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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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:

other: OECD Guideline for the Testing of Chemicals 422. Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test,

Version / remarks:

29 July 2016

GLP compliance:

yes

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 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

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 qs°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

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.

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.

Duration of treatment / exposure:

90 days: 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

Duration of test:

90 days

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.

Examinations

Maternal 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, 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/group (for 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/group (for 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/group shortly prior to sacrifice of the female rats. These determinations were performed on the (surviving) female animals 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

POST- MORTEM EXAMINATIONS
Sacrifice
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 performed 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.
- 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, 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. Thyroid glands in the pups were not 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:

Examinations included:
- Ovaries, paired (organ weight and tissue fixation)
- Uterus, including cervix (organ weight and tissue fixation)
- Number of implantations
- Vagina (organ weight and tissue fixation)

Fetal examinations:

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°C) 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.

Statistics:

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

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
- 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
- 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: maternal animals

General toxicity (maternal animals)

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. 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 females during premating, mating, gestation and lactation.
- Mean body weight change was statistically significantly lower 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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable in all groups for females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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

Description (incidence and severity):

- In females the relative mean heart weight was slightly, but statistically significantly lower in the mid and high dose females. No effects were observed on absolute mean heart weights.
- In females no changes were observed in absolute or relative mean kidney weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy no treatment related macroscopic changes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopic evaluation revealed histopathological changes in the kidneys, characterized by mild to moderate (multi)focal tubular dilatation 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

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

All pregnant females delivered live litters

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Prenatal loss was comparable between groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

All pregnant females delivered live litters, resulting in a gestation index of 100%.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

One female in the high dose group had stillborn pups and no litters showed complete litter loss.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Mean gestational length was comparable in all 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): Mean gestational length was comparable in all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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.

Other effects:

not examined

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

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.

Basis for effect level:

maternal abnormalities

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Pup weight and weight gain were comparable in all groups.
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): Pup weight and weight gain were comparable in all groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

All pregnant females delivered live litters, resulting in a gestation index of 100%.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio (percentage male or female pups) was comparable in all groups on days 0 and 13 of lactation.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of liveborn pups per litter was comparable in all groups.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

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.

External malformations:

no effects observed

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.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

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.
- 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.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

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

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 sacrifices 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.

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. There was no effect of the test substance on 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 in the females 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 NOAL forfertility and developmental toxicity was established at ≥ 45 mg/kg body weight per day.