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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-05 till 2016-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-05 till 2016-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March, 1996
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Han IGS
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: Males: 294-335 g; Females: 172-205 g
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of four per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females were housed in the order of animal number). After delivery, the cage containing the dam with litter was transferred to another cage rack, the location being determined by delivery date and animal number as described for mated females. On the day of FOB testing and motor activity assessment, animals were temporarily kept singly.
- Diet: ad libitum, from their arrival, the rats received a cereal-based (closed formula) powder rodent diet (VRF1(FG)) from a commercial supplier (SDS Special Diets Services, Witham, England).
- Water: ad libitum
- Acclimation period: > 5 days

DETAILS OF FOOD AND WATER QUALITY: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: two times during the study (on 28 December 2015 and on 22 January 2016)
- Mixing appropriate amounts with: cereal-based (closed formula) powder rodent diet (VRF1(FG))
- Storage temperature of food: ≤ -18 °C
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: until mating occurred or 1 week had elapsed
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually for the birth and rearing of their pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From both batches of diets prepared in the study (28 December 2015 and 22 January 2016), samples were taken and stored in a freezer (≤ -18 °C). Analyses to determine the stability, homogeneity and content of the test substance in the test diets were conducted as indicated below.
- Content: The content of the test substance at each dietary level was determined in the batches prepared on 28 December 2015 and 22 January 2016.
- Homogeneity: The homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (28 December 2015), by analyzing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analyzed.
- Stability: To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 28 December 2015 (low-dose diet, mid-dose diet and highdose diet) were analyzed at t=0¹ and reanalyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.
Duration of treatment / exposure:
- Males: from 5 January 2016 to 3 February 2016
- Not-pregnant females (1): from 5 January 2016 to 22 February 2016
- Pregnant females: from 5 January 2016 to between 15-22 February 2016
.
Frequency of treatment:
Continuously
Dose / conc.:
450 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
4 500 mg/kg diet
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale: the dose levels were selected based on the results of a 14-day dose-range finding study with this compound in rats.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/ DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays,
Sundays and public holidays only one check per day was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations: one day before the start of the treatment and at the start of the study. Males were weighed weekly, females were weighed once during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during gestation and on day 0 and 4 of lactation. Non-mated female 45 was weighed once per week after the mating period. All animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY/CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/O2
- Animals fasted: Yes, overnight
- How many animals: in 5 rats/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to the end of the premating period
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and then once weekly until sacrifice for males, or until delivery of the first lot for females. Arena testing was also conducted prior to sacrifice in the females concerned, as part of the Functional Observational Battery.
- Dose groups that were examined: all rats
- Battery of functions tested: Functional Observational Battery (FOB) and motor activity assessment
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups was evaluated on days 0 and 4 of lactation. Any abnormal behavior of pups was recorded on day 0 and 4 of lactation.

GROSS EXAMINATION OF (DEAD) PUPS: yes, macroscopic abnormalities were recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving males were sacrificed after the mating period on 3 February 2016
- Maternal animals: the dams were sacrificed on day 4 of lactation or one day thereafter (those selected for FOB/motor activity testing, and one mid-dose female). A not-pregnant female (showing only implantations) of the low-dose group was sacrificed on 22 February 2016. Pups were sacrificed by gradual CO2 anesthesia, followed by irreversible hypothermia on day 4 of lactation (or, for one mid-dose female, one day thereafter). The various lots were sacrificed between 15 – 22 February 2016.

GROSS NECROPSY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS:
In addition the following organs of five animals/sex/group (2, 2 and 1 surviving males with the lowest animal number in each cage, and females with a litter taking into account the day of delivery) were preserved. The following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying: adrenals, brain, heart, kidneys, liver, spleen and thymus.

Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin, except for sections of the testes which were stained with PAS hematoxylin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high-dose group (group 4). In addition, organs showing gross lesions were examined microscopically in animals of all groups. Reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of one low-dose male that failed to sire and a low-dose female that showed implantation but was not pregnant were also examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations macroscopic abnormalities.

- Pups were stored in a freezer (≤-18°C) for possible skeletal analyses, and were discarded after finalization of the report.
Statistics:
The data were analyzed using the methods mentioned below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**). Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
- Continuous data were subjected to a decision tree for continuous data.
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).
Reproductive indices:
- mating days until Day 0 pc = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- female mating index = (number of females inseminated/number of females placed with males) x 100
- male mating index = (number of males placed with females /number of females inseminated) x 100
- female fertility index = number of pregnant females*100/number of inseminated females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- gestation index = (number of females with live pups / number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites) / number of corpora lutea] 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.
Offspring viability indices:
- 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) x100
- sex ratio day 0 = [(number of live male or female pups on day 0 / number of live pups on day 0] x 100
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mid-dose male died as a result of anesthesia during blood collection before the start of the mating period. Hence the death of his rats was not treatment-related.
Another mid-dose male was found dead on day 4 of the mating period. The death of this single rat in the mid-dose group was considered an incidental finding, not related to the treatment. For mating, both males were replaced by other males of the same group.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption
From 5 to 9 February 2016 (gestation period), there was an unnoticed balance error resulting in unreliable recording of the food added to the cans. Because the day of gestation varied for the various lots, this error affected the individual food intake recordings in week 2 and/or 3 of gestation for many cages. However, all possibly suspect values were excluded from the calculation of the mean.
There were no marked differences in food consumption between the test groups and the controls. Mean food intake in the high-dose group tended to be somewhat lower in the premating period, in the in the post-mating period (males) and during lactation, but the differences with the controls were only slight (between 5%-9%) and no relevance was attached to these findings.

Test substance intake
Premating period and males postmating period
During the premating period, the overall mean intake of test substance was 34, 115 and 328 mg/kg bw/d in females of the low-, mid- and high-dose group, respectively. In males of these groups, the overall intake during the premating and the post mating period combined was 28, 93 and 265 mg/kg bw/d, respectively.
Gestation period
Because food intake recordings in week 2 and/or 3 of gestation were possibly unreliable (see above), test substance intake could not be calculated (mainly) in week 3 of gestation for many cages, especially in the low- and mid-dose groups. Because, in the authors' experience, test substance intake/kg bw does not significantly drop in the last week of gestation, in these groups the figures obtained in the first two weeks of the study were considered representative for the entire gestation period. During the gestation period, the overall mean intake of test substance in the females of the low-, mid- and high-dose group was 35, 115 and 337mg/kg body weight/day, respectively.
Lactation period
During the 4-day lactation period, the mean intake of test substance in females of the low-, mid- and high-dose group was 51, 177 and 507 mg/kg body weight/day, respectively.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Total white blood cell count and absolute lymphocyte count were statistically significantly increased in female of the high-dose group. These changes were within the range of historical control data and occurred in one sex only. Therefore they were not ascribed to treatment. The percentage of eosinophils was decreased in females of the low- and high-dose group but there was no dose-response relationship, the values in the test groups were well within the range of historical control data and these findings were not reflected in significant changes in absolute eosinophil counts. In males, there were no statistically significant differences in total or differential white blood cell counts.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cholesterol and phospholipids were statistically significantly increased in females of the high-dose group. These changes were within the range of historical control data and occurred in one sex only. Therefore they were not ascribed to treatment.
GGT activity was slightly though statistically significantly increased in females of the high-dose group. The relevance of this finding is doubtful because all values were well within the range of historical control data.
Calcium concentration was statistically significantly increased in females of the low- and high-dose group. There was no-dose response and this finding was probably due to a relatively low control value.
ASAT activity was statistically significantly decreased in males of the mid- and high-dose group. No relevance was attached to this finding because an increase rather than a decrease in this parameter is considered to represent a toxic effect.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic analysis of the rat that died as a result of anesthesia during blood collection revealed minimal basophilic tubules in the kidney and a minimal increase of proteinaceous droplets in the tubuli. In the liver, mild congestion and mild centrilobular hypertrophy was observed. In addition a mild focal mononuclear cell inflammation was present. Microhaemorrhages were observed in the thymus and ectopic thymus was observed in the thyroid.
Microscopic analysis of the male that was found dead on day 4 of the mating period showed already autolytic changes in a number of organs. The intestines and the lymphoid organs were to autolytic to evaluate. Close to the prostate, moderate inflammation was observed around the urethra. The presumed ileac lymph node turned out to be an inflamed urethra, surround by inflamed fat. In addition, a large necrotic area was visible next to the urethra, showing signs of early abcess formation. The urinary bladder showed no abnormalities but in agreement with its enlargement as a result of the urine build-up, the wall of the urinary bladder was stretched. The kidneys showed mild multifocal mineralization. In addition, diffuse moderate epithelial vacuolation was observed. With respect to the cause of death, we hypothesize that as a result of the necrosis and inflammation around the urethra, the passage of urine was blocked, leading to the accumulation of urine in the urinary bladder and subsequent swelling of the kidney. When the flow of urine is obstructed, stones (calculi) are more likely to form which might explain the observed mild multifocal mineralization. The elevated pressure due to the obstruction may ultimately result in kidney failure and subsequent death. Because similar findings were not observed in other rats at any dose level (see below) the above findings were considered incidental findings, not related to the treatment.
Microscopic examination of surviving animals revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Reproductive performance:
no effects observed
Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
1 stillborn pup in the control and 8 stillborn pups in the high-dose group. Such findings in individual dams are normal for this strain of rats.
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In this study the test substance did not induce adverse effects to reproductive performance or fertility parameters, so the NOAEL was placed at ≥4500 mg/kg diet (the highest dose tested, equivalent to an overall intake of at least 328 mg/kg bw/d in females and at least 265 mg/kg bw/d in males).
Executive summary:

In this GLP compliant study performed according to OECD422, the possible effects of the test substance on reproductive performance were examined in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 450, 1500 and 4500 mg/kg diet during a premating period of 2 weeks and during mating, gestation, until day 4 of lactation. These dietary levels provided an overall mean intake of at least 34, 115 and 328 mg/kg bw/d in females of the low-, mid- and high-dose group. In males (during the premating and the post mating period), the overall mean intake was 28, 93 and 265 mg/kg bw/d, respectively. Female animals and pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after the mating period. The content, homogeneity and stability of the test substance in the carrier were confirmed by analysis.

 

There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no relevant differences in body weights or food consumption during the premating period, during the post-mating period in males, or in dams during the gestation period and the lactation period.

 

Hematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. In females of the high-dose group, total white blood cell count, absolute lymphocyte count, cholesterol and phospholipids were increased compared to controls. Because these changes were within the range of historical control data and occurred in one sex only, they were not ascribed to treatment.

 

The weight of the liver was increased in males of the high-dose group. In the absence of histopathological alterations or clinical pathology evidence of liver toxicity, this finding was considered an adaptive and non-adverse reaction to the test substance. Macroscopic and microscopic examination did not reveal any treatment-related abnormalities There were no effects of the test substance on fertility and reproductive performance, litter data or pup observations in any group.

 

Because there were no adverse effects, and because fertility parameters and reproductive performance were not affected by the test substance, the NOAEL for reproduction was placed at ≥ 4500 mg/kg diet (the highest concentration tested; equivalent to an overall intake of at least 328 mg/kg bw/d in females and at least 265 mg/kg bw/d in males).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March, 1996
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Substance name in the test report: FRET 10-0367
- Physical state: clear, colorless liquid
- Storage conditions: 2-10 °C, protected from light

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han IGS
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: Males: 294-335 g; Females: 172-205 g
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the premating period, the animals were housed in groups of four per sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack. The location of the mated females in the new cage racks was determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females were housed in the order of animal number). After delivery, the cage containing the dam with litter was transferred to another cage rack, the location being determined by delivery date and animal number as described for mated females. On the day of FOB testing and motor activity assessment, animals were temporarily kept singly.
- Diet: ad libitum, from their arrival, the rats received a cereal-based (closed formula) powder rodent diet (VRF1(FG)) from a commercial supplier (SDS Special Diets Services, Witham, England).
- Water: ad libitum
- Acclimation period: > 5 days

DETAILS OF FOOD AND WATER QUALITY: 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.

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

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The food was provided to the rats as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the cans was replaced twice weekly with fresh portions from the freezer and topped up if needed.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: two times during the study (on 28 December 2015 and on 22 January 2016)
- Mixing appropriate amounts with: cereal-based (closed formula) powder rodent diet (VRF1(FG))
- Storage temperature of food: ≤ -18 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From both batches of diets prepared in the study (28 December 2015 and 22 January 2016), samples were taken and stored in a freezer (≤ -18 °C). Analyses to determine the stability, homogeneity and content of the test substance in the test diets were conducted as indicated below.
- Content: The content of the test substance at each dietary level was determined in the batches prepared on 28 December 2015 and 22 January 2016.
- Homogeneity: The homogeneity (and content) of the test substance in the experimental diets was assessed in the first batch (28 December 2015), by analyzing five samples (taken at different locations in the feed container) of each test diet in duplicate. One sample of the control diet was analyzed.
- Stability: To demonstrate the stability of the test substance under experimental conditions, samples of the batch of test diets prepared on 28 December 2015 (low-dose diet, mid-dose diet and highdose diet) were analyzed at t=0¹ and reanalyzed after storage in the animal room (in an open container) for 4 days, and after storage in a freezer (<-18 °C) for at least 5 weeks.
Duration of treatment / exposure:
- Males: from 5 January 2016 to 3 February 2016
- Females: from 5 January 2016 to 15-22 February 2016
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
450 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
4 500 mg/kg diet
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rationale: the dose levels were selected based on the results of a 14-day dose-range finding study with this compound in rats.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/ DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations: one day before the start of the treatment and at the start of the study. Males were weighed weekly, females were weighed once during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during gestation and on day 0 and 4 of lactation. Non-mated female 45 was weighed once per week after the mating period. All animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY/CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to the end of the premating period
- Anaesthetic used for blood collection: Yes, CO2/O2
- Animals fasted: Yes, overnight
- How many animals: in 5 rats/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to the end of the premating period
- Animals fasted: Yes, overnight
- How many animals: 5 rats/sex/group

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and then once weekly until sacrifice for males, or until delivery of the first lot for females. Arena testing was also conducted prior to sacrifice in the females concerned, as part of the Functional Observational Battery.
- Dose groups that were examined: all rats
- Battery of functions tested: Functional Observational Battery (FOB) and motor activity assessment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The data were analyzed using the methods mentioned below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results was p<0.05 (*) or p<0.01 (**). Non-mated females were excluded from mean data tables presenting data from the gestation and lactation periods.
- Continuous data were subjected to a decision tree for continuous data.
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One mid-dose male died as a result of anesthesia during blood collection before the start of the mating period. Hence the death of his rats was not treatment-related.
Another mid-dose male was found dead on day 4 of the mating period. The death of this single rat in the mid-dose group was considered an incidental finding, not related to the treatment. For mating, both males were replaced by other males of the same group.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption
From 5 to 9 February 2016 (gestation period), there was an unnoticed balance error resulting in unreliable recording of the food added to the cans. Because the day of gestation varied for the various lots, this error affected the individual food intake recordings in week 2 and/or 3 of gestation for many cages. However, all possibly suspect values were excluded from the calculation of the mean.
There were no marked differences in food consumption between the test groups and the controls. Mean food intake in the high-dose group tended to be somewhat lower in the premating period, in the in the post-mating period (males) and during lactation, but the differences with the controls were only slight (between 5%-9%) and no relevance was attached to these findings.

Test substance intake
Premating period and males postmating period
During the premating period, the overall mean intake of test substance was 34, 115 and 328 mg/kg bw/d in females of the low-, mid- and high-dose group, respectively. In males of these groups, the overall intake during the premating and the post mating period combined was 28, 93 and 265 mg/kg bw/d, respectively.
Gestation period
Because food intake recordings in week 2 and/or 3 of gestation were possibly unreliable (see above), test substance intake could not be calculated (mainly) in week 3 of gestation for many cages, especially in the low- and mid-dose groups. Because, in the authors' experience, test substance intake/kg bw does not significantly drop in the last week of gestation, in these groups the figures obtained in the first two weeks of the study were considered representative for the entire gestation period. During the gestation period, the overall mean intake of test substance in the females of the low-, mid- and high-dose group was 35, 115 and 337mg/kg body weight/day, respectively.
Lactation period
During the 4-day lactation period, the mean intake of test substance in females of the low-, mid- and high-dose group was 51, 177 and 507 mg/kg body weight/day, respectively.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Total white blood cell count and absolute lymphocyte count were statistically significantly increased in female of the high-dose group. These changes were within the range of historical control data and occurred in one sex only. Therefore they were not ascribed to treatment. The percentage of eosinophils was decreased in females of the low- and high-dose group but there was no dose-response relationship, the values in the test groups were well within the range of historical control data and these findings were not reflected in significant changes in absolute eosinophil counts. In males, there were no statistically significant differences in total or differential white blood cell counts.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cholesterol and phospholipids were statistically significantly increased in females of the high-dose group. These changes were within the range of historical control data and occurred in one sex only. Therefore they were not ascribed to treatment.
GGT activity was slightly though statistically significantly increased in females of the high-dose group. The relevance of this finding is doubtful because all values were well within the range of historical control data.
Calcium concentration was statistically significantly increased in females of the low- and high-dose group. There was no-dose response and this finding was probably due to a relatively low control value.
ASAT activity was statistically significantly decreased in males of the mid- and high-dose group. No relevance was attached to this finding because an increase rather than a decrease in this parameter is considered to represent a toxic effect.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver weights were statistically significantly increased in males of the high-dose group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The mid-dose male that died as a result of anesthesia during blood collection before the start of the mating period did not show any macroscopic findings. The mid-dose male that was found dead on day 4 of the mating period showed red fluid in the thoracic cavity at necropsy. The abdominal cavity was filled with a clear fluid. The urinary bladder was strongly enlarged and contained red opaque fluid. The kidneys were enlarged. Prostate and seminal vesicles were enlarged showed red discolouration and looked edematous. The adrenals were enlarged as well. The intestines were empty and looked pale. The patches of Peyer could not be distinguished. The stomach showed no mucosal folds. A presumed iliac lymph node was enlarged and brown. The right and left inguinal regions looked edematous. The thymus was small, flabby and showed red discolouration.
Macroscopic observations of surviving animals revealed no treatment-related abnormalities. The findings were considered unremarkable and part of the background pathology of rats of this strain and age.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic analysis of the rat that died as a result of anesthesia during blood collection revealed minimal basophilic tubules in the kidney and a minimal increase of proteinaceous droplets in the tubuli. In the liver, mild congestion and mild centrilobular hypertrophy was observed. In addition a mild focal mononuclear cell inflammation was present. Microhaemorrhages were observed in the thymus and ectopic thymus was observed in the thyroid.
Microscopic analysis of the male that was found dead on day 4 of the mating period showed already autolytic changes in a number of organs. The intestines and the lymphoid organs were to autolytic to evaluate. Close to the prostate, moderate inflammation was observed around the urethra. The presumed ileac lymph node turned out to be an inflamed urethra, surround by inflamed fat. In addition, a large necrotic area was visible next to the urethra, showing signs of early abcess formation. The urinary bladder showed no abnormalities but in agreement with its enlargement as a result of the urine build-up, the wall of the urinary bladder was stretched. The kidneys showed mild multifocal mineralization. In addition, diffuse moderate epithelial vacuolation was observed. With respect to the cause of death, we hypothesize that as a result of the necrosis and inflammation around the urethra, the passage of urine was blocked, leading to the accumulation of urine in the urinary bladder and subsequent swelling of the kidney. When the flow of urine is obstructed, stones (calculi) are more likely to form which might explain the observed mild multifocal mineralization. The elevated pressure due to the obstruction may ultimately result in kidney failure and subsequent death. Because similar findings were not observed in other rats at any dose level (see below) the above findings were considered incidental findings, not related to the treatment.
Microscopic examination of surviving animals revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain and age.

Effect levels

Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analytical results

The test substance was homogeneously distributed in the diet at each dose level. The test substance was stable in the test diets under the experimental conditions (4-days storage in the animal room, or storage in a freezer for more than 5 weeks). The concentration of the test substance was close to intended (90-110%) for all diets at all dose levels, except for the mid dose diet (1500 mg/kg) prepared on 28 December 2015 that differed +14% from the nominal concentration and the high dose diet (4500 mg/kg) prepared on 22 January 2016 that differed -14% from the nominal concentration. Although the latter findings did not meet the acceptance criteria, these slight differences between the determined and the accepted levels are considered negligible.

Applicant's summary and conclusion

Conclusions:
In this repeated dose toxicity test, based on the absence of adverse effects the NOAEL was determined to be 4500 mg/kg diet for male and female rats (equivalent to an overall intake of at least 265 mg/kg bw/d in males and at least 328 mg/kg bw/d in females).
Executive summary:

In this GLP-compliant study performed according to OECD422, the possible effects of the test substance on general toxicity was examined in groups of 12 male and 12 female Wistar rats. The test substance was administered at constant concentrations in the diet at levels of 0 (control), 450, 1500 and 4500 mg/kg diet during a premating period of 2 weeks and during mating, gestation, until day 4 of lactation. These dietary levels provided an overall mean intake of at least 34, 115 and 328 mg/kg bw/d in females of the low-, mid- and high-dose group. In males (during the premating and the post mating period), the overall mean intake was 28, 93 and 265 mg/kg bw/d, respectively. Female animals were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after the mating period.

 

The content, homogeneity and stability of the test substance in the carrier were confirmed by analysis. There was no treatment-related mortality. Daily clinical observations did not reveal any

treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential of the test substance. There were no relevant differences in body weights or food consumption during the premating period, during the post-mating period in males, or in dams during the gestation period and the lactation period.

 

Haematology and clinical chemistry was conducted in 5 rats/sex/group at the end of the premating period. In females of the high-dose group, total white blood cell count, absolute lymphocyte count, cholesterol and phospholipids were increased compared to controls. Because these changes were within the range of historical control data and occurred in one sex only, they were not ascribed to treatment. The weight of the liver was increased in males of the high-dose group. In the absence of histopathological alterations or clinical pathology evidence of liver toxicity, this finding was considered an adaptive and non-adverse reaction to the test substance.

Macroscopic and microscopic examination did not reveal anytreatment-related abnormalities.

 

Because there were no adverse effects the NOAEL was placed at ≥ 4500 mg/kg diet (the highest concentration tested; equivalent to an overall intake of at least 328 mg/kg bw/d in females and at least 265 mg/kg bw/d in males).