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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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

Cross-reference

Reason / purpose for cross-reference:

reference to same 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:

other: 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)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males: 8 weeks; females: 7 weeks
- Weight at study initiation: Males: 259.77-260.49 g; Females:237.73-240.89 g
- Fasting period before study: animals were fasted overnight before examinations
- Housing: in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment.
- Diet: cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England); ad libitum.
- Water: domestic mains tap-water suitable for human consumption; ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45 - 65
- Air changes (per hr): 10

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on 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.

Details on mating procedure:

After the 10-week pre-mating period, each female was caged with one male from the same dose group. Animals were caged together for maximally 4 days. In this period, the required number of animals was mated. Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day (GD) 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Males, females that delivered and females that appeared not pregnant were sacrificed.

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)

Duration of test:

90 days in total

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.

Examinations

Maternal examinations:

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 females. In the week prior to sacrifice, FOB tests and (MAA) were performed 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 animals were fasted overnight (water was freely available). At sacrifice, blood was taken from the abdominal aorta of 5 adult ratsfor the control, low-dose and high dose groups (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/group for the control, low and mid-dose (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/group blood (those with the lowest identification numbers) 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.

POST- MORTEM EXAMINATIONS
Sacrifice: Before necropsy all animals were fasted overnight (water was freely available). All surviving parental female animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then subjected to macroscopic examination for pathological changes. Parental female animals were sacrificed on day 13 of lactation or shortly thereafter.

Gross necropsy: Macroscopic examination for pathological changes was perfromed on all animals

Histopathology/organ weights:
- At scheduled necropsy the organs of the adult animals, listed in Section “Any other information on materials and methods incl. tables”, were weighed (paired organs together) as soon as possible after dissection to avoid drying. Samples of the tissues and organs of the adult animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, epididymides and ovaries, which were preserved in Bouin’s fixative. The reproductive organs of all female animals were preserved. The number of implantation sites in the uterus were counted. In case of the other organs/tissues (see table in Section “Any other information on materials and methods incl. tables”) five adult animals/sex/group (females with a litter were selected) were preserved.
- Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin. Microscopic examination was performed on the preserved organs of all animals of the control (group 1) and high-dose group (group 4). The levator ani and bulbocavernosus complex was preserved, but not histopathologically examined. Upon treatment-related changes in the kidney observed in the high-dose group, the evaluation of this organ was extended to the intermediate-dose groups (2 and 3).
- In addition, reproductive organs (ovaries, uterus) of females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

PUP SURVIVAL, SEX AND WEIGHT
Litters were evaluated on the day of birth (i.e. day 0 of lactation). To keep nest disturbance to a minimum the litters were examined only once daily for dead pups. The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups, runts (pups that are smaller than control pups) and grossly malformed pups was evaluated on days 0, 4, 7 and 13 of lactation. The alive pups were individually weighed on days 0, 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both sexes combined per dose group.

ANOGENITAL DISTANCE IN PUPS
At day lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as such, corrected for body weight and for the cube root of body weight.

CULLING AND BLOOD COLLECTION FOR HORMONE ANALYSIS
On lactation day 4 the litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment was applied. Preference was to retain four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13. Blood samples of the culled pups were collected.

NIPPLE RETENTION IN MALE PUPS
On postnatal day 13 all surviving male pups were examined for the presence of nipples or areolas.

GROSS EXAMINATION OF DEAD PUPS:
Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups that died during the study and macroscopic abnormalities of these pups were recorded.

SIGNS AND SACRIFICE, GROSS NECROPSY AND PATHOLOGY OF PUPS
Any abnormal behavior of pups was recorded on day 0, 4, 7 and 13 of lactation. Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups that died during the study and macroscopic abnormalities of these pups were recorded. At necropsy of the surviving dams and their litter, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by decapitation (except for the two pups per litter of which blood was collected for hormone analysis. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development

HORMONE DETERMINATIONS (T4)
The serum samples taken from two pups per litter (LD13) were analysed for T4 hormone levels. Analysis were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method.

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.

Indices:

Reproductive indices
- Female mating index = no. of females mated*100/no. of females placed with males
- Female fertility index = no. of females pregnant*100/no. of females placed with males
- Female fecundity index = no. of females pregnant*100/no. of females mated
- Gestation index = no. of females with a viable litter *100/ no. of females pregnant

Offspring viability indices:
- Live birth index = no. of liveborn pups *100/no. of newborn pups
- Stillborn index = no. of stillborn pups *100/no. of newborn pups
- Viability index 0 - 4 = no. of live pups on day 4 *100/no. of liveborn pups
- Viability index 4 - 13 = no. of pup live pups on day 13 *100/no. of live pups post cull day 4
- Sex ratio (day n) = no. of live male pups on day n *100/no. of live pups on day

Historical control data:

Historical control data for clinical chemistry

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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. 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.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not relevant

Mortality:

mortality observed, treatment-related

Description (incidence):

In the high dose group 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 maternal animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was statistically significantly decreased in 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 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 ( females), and increased only at 49-56 (females). In the low-dose group body weight change was only statistically significantly increased at 49 – 56 (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 females of the high dose group for all time-points, measured until 56 days of treatment. 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:

effects observed, non-treatment-related

Description (incidence and severity):

Of the red blood cell parameters only mean MCV was slightly, but significantly higher in females in the mid-dose group. Of the white blood cell parameters, only the percentage eosinophils were slightly, but statistically significantly higher in females in the mid-dose group. These findings were considered incidental findings of no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 mid dose 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.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Effects of treatment with the test substance 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 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, non-treatment-related

Description (incidence and severity):

Also no statistically significant differences were observed in the mean absolute organ weights between the control and treatment groups in females. 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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Day 37, 51 and 52 (Necrospy moribund high-dose animals). A female, 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.
- Day 57 (Necrospy interim-killed high-dose animals). Macroscopic analysis at necropsy revealed the following:
Liver: One out of 11 remaining female animals showed yellow spots on the liver.
Lungs: Eight out of 11 remaining female animals showed pale/yellow nodules on the lungs.
Spleen: 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 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:

no effects observed

Description (incidence and severity):

Minimal accumulation of alveolar macrophages 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 females, revealed no pathology.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

All females delivered live born pups, there were no females with all stillborn pups.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

no statistically significant differences were observed on prenatal loss between the control and treatment groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed on prenatal loss between the control and treatment groups

Early or late resorptions:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed on prenatal loss between the control and treatment groups. The incidences of live and stillborn pups and perinatal loss indices were also comparable among the groups

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The number of stillborn pups accounted 0, 1 and 1 for the control, low-, and mid-dose groups, respectively

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was comparable among the groups
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy was not affected by treatment, except for one female all females were pregnant and the mean duration of gestation was comparable among the groups and the gestation index was 100% in all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females except for one female in the low-dose group were mated. Another female in the low dose group was misjudged to be mated, and therefore was not pregnant.

Other effects:

not examined

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

maternal systemic effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

2

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental effects

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no effects at highest analysable dose

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related differences in mean pup weights of male and female pups (per sex and together) between the test groups and the controls on day 0, 4, 7 and 13.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no treatment-related differences in mean pup weights of male and female pups (per sex and together) between the test groups and the controls on day 0, 4, 7 and 13.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of pups delivered per litter and the incidences of live born pups were comparable among the groups. The number of stillborn pups accounted 0, 1 and 1 for the control, low-, and mid-dose groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No differences were observed on sex ratio on day 0 and day 13 among the various groups

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of pups delivered per litter were comparable among the groups

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number of number of pups that died or were missing between days 0- 4 accounted 1, 5 and 0 for the control, low-, and mid-dose, respectively. After culling on day 4, 1 pup was lost in the low-dose group.

External malformations:

no effects observed

Description (incidence and severity):

Macroscopic examinations of stillborn pups and pups that died did not reveal treatment-related effects. Macroscopic observations of pups at necropsy on postnatal day 13 revealed no treatment-related abnormalities

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Results of T4 hormone analysis in male and female pups of postnatal day 13 did not show any significant effects between the groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects up to highest analysable dose

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no