Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
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 (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch identification: Lab-sample from Dec 2016
- Purity test date: Jan 26 - May 11, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 10-11 weeks (male animals); about 9 weeks (female animals)
- Weight at study initiation: males: 382.2 g (mean); females: 224.6 g (mean)
- Housing: individually
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 01 Aug 2017 To: 31 Sep 2017 (parental males); 24 Oct 2017 (parental females); PND 4 or 13 (pups)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
ultra-pure
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed out depending on the desired concentration. Then, ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At these time points, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed via capillary electrophoresis with internal standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.

The test substance concentrations in ultra-pure water were found to be in the range of 90-110% of the nominal concentration.
Duration of treatment / exposure:
males: 30 days; females: 55 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 1972 mg/kg bw/d.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
mid-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 592 mg/kg bw/d.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low-dose group;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 197 mg/kg bw/d.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• During the premating phase, body weight was determined twice a week, i.e. on study days 3, 7, 10 and 14.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of administration volume).
• Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 27 (males) and 52 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/
stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response),
coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined.
Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in parental males:
testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides, prostate gland, seminal vesicles, testes; spermatogenic staging profiles
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, postnatal mortality, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, blood thyroid hormone concentrations, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on study day 31
- Maternal animals: All surviving animals, on study day 56

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.
Statistics:
see table 5
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Offspring viability indices:
- viability index, survival index (for formulas see "Any other information on materials and methods")
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- mean body weight value was significantly increased in female animals of test group 3 (1000 mg/kg bw/d) on lactation day 13
- mean body weight change values were significantly increased in female animals of test group 2 (300 mg/kg bw/d) between pre-mating days 7-13 and 0-13
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test group 3 (1000 mg/kg bw/d) mean corpuscular volume (MCV) was significantly increased, but the mean was within the historical control range
- In females of test group 2 (300 mg/kg bw/d) absolute basophil counts were significantly increased, but this alteration was not dose-dependent.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) chloride values were significantly higher compared to controls, but the values were within the historical control range.
- In females of test group 2 (300 mg/kg bw/d) creatinine values were significantly decreased and in females of test group 1 (100 mg/kg bw/d) triglyceride levels were significantly lower compared to controls. Both latter parameters were not dose-dependently changed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.

Foot splay test was significantly decreased in male animals of test group 3 (1000 mg/kg bw/d). The change was assessed as being spontaneous in nature and not related to treatment. The value was well within the historical control data.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- minimal, focal superficial erosion in the glandular stomach of a single female of test group 1 (100 mg/kg bw/d) and in a single male and 2 females of the test group 2 (300 mg/kg bw/d); lack of a dose-depending relationship and therefore considered incidental and not test-item related; the incidences of erosions in the glandular stomach in this study also felt within the range of historical control data on the glandular stomach for this lesion
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
- Male animal Nos. 21 and 28 of test group 2 (300 mg/kg bw/d), which were mated with female animal Nos. 121 and 128, and male animal Nos. 36, 38 and 40 of test group 3 (1000 mg/kg bw/d), which were mated with female animal Nos. 136, 138 and 140, did not generate F1 pups and no implants were found at necropsy although the female animals had sperm in vaginal smear. Thus, the male and female fertility index was 100% in test groups 0 (control; 0 mg/kg bw/d) and 1 (100 mg/kg bw/d), 80% in test group 2 (300 mg/kg bw/d) and 70% in test group 3 (1000 mg/kg bw/d).
The values for test groups 0, 1, 2 and 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within or marginally below the range of the historical control data.

- The mean duration until sperm was detected (GD 0) was 3.8 days for test group 0, 2.5 days for test group 1, 2.1 days for test group 2 and 2.4 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

- The postimplantation loss was 8.7% in test group 0 (control; 0 mg/kg bw/d), 6.9% in test group 1 (100 mg/kg bw/d), 6.4% in test group 2 (300 mg/kg bw/d) and 10.5% in test group 3 (1000 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Three male and 2 female pups of test group 0 (control; 0 mg/kg bw/d), 1 female pup of test groups 1 and 2 (100 and 300 mg/kg bw/d) and 1 female pup of test group 3 (1000 mg/kg bw/d) died prematurely.
Female pup No. 126-14 of test group 2 (300 mg/kg bw/d) showed loss of both hindlimbs (due to cannibalization) on the day of death (PND 0).
These changes were assessed to be incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- Mean body weight of male pups in test group 3 (1000 mg/kg bw/d) was significantly higher on lactation day 1 (+14.5%) when compared to control. Also, mean of means of body weights in both sexes of test group 3 was significantly higher on lactation day 1 (+13.2%) when compared to control.
These deviations to the control were assessed to be incidental and not related to the test item.

- Five male runts were found in 1 litter and 5 female runts were found in 2 litters of test group 0 (control; 0 mg/kg bw/d). One female runt was found in a litter of test group 1 (100 mg/kg bw/d).
A relation to dosing was not observed, test substance-related effects did not occur.

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:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- One male and 2 female pups of test group 0 (control; 0 mg/kg bw/d) and 1 female pup of test group 2 (300 mg/kg bw/d) showed post mortem autolysis. These findings were observed as the pups were found dead and the autolysis was in progress before the daily clinical observations started.
One female pup of test group 2 (300 mg/kg bw/d) was partly cannibalized (both hindlimbs) on the day of death (see 4.2.2.2.).
These findings were assessed to be incidental and not related to the test substance.

- Two male pups from different litters of test group 0, one female pup of test group 1 and test group 2 were not assessed after death, because their bodies were missing (cannibalization).
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000 mg/kg bw/d.

The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test substance was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at effective dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2)and 1000 mg/kg bw/d (test group 3). Ultra-pure water served as vehicle, control animals were dosed daily with the vehicle only.

Considering the content of the test substance of 50.7 g/100 g (100 g/100 g minus water content), the dose levels of the product were chosen to be 197, 592 and 1972 mg/kg bw/d to achieve an intake of 100, 300 and 1000 mg/kg bw/d, respectively.

 

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and

one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental animals per sex and test group.

Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: 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 (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch identification: Lab-sample from Dec 2016
- Purity test date: Jan 26 - May 11, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 10-11 weeks (male animals); about 9 weeks (female animals)
- Weight at study initiation: males: 382.2 g (mean); females: 224.6 g (mean)
- Housing: individually
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 01 Aug 2017 To: 31 Sep 2017 (males); 24 Oct 2017 (females)
Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed out depending on the desired concentration. Then, ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At these time points, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed via capillary electrophoresis with internal standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.

The test substance concentrations in ultra-pure water were found to be in the range of 90-110% of the nominal concentration.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Duration of treatment / exposure:
males: 30 days; females: 55 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor)
Duration of test:
56 days
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 1972 mg/kg bw/d.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
mid-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 592 mg/kg bw/d.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low-dose group;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 197 mg/kg bw/d.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• During the premating phase, body weight was determined twice a week, i.e. on study days 3, 7, 10 and 14.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of administration volume).
• Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined during the mating period.
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on study day 56
- Organs examined: see table 3

OTHER
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at PND 14
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

Functional observational battery (FOB):
- A functional observational battery was performed in the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 52.
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/
stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response),
coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five surviving females with litter (in order of delivery) per group
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
see table 4
Indices:
Viability index of pups, Survival index of pups (for detailed formulas see "Any other information on materials and methods"
Historical control data:
Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: maternal body weights, mating data, delivery data, litter data, pup weights, pup necropsy observations, anogenital distance, presence of areolas/nipples, incidence of erosion/ulceration in the forestomach of female control animals
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- mean body weight value was significantly increased in female animals of test group 3 (1000 mg/kg bw/d) on lactation day 13
- mean body weight change values were significantly increased in female animals of test group 2 (300 mg/kg bw/d) between pre-mating days 7-13 and 0-13
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test group 3 (1000 mg/kg bw/d) mean corpuscular volume (MCV) was significantly increased, but the mean was within the historical control range
- In females of test group 2 (300 mg/kg bw/d) absolute basophil counts were significantly increased, but this alteration was not dose-dependent.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) chloride values were significantly higher compared to controls, but the values were within the historical control range.
- In females of test group 2 (300 mg/kg bw/d) creatinine values were significantly decreased and in females of test group 1 (100 mg/kg bw/d) triglyceride levels were significantly lower compared to controls. Both latter parameters were not dose-dependently changed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- statistically significantly increased absolute and relative mean uterine weights present in females of all test-item treated groups. The mean absolute and relative values in the group treated with 1000 mg/kg bw/d (0.883 g and 0.366%) just exceeded the upper limits of the historical control range (0.824 g and 0.339%). All other values felt within the historical control range. The mean values in the control group were low and felt just above the lower limits of the historical control range resulting in an apparent big difference between treated groups and control. The uterine weight increase in females of the test group 3 (1000 mg/kg bw/d) was considered attributed to the relative high number of females (5 out of 10) in this group showing dilated (fluid-filled) uterus horns and therefore was considered to be not treatment-related.

- statistically significantly decreased (91%) mean absolute liver weight in females of test group 1 (100 mg/kg bw/d); not related to histopathologic changes and considered to be attributed to the normal biological variation in animals of this strain and age

- statistically significantly increased (104%) mean absolute kidney weight and the decreased (91%) mean relative brain weight in females of test group 3 (1000 mg/kg bw/d); not related to histopathologic changes and considered to be attributed to the normal biological variation in animals of this strain and age
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- A red/black focus on the glandular mucosa noted in the glandular stomach of a single female of test group 1 (100 mg/kg bw/d) and in a single male and 2 females of the test group 2 (300 mg/kg bw/d); lack of a dose-depending relationship and therefore considered incidental and not test-item related
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- minimal, focal superficial erosion in the glandular stomach of a single female of test group 1 (100 mg/kg bw/d) and in a single male and 2 females of the test group 2 (300 mg/kg bw/d); lack of a dose-depending relationship and therefore considered incidental and not test-item related; the incidences of erosions in the glandular stomach in this study also felt within the range of historical control data on the glandular stomach for this lesion
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The postimplantation loss was 8.7% in test group 0 (control; 0 mg/kg bw/d), 6.9% in test group 1 (100 mg/kg bw/d), 6.4% in test group 2 (300 mg/kg bw/d) and 10.5% in test group 3 (1000 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
The mean duration of gestation was 22.6 days in test groups 0 and 3. In test groups 1 and 2, the mean duration of gestation was 22.0 days and significantly reduced when compared to the controls. The shortened mean gestation period was not considered to be related to treatment. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was 22.6 days in test groups 0 and 3. In test groups 1 and 2, the mean duration of gestation was 22.0 days and significantly reduced when compared to the controls. The shortened mean gestation period was not considered to be related to treatment. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): - Mean body weight of male pups in test group 3 (1000 mg/kg bw/d) was significantly higher on lactation day 1 (+14.5%) when compared to control. Also, mean of means of body weights in both sexes of test group 3 was significantly higher on lactation day 1 (+13.2%) when compared to control.
These deviations to the control were assessed to be incidental and not related to the test item.

- Five male runts were found in 1 litter and 5 female runts were found in 2 litters of test group 0 (control; 0 mg/kg bw/d). One female runt was found in a litter of test group 1 (100 mg/kg bw/d).
A relation to dosing was not observed, test substance-related effects did not occur.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
for litter weights see "Fetal/pup body weight changes"
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
Three male and 2 female pups of test group 0 (control; 0 mg/kg bw/d), 1 female pup of test groups 1 and 2 (100 and 300 mg/kg bw/d) and 1 female pup of test group 3 (1000 mg/kg bw/d) died prematurely.
One female pup of test group 2 (300 mg/kg bw/d) showed loss of both hindlimbs (due to cannibalization) on the day of death (PND 0).
These changes were assessed to be incidental.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000 mg/kg bw/d.

The NOAEL for developmental toxicity was set to 1000 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test substance was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at effective dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Ultra-pure water served as vehicle, control animals were dosed daily with the vehicle only.

Considering the content of the test substance of 50.7 g/100 g (100 g/100 g minus water content), the dose levels of the product were chosen to be 197, 592 and 1972 mg/kg bw/d to achieve an intake of 100, 300 and 1000 mg/kg bw/d, respectively.

 

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and

one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental animals per sex and test group.

Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats.

The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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 (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-hydroxyethanesulphonate
EC Number:
213-037-4
EC Name:
Sodium 1-hydroxyethanesulphonate
Cas Number:
918-04-7
Molecular formula:
C2H6O4S.Na
IUPAC Name:
sodium 1-hydroxyethane-1-sulfonate
Test material form:
liquid
Details on test material:
- Batch identification: Lab-sample from Dec 2016
- Content: 50.7 g/100 g sodium 1-hydroxyethanesulphonate
- Physical state / color: liquid / colorless to yellowish
- Water: 46.1 g/100g
- Molecular weight: 148.1 g/mol
- LOG KOW: -5.58 (calculated)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch identification: Lab-sample from Dec 2016
- Purity test date: Jan 26 - May 11, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The appropriate amount of test substance was weighed out depending on the desired concentration. Ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were prepared in such a manner that the stability was guaranteed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in ultra-pure water

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 10-11 weeks (male animals); about 9 weeks (female animals)
- Weight at study initiation: males: 382.2 g (mean); females: 224.6 g (mean)
- Housing: individually
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 01 Aug 2017 To: 31 Sep 2017 (males); 24 Oct 2017 (females)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
water
Remarks:
ultra-pure
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed out depending on the desired concentration. Then, ultra-pure water was filled up to the desired volume and subsequently released with a magnetic stirrer.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At these time points, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed via capillary electrophoresis with internal standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.

The test substance concentrations in ultra-pure water were found to be in the range of 90-110% of the nominal concentration.
Duration of treatment / exposure:
males: 30 days; females: 55 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
Frequency of treatment:
daily at the same time in the morning (exception: no administration to animals being in labor)
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
high-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 1972 mg/kg bw/d.
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
mid-dose level;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 592 mg/kg bw/d.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
low-dose group;
Considering a purity of the test substance of 50.7% plus water, the dose level of the whole product was 197 mg/kg bw/d.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• During the premating phase, body weight was determined twice a week, i.e. on study days 3, 7, 10 and 14.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of administration volume).
• Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of administration volume).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 27 (males) and 52 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Estrous cycle:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
see table 5

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- mean body weight value was significantly increased in female animals of test group 3 (1000 mg/kg bw/d) on lactation day 13
- mean body weight change values were significantly increased in female animals of test group 2 (300 mg/kg bw/d) between pre-mating days 7-13 and 0-13
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test group 3 (1000 mg/kg bw/d) mean corpuscular volume (MCV) was significantly increased, but the mean was within the historical control range
- In females of test group 2 (300 mg/kg bw/d) absolute basophil counts were significantly increased, but this alteration was not dose-dependent.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- After the administration period, in females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) chloride values were significantly higher compared to controls, but the values were within the historical control range.
- In females of test group 2 (300 mg/kg bw/d) creatinine values were significantly decreased and in females of test group 1 (100 mg/kg bw/d) triglyceride levels were significantly lower compared to controls. Both latter parameters were not dose-dependently changed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.

Foot splay test was significantly decreased in male animals of test group 3 (1000 mg/kg bw/d). The change was assessed as being spontaneous in nature and not related to treatment. The value was well within the historical control data.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- statistically significantly increased absolute and relative mean uterine weights present in females of all test-item treated groups. The mean absolute and relative values in the group treated with 1000 mg/kg bw/d (0.883 g and 0.366%) just exceeded the upper limits of the historical control range (0.824 g and 0.339%). All other values felt within the historical control range. The mean values in the control group were low and felt just above the lower limits of the historical control range resulting in an apparent big difference between treated groups and control. The uterine weight increase in females of the test group 3 (1000 mg/kg bw/d) was considered attributed to the relative high number of females (5 out of 10) in this group showing dilated (fluid-filled) uterus horns and therefore was considered to be not treatment-related.

- statistically significantly decreased (91%) mean absolute liver weight in females of test group 1 (100 mg/kg bw/d); not related to histopathologic changes and considered to be attributed to the normal biological variation in animals of this strain and age

- statistically significantly increased (104%) mean absolute kidney weight and the decreased (91%) mean relative brain weight in females of test group 3 (1000 mg/kg bw/d); not related to histopathologic changes and considered to be attributed to the normal biological variation in animals of this strain and age
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- A red/black focus on the glandular mucosa noted in the glandular stomach of a single female of test group 1 (100 mg/kg bw/d) and in a single male and 2 females of the test group 2 (300 mg/kg bw/d); lack of a dose-depending relationship and therefore considered incidental and not test-item related
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- minimal, focal superficial erosion in the glandular stomach of a single female of test group 1 (100 mg/kg bw/d) and in a single male and 2 females of the test group 2 (300 mg/kg bw/d); lack of a dose-depending relationship and therefore considered incidental and not test-item related; the incidences of erosions in the glandular stomach in this study also felt within the range of historical control data on the glandular stomach for this lesion
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

The various analyses confirmed

·     the stability of the test-substance preparations for a period of 24 hours at room temperature,

·     the homogeneous distribution of the test substance in the vehicle,

·     the correctness of the prepared concentrations.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000 mg/kg bw/d.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.
Executive summary:

The test substance was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at effective dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). Ultra-pure water served as vehicle, control animals were dosed daily with the vehicle only.

Considering the content of the test substance of 50.7 g/100 g (100 g/100 g minus water content), the dose levels of the product were chosen to be 197, 592 and 1972 mg/kg bw/d to achieve an intake of 100, 300 and 1000 mg/kg bw/d, respectively.

 

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1 after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and

one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental animals per sex and test group.

Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group towards the end of the administration period.

All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of toxicity in male and female animals up to a dose level of 1000mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d formale and female Wistar rats.

The NOAEL for developmental toxicity was 1000 mg/kg bw/d.