3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Nov 2016 to 07 Mar 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:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonate

EC Number:

261-818-3

EC Name:

Potassium 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonate

Cas Number:

59587-38-1

Molecular formula:

C8H5F13O3S.K

IUPAC Name:

potassium 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1-sulfonate

Test material form:

solid

Details on test material:

Purity: 97.1%

Specific details on test material used for the study:

Purity: 97.1%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: female rats were about 10 weeks old and the male rats about 9 weeks old
- Weight at study initiation: Males: 244 g to 302 g, Females: 171 g to 203 g
- Fasting period before study: no
- Housing: 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: From their arrival, the rats received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum. The food was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was refreshed at least once weekly.
- 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, which were cleaned weekly and filled as needed
- Acclimation period: Upon arrival, the animals were housed in quarantine and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was carried out in a few randomly chosen rats of the lot delivered. Upon satisfactory results of the serology the quarantine room was cleared for use as experimental room.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

as a suspension

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in a refrigerator (2-10°C) in aliquots sufficient for one day. 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: 25, 75 and 225 mg/mL is corresponding to dose levels of 5, 15 and 45 mg/kg bw/day, respectively.
- Amount of vehicle: 5 mL/ kg bw

Details on mating procedure:

After a 10-week premating period, during which the male and female animals were dosed by daily gavage, each female was caged with one male from the same group. Animals were caged together until mating occurred. Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears was 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the second weekly batch of dosing formulations homogeneity and content was determined according to a validated method of analysis. In three batches throughout the study the content was determined. Data on homogeneity and stability obtained from the DRF study were considered as well.

ANALYTICAL METHOD VALIDATION:
Principle of analytical method
The analytical method is based on separation with ultra performance liquid chromatography and detection with mass spectrometry (UPLC-MS/MS).

Preparation of validation samples
Validation samples with nominal concentrations of 2, 10 and 20 mg/mL were prepared by accurately weighing 2 mg, 10 mg and 20 mg into 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 low dose validation samples were diluted 20,000-fold before analysis. The mid- dose and high- dose validation samples were diluted 200,000-fold before analysis. The samples were diluted in autosampler vials, appropriate for LC-MS analysis. Following this procedure the theoretical concentration of the diluted validation samples was approx. 0, 100 ng/ml, 50 ng/mL and 100 ng/mL for the blank, low-dose-, mid-dose and high-dose extracts, respectively.

Calibration
On each day, 7 calibration solutions were prepared by accurately diluting two stock solutions (1 mg/mL) to concentrations ranging from 5 ng/mL to 250 ng/mL in Milli-Q water. Calibration graphs were constructed by plotting the peak areas against the concentration of in ng/ml.

Calculation
The concentration of test substance in the (diluted) samples was calculated using the calibration curve.

Duration of treatment / exposure:

90 days: Male animals were dosed during a 10-week premating period, during mating and up to sacrifice. The female animals were dosed with the test substance during a 10-week premating period, and during mating, gestation and lactation up to the day before sacrifice (approx. day 14 of lactation).

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: An oral (gavage) 14-day dose range finding study with the test substance in rats.

An oral (gavage) 14-day dose range finding study with test substance was performed in Wistar Han IGS rats (Crl:WI(Han)). The objective of this study was to provide data for selection of the dose levels to be used in the combined repeated dose and reproduction toxicity screening study.
In the two week dose range finding study, 4 groups of 5 males and 5 females each were dosed with 0, 10, 50 and 100 mg/kg body weight per day by oral gavage. A dosing volume of 10 mL/kg body weight was used in all groups. Drinking water was used as vehicle. The vehicle was used for dosing the control group and was used for diluting the test item to the appropriate concentrations.
Daily oral (gavage) administration of test substance to male and female Wistar rats for fourteen consequtive days resulted in:
• No mortalities and treatment-related clinical signs;
• Dose related decrease in body weight gain in the males of the 50 and 100 mg/kg bw groups and the females of the 100 mg/kg bw group;
• Decrease in food consumption in the males of the 50 and 100 mg/kg bw groups and the females of the 100 mg/kg bw group;
• Dose related effects on clinical chemistry parameters (creatinine levels and urea levels) in males in the 50 and 100 mg/kg group and females in the 100 mg/kg group;
• Dose related effects on organ weight (increased kidney weight) in males in the 50 and 100 mg/kg group and females in the 100 mg/kg group;
• No treatment related macroscopic changes.
Analysis of the test formulations showed that the test substance was a homogeneous suspension and stable in the refrigerator for 8 days.

Based on the above results treatment-related effects were observed at 50 and 100 mg/kg body weight test substance after 14 consecutive days of oral (gavage) administration. In view of the longer duration of dosing in the subsequent combined toxicity and reproduction screening study, which will be at least 10 weeks for males and 15 weeks for females, a dose level of approximately 50 mg/kg is suggested as high dose. This dose level is anticipated to induce effects on clinical chemistry parameters, organ weights and possibly body weight and is therefore considered an appropriate dose level for the high dose group in an OECD 422 study.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Each adult animal was observed daily in the morning hours by cage-side observations

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: morning and afternoon
The observations included, but were not restricted to general clinical signs, signs related to behaviour, respiration, head, nose, eyes, ears, mouth, skin/fur, perineum, extremities (legs), tail, abdomen, faeces, injection site, penis, testes, urethra and urine. All abnormalities, signs of ill health or reactions to treatment were recorded. Any animal showing signs of severe debility or intoxication, particularly if death appears imminent, was humanely killed to prevent loss of tissues by cannibalism or autolytic degeneration.

BODY WEIGHT:
- Time schedule for examinations: The body weight of each adult animal was recorded at least once during the acclimatization period and at initiation of treatment (day 0).
Subsequently, males were weighed weekly. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. In addition, the adult animals were weighed on their scheduled necropsy date in order to calculate the correct 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 recorded. The results are expressed in g per animal per day.

HAEMATOLOGY:
Before necropsy all animals were fasted overnight (water was freely available) and from 5 adult rats/sex/group (for males those with the lowest identification numbers in each cage and females with a litter will be selected) blood will be taken from the aorta whilst under CO2/O2 anaesthesia.
In the low dose group blood was taken from 6 females. For prothrombin time citrate was used as anticoagulant. For the other parameters EDTA was 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 cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts*, prothrombin time and thrombocyte count. Additionally, the following parameters were calculated:
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)

* neutrophils, lymphocytes, eosinophils, basophils, monocytes

CLINICAL CHEMISTRY:
Before necropsy all animals were fasted overnight (water was freely available) and from 5 adult rats/sex/group (for males those with the lowest identification numbers in each cage and females with a litter were selected) and blood was taken from the aorta whilst under CO2/O2 anaesthesia.
Blood was collected in tubes filled with heparin (used as anticoagulant) and plasma was prepared by centrifugation. Plasma samples were discarded after analysis. The following clinical chemistry parameters were made in the plasma:
glucose (fasting), alkaline phosphatase (ALP), alanine aminotransferase (ALAT)/ glutamic-pyruvic transaminase (GPT), aspartate aminotransferase (ASAT)/ glutamic-oxalacetic transaminase (GOT), gamma glutamyl transferase (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, Inorganic phosphate, Calcium (Ca), Sodium (Na), Potassium (K), Chloride (Cl), inorganic phosphate (PO4) and bile acids.

NEUROBEHAVIOURAL EXAMINATION – Detailed clinical examinations:
Detailed clinical examinations were conducted in an arena outside the home cage in all adult rats of all groups prior to the first treatment and then once weekly (arena was not performed during the last days of pregnancy and during lactation).
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 behaviour.

NEUROBEHAVIOURAL EXAMINATION – Functional Observational Battery (FOB) and spontaneous motor activity
FOB and motor activity testing were performed in 5 adult animals/sex/group shortly prior to sacrifice of the male and female rats. These determinations were performed on the (surviving) male animals with the lowest identification numbers in each cage, and on females with a litter.
The FOB used in the laboratory was adapted from the WHO/IPCS Functional Observational Battery that was used in the Collaborative Study on Neurotoxicity Assessment sponsored by the International Programme on Chemical Safety of the World Health Organization

HORMONE DETERMINATIONS (T4):
The blood samples taken from the adult males were analysed for T4 hormone levels. Analysis was performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA452Ge). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

Oestrous cyclicity (parental animals):

Vaginal smears to evaluate the estrus cycle length and normality were made daily for fourteen days during the pre-treatment period. Cyclicity was used as an allocation criterium in the study. At the end of the ten week pre-mating period again vaginal smears were made until confirmation of mating. Smears were made, stained and examined in all females. An additional vaginal smear was made at the day of sacrifice.

Sperm parameters (parental animals):

Parameters examined in all males: epididymides (paired) weight, prostate (dorsolateral and ventral) weight, seminal vesicles and coagulation glands (paired) weight, testes weight
Additionally, testes, epididymides, seminal vesicles and prostate of males that failed to sire (did not mate or mated females were not pregnant) of the low- and mid-dose groups, were microscopically examined.

Litter observations:

STANDARDISATION OF LITTERS
- 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 prevents having four of each sex per litter, partial adjustment was acceptable. Preference was to retain at least four male pups in order to have sufficient male pups for nipple retention determinations on lactation day 13.

PARAMETERS EXAMINED
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.
- Any abnormal behaviour of pups was recorded on day 0, 4, 7 and 13 of lactation.
- At day lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter.
- On postnatal day 13 all surviving male pups were examined for the presence of nipples or areolas.
- Individual blood samples were collected from two pups per litter at sacrifice on or shortly after lactation day 13 (collected from the heart whilst under CO2/O2 anaesthesia). Serum was prepared and stored in a freezer (at ≤ 18°C) for determination of serum T4 levels. In addition blood samples were collected from two of the surplus pups per litter, and serum waspooled and stored in a freezer (at ≤ 18 oC) for possible determination of serum T4 levels. Thyroid gland weight was examined in 2 selected pups per litter on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
- Grossly malformed pups were sacrificed and examined.
- A necropsy was performed on stillborn pups and pups dying during the study and macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
Before necropsy all animals were fasted overnight (water was freely available). All surviving adult male and 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.
- Adult male animals were sacrificed after at least 90 days of treatment.
- 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 male and 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 (surviving males with the lowest identification numbers in each cage; females with a litter were selected) were preserved.
- Tissues for microscopic examination were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. 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, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and 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.

Postmortem examinations (offspring):

SACRIFICE, GROSS NECROPSY AND PATHOLOGY OF PUPS
Any abnormal behaviour 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 dying during the study and macroscopic abnormalities were recorded. At necropsy of the dams and litter, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques and necropsied.

Statistics:

Please see Section "Any other information on materials and methods incl. tables"

Reproductive indices:

- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated / number of females placed with males) x 100
- male fertility index = number of males with pregnant females * 100 / number of males placed with females
- female fertility index = number of pregnant females * 100 / number of inseminated females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- sex ratio day 0 = [(number of live male or female pups on day 0 / number of live pups on day 0] x 100

Offspring viability indices:

- prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
- perinatal loss = (Total number of pups delivered - Total number of alive pups delivered) x 100 / Total number of pups delivered
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x 100
- viability index day 4 - 13 = (number of pup surviving 13 days/number of live pups at day 4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs observed were related to the skin (encrustations, sparsely haired areas and eyes (encrustations). One animal in the low dose group showed blepharospasm of the eye and two males in the high dose group showed a sniffing respiration, which might be related to the route of administration (oral gavage). 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

Mortality:

no mortality observed

Description (incidence):

No mortalities occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weight was comparable in all groups in males and females during premating, mating, gestation and lactation.
- Mean body weight change was statistically significantly lower in the high dose group males from treatment day 49 to 70 and in the high dose females at the start of dosing (interval treatment day 0-7) and from treatment days 21-28 and 56-70. This was considered to be treatment related.
- During gestation and lactation mean body weight changes were comparable in all females.
- During the post-mating period mean body weight change was lower in the mid dose males from day 7-14 postmating (being day 84-91 related to the start of treatment). In view of the single occurrence and in absence of a dose response, this was considered to be a chance finding.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable in all groups for males and females.

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):

- The mean MHC was statistically significantly higher in the mid dose group males. In absence of a dose response relation this was considered a chance finding.
- The mean percentage monocytes was statistically significantly lower in the low dose males. In absence of a dose response relation this was considered a chance finding. No treatment-related effects on red blood cell and white blood cell parameters were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- The mean total protein and albumin levels were statistically significantly lower in the low dose and high dose males. In comparison to the historical control data these levels were slightly lower and therefore a relationship to treatment could not be ruled out. However, in absence of a dose-response relationship and in view of the limited effect, this effect is not considered adverse.
- Mean urea levels were slightly, but statistically significantly increased in the high dose males. This was considered related to treatment.

Urinalysis findings:

not examined

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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopic evaluation revealed treatment related histopathological changes in the kidneys, characterized by mild to moderate (multi)focal tubular dilatation in 5/12 high-dose males and in 1/12 high-dose females. Because of this finding, the kidneys of the low- and mid-dose animals were also processed and examined microscopically. In these groups tubular dilatation was not observed.
- The other histopathological changes observed were about equally distributed amongst the different treatment groups 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

Other effects:

no effects observed

Description (incidence and severity):

No statistical significant differences were observed in the T4 levels of the adult males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females showed regular estrus cycles. There were no females showing prolonged estrus cycles. The mean number of complete cycles during the 14-day pre-treatment phase and the 15-day premating phase was comparable in all groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Twelve females in each group were placed with males and all animals except for one female in the control group were mated. One female in the low dose group (animal 35) was misjudged to be not mated, but was pregnant. Therefore the mating date was not exactly known for this female. One mated female in the mid dose group was not pregnant. This resulted in mating indices of 91.7% for the control group and 100% for the treatment groups. The male fertility index was 91.7% for the control group and mid dose group and 100% for the low dose and high dose group, respectively. The mean number of mating days until successful copulation was comparable in all groups. Mean gestational length was also comparable in all groups.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pup observations included individual cases of subcutaneous haemorrhage, a wound, kink tail and pale skin. These findings were not related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of liveborn pups per litter was comparable in all groups. Pup survival was comparable in all groups: in the control group no pups were lost postnatal, in the low and mid dose groups 1 pup was missing or dead and in the high dose group 2 pups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup weight and weight gain were comparable in all groups.

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

- The mean anogenital distance corrected for body weight was statistically significantly increased in the female pups in the mid dose group. In absence of a dose response relationship this was considered a chance finding. No treatment-related effects were observed in the male and female pups on anogenital distance.
- In the male pups no nipples were present on lactation day 13. There were no effects on nipple retention.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects were observed on absolute or relative mean thyroid weight in male and female pups on lactation day 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination confirmed the stillbirth of one pup in the high dose group (lungs not distended) and the nurture status of the found dead pups. No macroscopic changes were observed on the 13-day old pups.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- In the 13 day old pups no statistical significant differences were observed in T4 levels female pups.
- In 13 day old male pups the T4 levels were statistically significantly higher in the low and mid dose group pups. However, in comparison with the historical control data the standard deviation in this study are large. The increased mean levels could be attributed to one or two high values per group. In addition, in the high dose male pups no statistical significance was reached. In absence of a dose response curve and in absence of an effect in female pups and the fact
that the increased mean could be attributed to one or two pups per group, these results were considered not related to treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical verification of dosing solutions

The test substance was considered to be homogeneously distributed in the diets and was considered to be stable in the vehicle (tap water) when stored at 2-10°C for 10 days. The concentration of the test substance was close to intended (85-115%) for all diets at all dose levels, except for the lowest level measured on 9 March where a slightly higher deviation of 16.4% was found.

Applicant's summary and conclusion

Conclusions:

Based on the histopathological changes in the kidney in the high dose males the NOAEL for parental toxicity was considered to be 15 mg/kg body weight per day.
Based on the absence of effects on fertility and developmental parameters, the NOAEL for fertility and developmental toxicity was established at ≥ 45 mg/kg body weight per day.

Executive summary:

The study was performed according to OECD TG 422 and GLP principles. The test substance was administered once daily by oral gavage, as a suspension in tap water. Controls were treated with vehicle (tap water) only. The study comprised 4 groups of 12 male and 12 female Wistar Han IGS rats (Crl:WI(Han)) each, viz. one control group that received the vehicle and three groups that received the test substance. The dose levels have been selected in consultation with the sponsor and are based on the results of a 2-weeks dose-range finding study with the test item in rats. The test substance was administered at dose levels of 5, 15 or 45 mg/kg body weight/day 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 90 days of exposure. A dosing volume of 5 mL/kg body weight was applied in all groups.

In life parameters included clinical observations, body weight, food consumption, mating,gestation and delivery parameters and individual pup parameters up to lactation day 13. Atnecropsy animals were macroscopically examined and several organs were examinedmicroscopically.The oral administration of test substance was well tolerated. There were no mortalities orchanges in clinical signs, neurobehavioural observations, growth, food intake, red blood cellvariables, clotting potential and results on macroscopy.A treatment-relationship could not be ruled out for the lower mean total protein levels and mean albumin levels in the low dose and high dose males. However, in absence of a dose-response relationship and in view of the limited effect, this was not considered adverse. In addition the higher mean urea levels in high dose males were considered related to treatment.Microscopic evaluation revealed treatment related histopathological changes in the kidneys,characterized by mild to moderate (multi)focal tubular dilatationin 1/12 high-dose females. Because of this finding, the kidneys of the low- and mid-dose animals were also processed and examined microscopically. In these groups tubular dilatation was not observed. There was no effect of the test substance on male and female fertility or reproductive performance. There were no effects on the litter data in the number of pups, pup survival, growth, sex ratio and developmental parameters.

Based on the histopathological changes in the kidney the NOAEL for maternal toxicity was considered to be 15 mg/kg body weight per day.Based on the absence of effects on fertility and developmental parameters, the NOAEL forfertility and developmental toxicity was stablished at ≥ 45 mg/kg body weight per day.

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