male and female dosed groups were increased (up to 89%) compared to their respective controls.

Heart
Absolute and relative heart weights were increased approximately 14% in the 50% and 100% OTNE females
Relative heart weights of 25% and 100% OTNE males were also increased, but this may be due to the decreased body weights noted in the males.

Thymus
Absolute thymus weights of males and females as well as the relative thymus weight of females were decreased by approximately 22% in the 100% OTNE groups compared to untreated controls

Testis
In males, the absolute testis weight was decreased by 10% in the 50% OTNE group but not in the high dose and therefore not considered relevant.

Lung
In females, the relative lung weight of the 100% OTNE group was decreased by 21% compared to untreated controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- At the site of application, the incidences of minimal to moderate hyperplasia and chronic active inflammation were significantly increased in all dosed groups of males and females.
- In the liver, the incidence of centrilobular hypertrophy in 100% OTNE males was significantly greater than that in the untreated controls. There were no hepatic lesions in female mice. Centrilobular hypertrophy was of mild severity; affected hepatocytes were larger than the surrounding hepatocytes and had increased amounts of lightly eosinophilic to granular cytoplasm and loss of cytoplasmic glycogen.

Minimal to mild hyperkeratosis
Males: 250 mg/kg bw/day (5/10), 500 mg/kg bw/day (5/10), 1000 mg/kg bw/day (8/10), 2000 mg/kg bw/day (10/10)
Females: (5/10) for 125 and 250 mg/kg bw/day, (6/10) for 1000 mg/kg bw/day, (10/10) for 500 mg/kg bw/day, 2000 mg/kg bw/day

Fibrosis
Males: 125 mg/kg bw/day (4/10), 500 mg/kg bw/day (8/10), 1000 mg/kg bw/day (8/10), 2000 mg/kg bw/day (10/10)
Females : 250 mg/kg bw/day (10/10), 500 mg/kg bw/day (10/10), 1000 mg/kg bw/day (4/10), 2000 mg/kg bw/day (9/10)

Epidermis suppurative inflammation
Males: 2000 mg/kg bw/day (5/10)
Females: 500 mg/kg bw/day (4/10), 1000 mg/kg bw/day (4/10), 2000 mg/kg bw/day (7/10)

Hair follicle hyperplasia
Males: 1000 mg/kg bw/day (7/10), 2000 mg/kg bw/day (8/10)
Females: 500 mg/kg bw/day (4/10), 1000 mg/kg bw/day (3/10), 2000 mg/kg bw/day (10/10)

In the skin adjacent to the site of application, the incidences of hyperplasia, hyperkeratosis, chronic active inflammation, fibrosis, and epidermis suppurative inflammation were significantly increased, however these lesions were considered secondary to the spread of the test material from the site of application rather than a systemic effect.

Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reproductive effects
- Male mice administered 2000 mg/kg bw/day OTNE displayed fewer caudal sperm and caudal sperm/mg; these findings were not associated with any histopathologic changes in the testes or epididymides
- Male mice in the 2000 mg/kg bw/day OTNE group also exhibited lower sperm motility (~11%)

- Female mice administered 2000 mg/kg bw/day OTNE exhibited an increase in cycle length of approximately 1 day and displayed an increased probability of extended estrus as compared to the untreated controls.
- The increased probability of extended estrus was also observed in the 500 mg/kg bw/day and 1000 mg/kg bw/day OTNE groups as compared to the vehicle controls.
- When displayed as days in any given stage and presented as a percentage, the 500 mg/kg bw/day, 1000 mg/kg bw/day, and 2000 mg/kg bw/day OTNE groups exhibited more time in estrus than the respective control groups.
Dose descriptor:
other: NOAEC
Remarks:
Local
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Dermal local effects are confounded by grooming
Dose descriptor:
NOAEL
Remarks:
Systemic
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Systemic effects are confounded by grooming
Critical effects observed:
yes
Lowest effective dose / conc.:
6.25 other: %
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

The effects reported in this study need to be evaluated in light of the used route of exposure. The study was intended as a sub-chronic dermal study; however, the animals were dermally exposed to OTNE in ethanol without occlusion of the treated site. Oral absorption (voluntarily or involuntarily), is expected as a result of oral grooming by the animals. In the present study, the grooming could have a contributed to an unknown extent to the systemic exposure and toxicity. Furthermore, also the local effects observed as a result of dermal exposure may have been confounded, a , as grooming by the animals can have influenced the actual dermal dose, dermal exposure time and could have given te opportunity for the animals to scratch, rub or lick the exposed site. As a result of these methodological limitations, the derivation of a dose descriptor for systemic or local effects due to dermal exposure is deemed inappropriate.

Conclusions:
As a result of methodological limitations of this dermal repeated dose toxicity study (confounding of exposure route due to oral grooming), the derivation of a dose descriptor for systemic or local effects after dermal exposure is deemed inappropriate. However, the systemic (liver) and local dermal (skin irritant) effects reported in this study are considered to support the respective findings in the oral repeated dose toxicity, and the skin Irritation endpoint.
Executive summary:

Introduction

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.

Systemic effects:

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 finding.

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, the moderate to severe systemic effects seen at this 2000 mg/kg bw, 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

A local long-term repeated dose NOAELcould not be established because the concentration to which the animals are exposed 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 is NOAEL is 500 mg/kg bw considering the relative liver weight increase as an adaptive effect in absence of other liver findings.

Endpoint conclusion
Quality of whole database:
The information is not used for deriving a NOAEC because the dermal sites were not covered and grooming occurred. The dermal local effects cannot be interpreted correctly and therefore presented only as supporting information.

Additional information

Summary of the repeated dose toxicity studies

An oral 90-day via gavage in rats according to OECD TG 408 is available (IFF, 2017). Two dermal 90-day studies (similar to OECD TG 411) in rat and mice are carried out by NTP (2016). One rat 28 day oral gavage toxicity study is present according to OECD TG 407 (IFF, 1997).

The key study for all routes is the 90-day oral study. This is because in the dermal NTP 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 long-term dermal effects cannot be evaluated according the standard OECD 411 criteria.

The key systemic effects seen in all 4 studies are summarized in the table. In the 90 -day oral gavage study at 500 mg/kg bw and in the 90 -day dermal mouse study at 1000 mg/kg bw slight but significant effects are seen in spleen and haematology parameters. The liver is the key target organ in all studies. The relative liver weight increases up to 50% at 500 mg/kg bw in the 90 -day oral gavage study accompanied with liver hypertrophy. At this dose also some liver parameters are affected such as increase in cholesterol and phospholipids. These liver effects are likely due to high metabolic demand and can be considered an adaptive effect; the increase of relative liver weight > 30% is high and therefore considered an effect dose. In the 90 -day dermal (/oral) study in mice the liver is increased up to 90%. The relatively kidney weights are slightly increased, circa 10% at >=500 mg/kg bw in the repeated dose toxicity studies. In the oral gavage studies the key finding in the kidney are the eosinophilic droplets and casts due the accumulation of alpha-2u globulin specific for male rats and are not used for deriving the NOAEL.

There are no neurotoxic, immunotoxic or fertility effects seen. 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). In the absence of effects on reproductive organs and in the presence of 89% increase in liver weights at this dose (NTP, 2016 study) these effects are not considered adverse. 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.

 Table Summary of results for systemic toxicity of all repeated dose toxicity studies

Systemic toxicity

90-day oral rat

90-day dermal / oral rat

90-day dermal/oral mice

28-day oral rat

OECD Guideline

408

411

411

407

Lowest Effect dose

500

500

500

1000

NOAEL

120

250

250

150

Neurotoxicity and immunotoxic effects

No effects

No effect

No effects

No effects

Reproductive and/or Endocrine organs

No effects

No effect

No effects

No effects

Lowest Adverse Effects in

mg/kg bw

 

 

 

 

Body weight and body weight gain effect > 10%

500

500

1000

1000

Haematology

500

No effects

500

No effects

Spleen

500

No effects

 

 

Liver

Increase in weight and hypertrophy

500

500

2000

1000

Kidney

Increase in weight and Alpha-hydrocarbon nephropathy

500

No effects

No effects

1000

Male fertility: sperm

No effects

No effects

2000

Not determined

Female fertility: Oestrus cycle

No effects

No effects

2000

Not determined

 

Introduction

In an oral 90-day study via gavage with rats according to OECD TG 408 is available (IFF, 2017). Two dermal 90-day studies (similar to OECD TG 411) in rat and mice are carried out by NTP (2016). One rat 28 day oral gavage toxicity study is present according to OECD TG 407 (IFF, 1997). Summaries of the studies are presented below.

 

The 90-day oral gavage repeated dose toxicity (OECD TG 408, IFF, 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 alpha-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 alpha 2urinary 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.

 

Two supporting 90-day repeated dose dermal NTP studies are available, both with the same design in rat and mice.

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

Introduction: 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.

Systemic effects

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.

 

The 90-day dermal mouse repeated dose toxicity with oral exposure (OECD TG 411, NTP reported in 2016)

Introduction

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.

Systemic effects:

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

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.

The 28-day repeated gavage dose in rat (OECD TG 407, 1995, IFF)

Introduction

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 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 alpha 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. 

Justification for classification or non-classification

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