N-[3-(dimethylamino)propyl]-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonamide N-oxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Jun 2017 to 15 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

26 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonamide N-oxide

EC Number:

279-481-6

EC Name:

N-[3-(dimethylamino)propyl]-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonamide N-oxide

Cas Number:

80475-32-7

Molecular formula:

C13H17F13N2O3S

IUPAC Name:

N-[3-(dimethylamino)propyl]-3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonamide N-oxide

Specific details on test material used for the study:

Purity: 96.7%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The study was conducted with albino rats. The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. The Crl:WI(Han) rat strain was used because it is routinely used at the test facility for this type of studies.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Weekly, dilutions of the test substance in the vehicle were prepared and stored in a refrigerator (2-10°C) in aliquots sufficient for one day and one extra. Aliquots removed from the refrigerator were allowed to equilibrate to ambient temperature prior to dosing. Volumes for dosing were taken under constant stirring on a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.1, 2, 8 and 5 mg/mL is corresponding to dose levels of 1, 20, 80 and 50 mg/kg bw/day respectively.
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD OF VALIDATION

PRINCIPLE: The concentration of the test substance in gavage was determined using Ultra Performance Liquid Chromatography and Mass Spectrometric detection (UPLC-MS/MS).

VALIDATION CRITERIA; 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 ≥0.98
- Recovery: the mean recovery of the test item from the formulation 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 study, should be less than 10%.
- Specificity: the signal obtained for samples should be corrected for the signal obtained for blank samples in case the signal obtained for blank samples is > 5% of the signal obtained for low-dose samples.

PREPARATION OF VALIDATION SAMPLES: Validation samples with nominal concentrations were prepared in five-fold by accurately weighing 5 mg, 30 mg and 100 mg into 20-ml vials. Subsequently 1.0 ml tap water was added.

SAMPLE TREATMENT: The method of analysis consisted of two parts: dilution and UPLC-MS/MS. The solvent used for dilution is a mixture of dimethylsulfoxide/acetonitrile/MilliQ water (1:1:8, v:v:v). The samples were diluted in three steps according to the scheme shown in Table 1. The initial dilution factor for all samples is 20-fold, by adding 19.0 ml of solvent to 1.0 ml of sample contained in 20-ml vial. The follow-up step are performed in autosampler vials. The samples were analysed using the UPLC-MS method.

CALIBRATION: The solvent used for solutions was a mixture of dimethylsulfoxide/acetonitrile/ MilliQ water (1:1:8, v:v:v). Stock solutions (1 mg/ml) were prepared by dissolving 10 mg test substance in 10 ml of solvent. On each day, 7 calibration solutions were prepared by accurately diluting two the test substance stock solutions (1 mg/ml) to concentrations of 5, 20, 50, 100, 150, 200 and 250 250 ng/ml solvent. Four calibrators are from the first stock, three calibrators from the second stock. The calibration samples were analyzed as described in section 2.2.5. Each calibrator was analysed in duplicate, first at the start of the analytical run, second at the end. Calibration graphs were constructed by plotting the peak area of NON-TDFOS against the concentration of the test substance.

CALCULATION: The concentration of the test substance in the (diluted) samples was calculated using the calibration curve in the software Analyst version 1.6.2 (Waters).

HOMOGENEITY, STABILITY AND CONTENT OF THE TEST SUBSTANCE

HOMOGENEITY: The homogeneity of the test substance was assessed in gavage solutions prepared on 11 April 2017. Six samples (2 replicates x 3 locations) of each gavage solution were analysed. From the control solution (0 mg/ml), one sample was analysed. 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 container). The test substance was considered to be homogeneously distributed in the solution 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 solution that was stored in the refrigerator was examined in the batch of gavage prepared on 11 April 2017. Samples were analyzed at t = 0, and after storage for 7-8 days at 2-10°C. 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 measured concentrations at the start (t = 0) and at the end of the storage period differed statistically significantly). The substance was considered to be stable in the solution if p ≥ 1 and/if the relative decrease int eh mean concentration after the storage was ≤ 10%.

CONTENT: The content of the test substance was determined in the batch gavage solutions prepared on 11 April 2017. The content of the test substance in gavage 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:

- Males: Male animals of the control, low- and mid-dose group were dosed during a 10-week premating period, during mating and up to the day of sacrifice after, in total, at least 90 days of treatment.
- Females: The female animals of the control, low- and mid-dose group were dosed with the test substance during a 10-week premating period, and during mating, gestation and lactation up to the day before sacrifice (day 13 of lactation or shortly thereafter).

Frequency of treatment:

once per day (7 days / week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day (actual dose received)

Remarks:

Low-dose

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

Mid-dose

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Remarks:

High-dose until day 43

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

High-dose for days 44-56

No. of animals per sex per dose:

12 animals per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in consultation with the sponsor and were based on the results of a 2-weeks dose range finding study with the test substance in rats (Triskelion study V20934/01).
Dose-range finding stud The objective of this study was to select dose levels for the subsequent combined repeated dose and reproduction toxicity screening study with the test substance in rats. Daily oral (gavage) administration of 0, 50, 250 or 1000 mg/kg bw the test substance to male and female Wistar rats for fourteen consecutive days resulted in:
- The spontaneous death of 2 male and 3 females in the 1000 mg/kg bw group on day 6 of the study. The remaining animals were sacrificed moribund based on clinical signs
and body weight loss.
- The spontaneous death of 1 female in the 250 mg/kg bw group on day 9 of the study. The remaining animals were sacrificed moribund based on clinical signs and body weight loss.
- In the 50 mg/kg bw group all animals survived the 14-day dosing period and showed no treatment-related clinical signs.
- Decreased body weight and body weight gain in the 250 and 1000 mg/kg bw groups. No effect on body weight in the 50 mg/kg bw group was observed.
- Decreased food consumption in the 250 and 1000 mg/kg bw groups. No effect on the food consumption in the 50 mg/kg bw group was observed.
- Clinical chemistry parameters showed treatment-related effects on the liver parameters (ALP, ALAT, ASAT, GGT) in the 250 and 1000 mg/kg bw groups.
- Dose related increase in mean kidney and liver weight in the 250 and 1000 mg/kg bw groups. No statistical significant changes in kidney and liver weight were observed in the 50 mg/kg bw group.
- Macroscopic examination of the animals at the scheduled and and unscheduled necropsies showed changes in the liver.
- Histopathological evaluation of the livers revealed a dose-dependent pathology in the liver, characterized by hepatocellular microvacuolation, mixed inflammation and necrosis in the 250 and 1000 mg/kg bw groups. No treatment-related pathology was observed in the control and 50 mg/kg bw groups.
Analysis of the test formulations showed that the test substance was homogeneously distributed and stable in the refrigerator for 8 days. The concentrations were close to intended. Based on these results dose levels of 250 and 1000 mg/kg bw exceed the maximum tolerated dose. Based on the macroscopic effects, the increase in liver weight and the effects on liver parameters in clinical chemistry, the liver is a sensitive target organ. Taking into account the longer duration of dosing in the subsequent OECD 422 study, which will be at least 10 weeks for males and 15 weeks for females, a dose level lower between 50 and 250 mg/kg bw is suggested as high dose in the subsequent study.

-Other:
The high-dose level was reduced from day 44 because of mortality in the high-dose group.The remaining males and females in the high-dose group were killed moribund.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

GENERAL CLINICAL SIGNS
Each adult animal was observed daily in the morning hours by cage-side observations. All cages were checked again in the afternoon for dead or moribund animals to prevent loss of tissues by cannibalism or autolytic degeneration. All abnormalities, signs of ill health or reactions to treatment were recorded. Animals showing signs of severe debility or intoxication, particularly if death appeared imminent, were humanely killed.

BODY WEIGHT
Body weights of male and female animals were recorded just before the start of the treatment (to enable randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the pre-mating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on days 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. The animals were weighed on their scheduled necropsy date in order to calculate the organ to body weight ratios.

FOOD CONSUMPTION
The food consumption was measured per cage over the same periods as the body weight are measured, except during the mating period when food intake was not registered. The results are expressed in g per animal per day. Food intake of non-mated females was not recorded.

DETAILED CLINICAL EXAMINATIONS
In addition to the above daily general clinical observations, detailed clinical examinations were outside the home cage were performed on all surviving rats of the remaining groups prior to the first exposure and then once weekly throughout the study, except for the last days of pregnancy and during the initial phase of the lactation period. In the last week of the study the detailed clinical examinations were part of the Functional Observational Battery tests (FOB) in the animals concerned. Signs noted included but were not limited to changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behavior.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) AND SPONTANEOUS MOTOR ACTIVITY
No FOB and spontaneous motor activity assessment (MAA) was performed in high-dose males and females. In the week prior to sacrifice, FOB tests and (MAA) were performed in 5 males/group in the control, low- and mid-dose groups after 87 days of dosing and in 5 females with a litter/group on PN day 13 of the control, low- and mid-dose groups. For females both tests were performed after sacrifice of their pups on PN day 13. Because light reflections in the background were not removed, total distance moved was also recorded in empty boxes during motor activity assessment.

HEMATOLOGY
Prior to sacrifice, all surviving were fasted overnight (water was freely available). At sacrifice, blood was taken from the abdominal aorta of 5 adult rats/sex for the control, low-dose and high dose groups (for males those with the lowest identification numbers in each cage and females with a litter were selected) whilst under CO2/O2 anaesthesia. For prothrombin time citrate was used as anticoagulant. For the other parameters EDTA was used as anticoagulant. In each sample the following determinations were carried out: Hemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts, prothrombin time, thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY
Prior to sacrifice, animals of the control, low- and mid-dose were fasted overnight (water was freely available). The remaining high-dose rats that were killed moribund after 56 days of dosing were not fasted before sacrifice. At sacrifice, blood was taken from the abdominal aorta of 5 rats/sex/group for the control, low and mid-dose (for males those with the lowest identification numbers in each cage and females with a litter were selected) whilst under CO2/O2 anaesthesia. At day 57 after start of treatment, of the remaining high-dose rats also of 5 rats/sex/group blood (males and females those with the lowest identification numbers; for males rats 66 was replaced by rat 68) were taken for clinical chemistry, using the same procedure. Blood was collected in tubes filled with heparin (used an anticoagulant) and plasma was prepared by centrifugation. the following determinations were carried, in each plasma sample: alkaline phosphatase activity (ALP), bilirubin (total), aspartate aminotransferase activity (ASAT), cholesterol (total) alanine aminotransferase activity (ALAT) triglycerides, gamma glutamyl transferase activity (GGT), phospholipids,total protein, calcium (Ca),albumin, sodium (Na), ratio albumin to globulin (calculated), potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate (PO4), glucose (fasting), bile acids.

BLOOD SAMPLING FOR HORMONE DETERMINATIONS
During necropsy blood was taken from the aorta under CO2/O2 anaesthesia from all surviving adult male and female animals and serum was stored in a freezer (at ≤ 18°C).

HORMONE DETERMINATIONS (T4)
The serum samples taken from the surviving adult males were analysed for T4 hormone levels. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge).

Sacrifice and pathology:

SACRIFICE, BLOOD SAMPLES, GROSS NECROPSY AND HISTOLOGY

The remaining males and females of the high-dose group were killed in moribund condition after 56 days of dosing.
Prior to sacrifice, adult male and female parent animals of the control, low- and mid-dose groups were fasted overnight (water was freely available). All male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. A thorough necropsy was also performed on animals that had to be killed because they were moribund.
Males of the control, low- and mid-dose group were sacrificed after the mating period (after 92 days of dosing). At the day of necropsy, a vaginal smear was taken from each female. Adult female animals that failed to mate or were not pregnant were sacrificed 37 days after the last mating date.
At scheduled necropsy, the organs of the adult animals indicated below were weighed (paired organs together) as soon as possible after dissection to avoid drying. Samples tissues and organs which were preserved are listed in the "any other information  on materials and methods incl. tables" section. The tissues of the adult animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes and pididymides, which were preserved in Bouin’s fixative). The reproductive organs and gross lesion of all adult male and female animals were preserved.
In addition, a series of other organs/tissues (see in the table "List of tissues / organs that were examined after sacrifice", in the "any other information  on materials and methods incl. tables" section,) of five adult animals/sex/group were preserved but not microscopically examined:
- Male reproductive organs: Epididymides (paired), Prostate (dorsolateral and ventral), Seminal vesicles and coagulation glands (paired), Testes, Levator ani plus bulbocavernosus muscle complex Cowper’s glands and glans penis
- Female reproductive organs: Ovaries (paired), Uterus, including cervix, Vagina
- Other organs: All gross lesions, Adrenal glands (paired), Bone marrow (sternum), Brain (including sections of cerebrum, cerebellum, medulla/pons), Eye, Heart, Kidney (paired), Liver, Lungs with trachea and larynx, Mesenterial and axillary lymph nodes, Peripheral nerve (sciatic or tibial), Pituitary, Skeletal muscle, Small and large intestines (including Peyer’s patches), spinal cord, Spleen, Stomach, Thymus, Thyroid gland, Urinary bladder.

Other examinations:

ESTROUS CYCLE EVALUATIONS:
Vaginal smears to evaluate the estrus cycle length and normality were made daily for the 14 days during the pre-treatment period and during the last two weeks of the pre-mating period and until confirmation of mating. Smears were stained and evaluated for estrus cyclicity in all (allocated) females. An additional vaginal smear was made at the day of sacrifice but the results of the microscopic examination of the uterus and vagina did not give any indication for further examination of these smears.

Statistics:

- Body weight data collected after initiation of treatment: “AnCova & Dunnett’s Test” with automatic data transformation. Day 0 body weight data are used as covariate unless removed during data preprocessing
- Pretreatment body weight data, body weight changes, clinical pathology (hematology and clinical chemistry) and organ weight data: “Generalized Anova Test” with automatic data transformation.
- Post-hoc analysis. If the group test shows significant (p<0.05) non-homogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).
Food/ water consumption: Dunnett’s multiple comparison test.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square
analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
- Incidences of histopathological changes: Fisher’s exact probability test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed in the animal in high-dose group were related to the poor health status of these animals and included dyspnoea, thin, muscle weakness, lethargic, hunched posture, piloerection, soiled fur, encrustation of nose, eyes, ears and legs, discharge of the nose and eyes, macrophythalmia and swollen leg. One male in the control group and one male in the high-dose group and two males in the lowdose group showed a sniffing respiration, which might be related to the route of administration (oral gavage). Other clinical signs in the pre-mating and mating, gestation and lactation period were related to the skin (sparsely haired area(s), encrustations, wound). In view of the incidence and distribution of the observed clinical signs, these are considered not treatment-related.

Mortality:

mortality observed, treatment-related

Description (incidence):

3 male rats in the high-dose group were killed in moribund condition on day 37, 51 and 52, respectively, and one female was killed in moribund condition on day 51. On day 57, after 56 days of treatment (after 43 days of dosing with 80 mg/kg bw per day and 13 days of dosing with 50 mg/kg bw per day), the remaining animals of the high-dose group were killed because the objective related to the reproductive performance could not be met because of the deteriorating health status of the parental animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was statistically significantly decreased in males and females in the high-dose group (measured until 57 days of treatment). In control, low- and mid-dose animals no other changes were observed in pre-mating, mating, gestation and lactation, except for a statistically significantly decreased body weight of mid-dose males and females compared to controls at day 7. In this group, however, the differences with the controls were very slight (<2%) and the statistical significance was probably due to the slightly higher body weights in this group at the start of the study (day 0). Therefore, this (very slight) statistically significant change in body weight in females and males of the mid-dose group is not considered relevant. Except for the last measured time period (49-46), body weight change was statistically significantly decreased in males of the high-dose group at all measured time points. In females of the high-dose group body weight change was only statistically significantly decreased at the start of treatment (day 0-7). In the mid-dose group, body weight change was statistically significantly decreased only at treatment day 0 – 7 (males and females), 21 – 28 (males), and increased only at 49-56 (females) and 77 – 84 (males). In the low-dose group body weight change was only statistically significantly decreased at treatment day 0 – 7 (males) and increased at 49 – 56 (males and females). During gestation and lactation mean body weight changes were comparable in all females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was statistically significantly decreased in males and females of the highdose group for all time-points, measured until 56 days of treatment. At day 14 – 21, food consumption was slightly, but statistically significantly, decreased in males in the mid-dose group. During the gestation and lactation period food consumption was comparable in all groups

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, mean cholesterol and phospholipid levels were statistically significantly lower in the mid-dose group and mean urea and potassium levels were statistically significantly higher in the mid-dose group. In females mean glucose levels were statistically significantly decreased in low-dose and mid-dose females. In females mean glucose levels were statistically significantly decreased in low-dose and middose females Because of the lack of a dose response relationship and because the values are within the range of historical control values this is considered a chance finding. Due to the different necropsy date of the remaining animals in the high-dose group, no statistical analysis could be performed for clinical chemistry parameters for this group. However, also in males of the high-dose group mean cholesterol and phospholipid levels were decreased.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Effects of treatment with the test substnce were observed in animals of the high-dose groups during weekly detailed clinical examinations. The effects included slightly to moderately tiptoe walking, a hunched body position, rocking, hindlimb wiping, piloerection, skinny, soiled fur (general) and soiled and/or wet fur around the penis or vagina or in the abdominal region. Although treatment related, these observations do not point to a neurotoxic effect of the test substance. Therefore it is concluded that the results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In control, low- and mid-dose males and females no differences were observed in terminal body weight among the groups for both male and females. Also no statistically significant differences were observed in the mean absolute organ weights between the control and treatment groups in males and females. The relative weight of the spleen in males in the low-dose group was statistically significantly decreased. Because of an absence of dose-response relationship and the absence of this finding in females, this finding was considered to be a chance finding. Due to the different necropsy date of the remaining animals in the high-dose group, no statistical analysis could be performed for organ weights for this group. However, the relative weight of the liver and kidneys was increased in the males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Day 37, 51 and 52 (Necrospy moribund high-dose animals) At necropsy, macroscopic analysis of one male animal, killed moribund at day 37, showed white spots on the liver and adhesion and an enlarged mesenteric lymph node, a dark discolored lung, and an enlarged kidney with hite spots. One male, killed moribund at day 51, showed yellow spots on the liver, red discolored lungs, and an enlarged mesenteric lymph node. One female, also killed moribund at day 51, showed yellow spots on the liver and the lungs also showed a yellow nodule and were incompletely collapsed. The spleen was enlarged and also contained yellow nodules and the mesenteric lymph node was enlarged. The male, killed moribund at day 52, showed yellow spots on the liver and yellow nodules on the pancreas.

Day 57 (Necrospy interim-killed high-dose animals). Macroscopic analysis at necropsy revealed the following:
- Liver: Four out of 9 remaining male animals showed yellow/pale spots/nodules on the liver. In one male the liver was pale. One out of 11 remaining female animals showed yellow spots on the liver.
- Lungs: Five out of 9 remaining male animals showed pale/yellow/white spots/nodules on the lungs. Eight out of 11 remaining female animals showed pale/yellow nodules on the lungs.
- Spleen: Three out of nine remaining male animals showed yellow nodules on the spleen. In one male the spleen was enlarged. In nine out of eleven remaining female animals the spleen was enlarged. In eight out of eleven remaining females, yellow/pale nodules were observed.
- Mesenteric lymph node: In seven out of nine remaining male animals the mesenteric lymph node was enlarged. In nine out of eleven female animals, the mesenteric lymph node was enlarged. In one female, a yellow nodule was present on he mesenteric lymph node.

Necropsy of control, (low-dose and mid-dose groups): Macroscopic observations at necropsy revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic analysis revealed a treatment-related effect in the liver and the lungs of 4/5 males of the mid-dose groups. Four out of five males of the mid-dose group, showed minimal to moderate centrilobular vacuolation in the liver. In addition, minimal to mild accumulation of alveolar macrophages was found in four out of five males of the mid-dose group. Minimal accumulation was found in three out of five female animals of the mid-dose group, but did not reach statistical significance Microscopic analysis of the lungs of the low-dose males and females, revealed no pathology. Microscopic analysis of the livers of the low-dose males revealed no pathology.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- No statistical significant differences were observed in the T4 levels of the adult males.
- No statistically significant effects were observed on the estrus cycle of the allocated remaining (control, low-, and mid-dose) females (length, cyclicity and number of complete cycles) during the 2 weeks pre-treatment and during the pre-mating phase, up to and including the day the animals were mated.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

20 mg/kg bw (total dose)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Analytical results

- The test substance was considered to be homogeneously distributed in all the gavage liquids prepared for all dose levels.

- The concentration of the test substance was close to intended (90-110%) for all gavage liquids at the measured dose levels, except for the samples of the low-dose concentration prepared on 18 July 2017 (mean concentration +25% of intended) and 3 October 2017 (mean concentration -18% of intended) and for the sample of the mid-dose concentration prepared on 3 October 2017 (mean concentration -18% of intended) and for a samples of the high-dose prepared on 15 August 2017 (mean concentration of -13% of intended). However, the average concentration of all samples was within 10% of the intended concentration for all dose levels, indicating that, on average, the animals received the intended dose of the test substances during the study.

Applicant's summary and conclusion

Conclusions:

Based on the histopathological findings in the liver of the males, together with the clinical chemistry observations, the NOAEL for parental toxicity was conservatively placed at 1 mg/kg body weight per day. Microscopic analysis of the control and the mid-dose groups, revealed treatment-related pathology in the liver, characterized by minimal to moderate centrilobular hepatocellular microvacuolation in the males of the mid-dose group (20 mg/kg bw).

Executive summary:

A study was performed according to OECD guideline 422 and GLP principles. The objectives of this study were to provide data on general toxicity and the possible effects of the test substance on reproductive performance and development of pups. The test substance was administered daily by oral gavage at dose levels of 1, 20 or 80 mg/kg body weight during a premating period of 10 weeks and during mating (1 week), gestation and lactation until 14 days after delivery. Male animals were sacrificed after at least 90 days of treatment. Because of mortality in the high-dose group, the high-dose level was reduced to 50 mg/kg body weight/day at day 44 of treatment. After 56 days after the start of treatment (premating period) the high-dose group was sacrificed because of deteriorating health.

The results of test substance analysis in the carrier (water) showed that, on average, the test substance was homogenously distributed in the carrier and that the concentrations of the test substances in the carrier were close to intended.

Three males and one female in the high-dose group were killed in moribund condition. These deaths were treatment-related and also occurred after the high-dose level had been reduced from 80 to 50 mg/kg bw/day. After 56 days of treatment the remaining male and female animals were sacrificed because of deteriorating health. The observations in these remaining high-dose animals were: Clinical signs, up to day 57 of treatment, in the high-dose group included dyspnoea, thin, muscle weakness, lethargic, hunched posture, piloerection, soiled fur, encrustation of nose, eyes, ears and legs, discharge of the nose and eyes, macrophthalmia, and a swollen leg. Weekly detailed clinical examinations confirmed the above daily clinical observations in the high-dose group. Mean body weights and food consumption were decreased in the and high-dose groups in both sexes. A decrease in cholesterol levels phospholipid levels in high-dose males was noted after 56 days of treatment. The relative liver and kidney weights were higher in high-dose males. Macroscopically, in the high-dose group, enlarged spleens and mesenteric lymph nodes were found and pale/yellow spots/nodules were found in the liver and lungs.

The following observations were made in the control, low- and mid-dose groups: No treatment-related clinical signs were noted in the mid- and low-dose groups. The results of the neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance in rats. No changes were observed in body weight. Body weight change was only slightly decreased at the start of treatment in all groups. Except from a slightly lower food consumption in mid-dose males at the initial stage of the study there were no other differences in food consumption. Haematology was conducted in 5 rats/sex/group at necropsy. There were no toxicologically relevant changes noted. Clinical chemistry, conducted in 5 rats/sex/group at necropsy, showed a treatment related decrease in cholesterol levels and in phospholipid levels in mid-dose males. Urea and potassium levels were increased in mid-dose males. No treatment-related differences were observed in relative and absolute organ weights in both sexes. Microscopic analysis of the control and the mid-dose groups, revealed treatment-related pathology in the liver, characterized by minimal to moderate centrilobular hepatocellular microvacuolation in the males of the mid-dose group. No centrilobular microvacuolation was observed in the control and the low-dose groups. In addition to the centrilobular microvacuolation, minimal to mild accumulation of alveolar macrophages was observed in the lungs of both males and females of the mid-dose groups. No accumulation of alveolar macrophages was found in the control and low-dose groups. There were no effects of the test substance on estrous cycle and on fertility and reproductive performance. Results of T4 hormone analysis in male animals did not show any significant effects between the groups. Based on the histopathological findings in the liver of the males, together with the clinical chemistry observations, the NOAEL for parental toxicity was conservatively placed at 1 mg/kg body weight per day.