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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015 - March 2016
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
Remarks:
endpoint 7.5.1
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
with reproduction/developmental toxicity sceening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The information is used to read across to Hexalon
Reason / purpose for cross-reference:
reference to same study
Remarks:
endpoint 7.8.1
Reason / purpose for cross-reference:
reference to same study
Remarks:
endpoint 7.8.2
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males 142-184 grams, females 112-139 grams
- Fasting period before study: no
- Housing: during premating in groups of 5 animals/sex/cage in Macrolon plastic cages; during mating females were caged together with males on a one-to-one-basis in Macrolon plastic cages; during post-mating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed in Macrolon plastic cages; during lactation pups were kept with the dam until termination in Macrolon plastic cages
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY:
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage. The same diets remained in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 November 2015 To: 11 March 2016
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared up to 3 weeks in advance of first use.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 8 days for supplementing food during the respective food consumption measurement interval.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on diet samples taken on a single occasion during the treatment phase, according to a validated method. Samples of diet preparations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in diet over 8 days at room temperature and 3 weeks in the freezer were also determined for 250 ppm diets. Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15,000 ppm was previously confirmed for at least 3 weeks in the freezer (≤-15°C) and for at least 8 days at room temperature (method development and validation study). The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy for selected females and until the day of necropsy for non-selected females).
Frequency of treatment:
ad libitum
Dose / conc.:
250 ppm
Dose / conc.:
700 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose levels were based on results of a 14-day dose range finding study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of exposure and weekly thereafter; mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males were tested during Week 13 of treatment and the selected females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling)
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: General reproduction data, litter and pup examination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines
Other examinations:
the following organ weights from 5 animals/group were recorded:
Adrenal glands, Brain -cerebellum, midbrain, cortex, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid

from all remaining males; epididymides and testes
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only clinical finding noted consisted of scabs on the back in a single female at 250 ppm. This incidental finding was not related to treatment and within the range of background findings expected for rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
One female at 250 ppm was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pre-mating body weight gain of females at 2000 ppm was slightly lower compared to controls (percentual difference in mean body weight gain was about 10% in most weeks, being statistically significant at Days 50 and 71).
Throughout the post-coitum and lactation periods, mean body weights of females at 2000 ppm were statistically significantly lower compared to controls (relative differences from controls were about 10%). However, body weight gain values during the post-coitum and lactation periods at 2000 ppm were considered similar to controls.
No treatment-related changes in body weights or body weight gain were noted up to 2000 ppm in males and up to 700 ppm in females.
Overall the decrease in body weight (gain) can be attibuted to decreased food consumption and without any other signs, these were not considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Throughout the post-coitum and lactation period, females at 2000 ppm had an approximately 30-40% lower food intake than controls. Food consumption after allowance for body weight showed a similar pattern, but was not statistically significantly lower during lactation. During premating, food consumption before and after allowance for body weight was similar to control levels.
No treatment-related changes in food consumption before or after allowance for body weight were noted up to and including 2000 ppm in males and up to and including 700 ppm in females.

The mean daily intake of the test item per kg body weight during the different phases of the study:

Mean test item intake (mg test item/kg bw/day)
250 ppm 700 ppm 2000 ppm
Males
Pre-mating 19 52 153
Mating 16 42 137
Females
Pre-mating 21 59 168
Post-coitum 32 87 184
Lactation 44 129 280

Food efficiency:
not examined
Description (incidence and severity):
Effects on food consumption are observed, which are likely due to the palatability of the substance, which is not considered an adverse effect.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a doserelated response.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights were noted in the 2000 ppm treated males (absolute and relative to body weight). Mean relative weight was approximately 13% higher than controls.
Test item-related higher kidneys weights were noted in the 700 and 2000 ppm treated males (relative to body weight at 700 ppm and both absolute and relative to body weight at 2000 ppm). Mean relative weight was approximately 13% and 15% higher than controls at 700 and 2000 ppm, respectively.
Test item-related higher kidneys weights were also noted in the 250, 700 and 2000 ppm treated females (relative to body weight at 250 and 2000 ppm and both absolute and relative to body weight at 700 ppm). Mean relative weights were approximately 13%, 21% and 16% higher than controls at 250, 700 and 2000 ppm, respectively. No dose-related trend was apparent.
There were no other test item-related organ weight changes. All other organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of the liver was noted in 2/10 males at 2000 ppm. This macroscopic finding was considered to be test item-related.
The remainder of the recorded macroscopic findings were within the range of background findings encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: hepatocellular hypertrophy in 4/5 males and 4/5 females at 2000 ppm up to slight degree.
- Kidneys: increased incidence and severity of hyaline droplet accumulation in males at 2000 ppm up to moderate degree, accompanied by an increased incidence and severity of tubular basophilia up to slight degree at 2000 ppm and tubular granular casts in a single male treated at 2000 ppm at minimal degree.
- Urinary bladder: Minimal to slight hypertrophy of the urothelium in females at 2000 ppm.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
diet 700 ppm
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
137 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Accuracy of preparation: The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations, with mean accuracies between 87% and 114%. No test item was detected in the Group 1 diets.

Homogeneity: The diets at 250 and 2000 ppm were homogeneous with coefficient of variation of 2.0% and 5.4%, respectively.

Stability: Diets at 250 ppm were stable when stored at room temperature under normal laboratory light conditions in an open container for at least 8 days, with difference between the stored and freshly taken samples was -7.7%. The relative difference of diet samples at low level 250 ppm for 3 weeks in the freezer (≤ -15°C) stability test was slightly above the criterion (- 13%). The decrease was considered not to be due to instability of the samples since the mean accuracy of the stability samples was comparable to the accuracy of the quality control samples at the same concentration level which were analysed together with the 3-week stability samples. Furthermore, since the deviations were considered small and accuracies after storage were within the range of 80 and 120%, the diets were considered stable. Based on this, the diets from 250 ppm to 15000 ppm were found to be stable during storage at room temperature under normal laboratory light conditions with open container for at least 8 days and in a freezer (≤ -15°C) for at least 3 weeks.

Conclusions:
At the high and sometimes mid dose treatment related effects were noted on body weight (gain) food consumption (both decreased), adaptive liver changes were seen. In males hydrocarbon nephropathy in kidneys was seen and related granular casts. These effects were not considered adverse or not relevant for humans. The toxicological relevance of the slight to mild hypertrophy in urine bladder in females at 2000 ppm (actual, 137 mg/kg bw) was discussed and it was decided to use this effect for the overall parental NOAEL for systemic effects of 700 ppm (actual 42 mg/kg bw).
Executive summary:

Introduction and method: The substance was administered via the diet to male and female Wistar Han rats at dietary concentrations of 250, 700 and 2000 ppm (10 rats/sex/dose level) according to OECD 422 Guideline (nominal ca: 133, 47 and 17 mg/kg bw). Concurrent controls (10 rats/sex) received basal diet without test item. Males were exposed for 10 weeks prior to mating, during mating, and up to termination (for 91 days). The females were exposed for 10 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 103 - 114 days). Analysis of diet preparations showed that the diets were prepared accurately and homogenously, and were stable during storage at room temperature under normal laboratory light conditions in an open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

The food and test substance intake of 250, 700 and 2000 ppm resulted in 16, 42 and 137 mg/kg bw, using the lower and thus most conservative values of males and females.

Results:Parental effects: There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

Heamatology and clinical biochemistry parameters: No adverse effects were observed.

Organ effects: liver: Relative liver weight was increased in males and females with 13% and macroscopic liver enlargement was seen at the high dose in some males. Microscopically a treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

Kidney: In males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

At 2000 ppm (ca 137 mg/kg bw) an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats and not in higher mammals, including man (Ref. 6, see end of section). In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. The granular casts and the increase in tubular basophilia are considered adverse for rats.

Microscopic examination in female urinary bladder revealed minimal to slight hypertrophy of the urothelium treated at 2000 ppm (ca 137 mg/kg bw). This test item-related change was considered to be adverse in this 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

Conclusion

Repeated dose systemic effects: The effects on the bladder in the females were considered adverse and therefore the overall NOAEL is set at 42 mg/kg bw.

Fertility: No fertility effects were observed up to and including 2000 ppm, the highest dose level tested. Based on the absence of adverse effects on fertility parameter, the NOAEL on fertility is >=2000 ppm (>=137 mg/kg bw) were seen.

Developmental toxicity: Developmental toxicity parameters were not affected up and including 2000 ppm (equivalent to 168 mg/kg bw, based on females only).Based on the absence of adverse effects on developmental toxicity parameters, the NOAEL developmental toxicity in this study is at least >=2000 ppm (>=168 mg/kg bw).

Reason / purpose for cross-reference:
reference to same study
Remarks:
endpoint 7.8.1
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This information is used for read across to Hexalon.
Reason / purpose for cross-reference:
reference to same study
Remarks:
endpoint 7.5.1
Reason / purpose for cross-reference:
reference to same study
Remarks:
endpoint 7.8.2
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Health Effects Test Guideline OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/D evelopmental Toxicity Screening Test.
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males 142-184 grams, females 112-139 grams
- Fasting period before study: no
- Housing: during premating in groups of 5 animals/sex/cage in Macrolon plastic cages; during mating females were caged together with males on a one-to-one-basis in Macrolon plastic cages; during postmating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed in Macrolon plastic cages; during lactation pups were kept with the dam until termination in Macrolon plastic cages
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment
DETAILS OF FOOD AND WATER QUALITY:
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillag
e. The same diets remained in the food hopper for a maximum of one week. On the day of weighing the
remaining food in the food hopper, remaining diet was replaced with new diet retained from the freezer
acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 18 November 2015 To: 11 March 2016
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared up to 3 weeks in advance of first use.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from S SNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 8 days for supplementing food during the respective food consumption measurement interval.
Details on mating procedure:
- M/F ratio per cage: 1;1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged (how): females were individually housed in Macrolon plastic cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on diet samples taken on a single occasion during the treatment phase, according to a validated method. Samples of diet preparations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in diet over 8 days at room temperature and 3 weeks in the freezer were also determined for 250 ppm diets. Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15,000 ppm was previously confirmed for at least 3 weeks in the freezer (≤-15°C) and for at least 8 days at room temperature (method development and validation study). The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination.
Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy for selected females and until the day of necropsy for non-selected females).
Frequency of treatment:
ad libitum
Dose / conc.:
250 ppm
Dose / conc.:
700 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose levels were based on results of a 14-day dose range finding study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of exposure and weekly thereafter; mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males were tested during Week 13 of treatment and the selected females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling)
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
Parameters examined in all/P male parental generations:
testis weight, epididymis weight, sperm motility and progressive motility were assessed, sperm smears were fixed from all samples and stained for possible future morphology analysis based on histopathology and sperm motility assessments. Based on the results of this range finding study, and in consultation with the sponsor, these samples were not analysed.
One testis and one epididymis was removed and kept in the freezer at ≤-15°C for possible future determination of sperm numbers based on histopathology and sperm motility assessments. Based on the results of this range finding study, and in consultation with the sponsor, sperm numbers were not determined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
-histopathology was performed according to Guidelines

the following organ weights from 5 animals/group were recorded:
Adrenal glands, Brain -cerebellum, midbrain, cortex, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid

from all remaining males; epididymides and testes
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic); mortality/viability, clinical signs, body weight, sex,

GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine in tergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups before planned necropsy / number of pups born alive) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only clinical finding noted consisted of scabs on the back in a single female at 250 ppm. This incidental finding was not related to treatment and within the range of background findings expected for rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
One female at 250 ppm was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pre-mating body weight gain of females at 2000 ppm was slightly lower compared to controls (percentual difference in mean body weight gain was about 10% in most weeks, being statistically significant at Days 50 and 71).
Throughout the post-coitum and lactation periods, mean body weights of females at 2000 ppm were statistically significantly lower compared to controls (relative differences from controls were about 10%). However, body weight gain values during the post-coitum and lactation periods at 2000 ppm were considered similar to controls.
No treatment-related changes in body weights or body weight gain were noted up to 2000 ppm in males and up to 700 ppm in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Throughout the post-coitum and lactation periods, mean body weights of females at 2000 ppm were statistically significantly lower compared to controls (relative differences from controls were about 10%). However, body weight gain values during the post-coitum and lactation periods at 2000 ppm were considered similar to controls.
No treatment-related changes in food consumption before or after allowance for body weight were noted up to and including 2000 ppm in males and up to and including 700 ppm in females.

The mean daily intake of the test item per kg body weight during the different phases of the study:
Mean test item intake (mg test item/kg bw/day)
250 ppm 700 ppm 2000 ppm
Males
Pre-mating 19 52 153
Mating 16 42 137
Females
Pre-mating 21 59 168
Post-coitum 32 87 184
Lactation 44 129 280
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a dose related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: hepatocellular hypertrophy in 4/5 males and 4/5 females at 2000 ppm up to slight degree.
- Kidneys: increased incidence and severity of hyaline droplet accumulation in males at 2000 ppm up to moderate degree, accompanied by an increased incidence and severity of tubular basophilia up to slight degree at 2000 ppm and tubular granular casts in a single male treated at 2000 ppm at minimal degree.
- Urinary bladder: Hypertrophy of the urothelium in females at 2000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The mating, fertility and conception indices, precoital time and number of corpora lutea and implantation sites were unaffected by treatment. All paired females had mated. One female at at 250 ppm, two females at 700 ppm and one female at 2000 ppm were not pregnant (as confirmed by negative Salewski staining). The incidence of non-pregnancy did not show any apparent dose-related trend, and as such this was considered to be unrelated to treatment.
The mean number of implantation sites at 2000 ppm appeared slightly lower than that in the control group. This finding was not considered toxicologically relevant because the difference was minor and not statistically significant and all pregnant females at 2000 ppm had normal numbers of implantation sites (between 7 and 13).
For 1 control female, i female from dose group 250 ppm and 1 female from dose group 2000 ppm the number of pups born was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 5 or 7 of lactation.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
reproductive toxicity
Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
diet 700 ppm
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs noted in pups that were found dead or went missing consisted of no or less milk in the stomach, dehydration, slow breathing and cold. Incidental clinical signs in surviving pups consisted of scabs or a wound on the snout or shoulder and a blue spot on the abdomen. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
One pup at 250 ppm was found dead at first litter check. During lactation, one pup of the control group, six pups at 250 ppm (from 3 litters), one pup at 700 ppm and two pups at 2000 ppm (2 litters) were found dead or went missing. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings in pups that were found dead included absence of milk in the stomach, dehydration, and beginning autolysis. Macroscopic findings in surviving pups were limited to scabs on the shoulder in one pup at 2000 ppm. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Generation:
F1
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no

Accuracy of preparation: The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations, with mean accuracies between 87% and 114%. No test item was detected in the Group 1 diets.

Homogeneity: The diets at 250 and 2000 ppm were homogeneous with coefficient of variation of 2.0% and 5.4%, respectively).

Stability: Diets at 250 ppm were stable when stored at room temperature under normal laboratory light conditions in an open container for at least 8 days, with difference between the stored and freshly taken samples was -7.7%. The relative difference of diet samples at low level 250 ppm for 3 weeks in the freezer (≤ -15°C) stability test was slightly above the criterion (- 13%). The decrease was considered not to be due to instability of the samples since the mean accuracy of the stability samples was comparable to the accuracy of the quality control samples at the same concentration level which were analysed together with the 3-week stability samples. Furthermore, since the deviations were considered small and accuracies after storage were within the range of 80 and 120%, the diets were considered stable. Based on this, the diets from 250 ppm to 15000 ppm were found to be stable during storage at room temperature under normal laboratory light conditions with open container for at least 8 days and in a freezer (≤ -15°C) for at least 3 weeks.

Conclusions:
For the substance a parental NOAEL for systemic effects of 700 ppm was derived, based on hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight). No reproduction toxicity was observed up to and including 2000 ppm, the highest dose level tested. Based on the absence of adverse effects on reproduction parameters, the NOAEL reproduction in this study is at least 2000 ppm.

Executive summary:

This summary is the same as presented in the repeated dose section.

Introduction and method: The substance was administered via the diet to male and female Wistar Han rats at dietary concentrations of 250, 700 and 2000 ppm (10 rats/sex/dose level) according to OECD 422 Guideline (nominal ca: 133, 47 and 17 mg/kg bw). Concurrent controls (10 rats/sex) received basal diet without test item. Males were exposed for 10 weeks prior to mating, during mating, and up to termination (for 91 days). The females were exposed for 10 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 103 - 114 days). Analysis of diet preparations showed that the diets were prepared accurately and homogenously, and were stable during storage at room temperature under normal laboratory light conditions in an open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

The food and test substance intake of 250, 700 and 2000 ppm resulted in 16, 42 and 137 mg/kg bw, using the lower and thus most conservative values of males and females.

Results:Parental effects: There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

Heamatology and clinical biochemistry parameters: No adverse effects were observed.

Organ effects: liver: Relative liver weight was increased in males and females with 13% and macroscopic liver enlargement was seen at the high dose in some males. Microscopically a treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

Kidney: In males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

At 2000 ppm (ca 137 mg/kg bw) an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats and not in higher mammals, including man (Ref. 6, see end of section). In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. The granular casts and the increase in tubular basophilia are considered adverse for rats.

Microscopic examination in female urinary bladder revealed minimal to slight hypertrophy of the urothelium treated at 2000 ppm (ca 137 mg/kg bw). This test item-related change was considered to be adverse in this 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

Conclusion

Repeated dose systemic effects: The effects on the bladder in the females were considered adverse and therefore the overall NOAEL is set at 42 mg/kg bw.

Fertility: No fertility effects were observed up to and including 2000 ppm, the highest dose level tested. Based on the absence of adverse effects on fertility parameter, the NOAEL on fertility is >=2000 ppm (>=137 mg/kg bw) were seen.

Developmental toxicity: Developmental toxicity parameters were not affected up and including 2000 ppm (equivalent to 168 mg/kg bw, based on females only).Based on the absence of adverse effects on developmental toxicity parameters, the NOAEL developmental toxicity in this study is at least >=2000 ppm (>=168 mg/kg bw).

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Health Effects Test Guideline OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
July 1995
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/D evelopmental Toxicity Screening Test.
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
EC Number:
944-482-9
Molecular formula:
C13H20O
IUPAC Name:
Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males 142-184 grams, females 112-139 grams
- Fasting period before study: no
- Housing: during premating in groups of 5 animals/sex/cage in Macrolon plastic cages; during mating females were caged together with males on a one-to-one-basis in Macrolon plastic cages; during postmating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed in Macrolon plastic cages; during lactation pups were kept with the dam until termination in Macrolon plastic cages
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access
to food for a maximum of 2 hours.
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY:
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage. The same diets remained in the food hopper for a maximum of one week. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 November 2015 To: 11 March 2016

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared up to 3 weeks in advance of first use.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 8 days for supplementing food during the respective food consumption measurement interval.
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on diet samples taken on a single occasion during the treatment phase, according to a validated method. Samples of diet preparations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in diet over 8 days at room temperature and 3 weeks in the freezer were also determined for 250 ppm diets. Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15,000 ppm was previously confirmed for at least 3 weeks in the freezer (≤-15°C) and for at least 8 days at room temperature (method development and validation study). The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy for selected females and until the day of necropsy for non-selected females).
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
ad libitum
Duration of test:
up to 114 days
Doses / concentrationsopen allclose all
Dose / conc.:
250 ppm
Dose / conc.:
700 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose levels were based on results of a 14-day dose range finding study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of exposure and weekly thereafter; mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked according to guidelines.

URINALYSIS: No

- Time schedule for examinations: selected males were tested during Week 13 of treatment and the select ed females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling)
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity

IMMUNOLOGY: No

OTHER: General reproduction data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test)
based on a pooled variance estimate was applied for the comparison of the treated groups and the co
ntrol groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal
distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine in
tergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Indices:
Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups before planned necropsy / number of pups born alive) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only clinical finding noted consisted of scabs on the back in a single female at 250 ppm. This incidental finding was not related to treatment and within the range of background findings expected for rats of this age and strain.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
One female at 250 ppm was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pre-mating body weight gain of females at 2000 ppm was slightly lower compared to controls (percentual difference in mean body weight gain was about 10% in most weeks, being statistically significant at Days 50 and 71).
No treatment-related changes in body weights or body weight gain were noted up to 2000 ppm in males and up to 700 ppm in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Throughout the post-coitum and lactation period, females at 2000 ppm had a notably lower food intake than controls (approximately 30-40% lower than controls). Food consumption after allowance for body weight showed a similar pattern, but was not statistically significantly lower during lactation. During premating, food consumption before and after allowance for body weight was similar to control levels.
No treatment-related changes in food consumption before or after allowance for body weight were noted up to 2000 ppm in males and up to 700 ppm in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a dose related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights were noted in the 2000 ppm treated males (absolute and relative to body weight). Mean relative weight was approximately 13% higher than controls.
Test item-related higher kidneys weights were noted in the 700 and 2000 ppm treated males (relative to body weight at 700 ppm and both absolute and relative to body weight at 2000 ppm). Mean relative weight was approximately 13% and 15% higher than controls at 700 and 2000 ppm, respectively.
Test item-related higher kidneys weights were also noted in the 250, 700 and 2000 ppm treated females (relative to body weight at 250 and 2000 ppm and both absolute and relative to body weight at 700 ppm). Mean relative weights were approximately 13%, 21% and 16% higher than controls at 250, 700 and 2000 ppm, respectively. No dose-related trend was apparent.
There were no other test item-related organ weight changes. All other organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of the liver was noted in 2/10 males at 2000 ppm. This macroscopic finding was considered to be test item-related.
The remainder of the recorded macroscopic findings were within the range of background findings encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: hepatocellular hypertrophy in 4/5 males and 4/5 females at 2000 ppm up to slight degree.
- Kidneys: increased incidence and severity of hyaline droplet accumulation in males at 2000 ppm up to moderate degree, accompanied by an increased incidence and severity of tubular basophilia up to slight degree at 2000 ppm and tubular granular casts in a single male treated at 2000 ppm at minimal degree.
- Urinary bladder: Hypertrophy of the urothelium in females at 2000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was 1/10 couple at 250 ppm with total litter loss.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were four couples without offspring: 1/10 couple of the 250 ppm group, 2/10 couples of the 700 ppm group and 1/10 couple of the 2000 ppm group. No abnormalities were seen in the reproductive organs which could account for their lack of healthy offspring. The incidence of couples without offspring did not show a dose-related trend, and as such this was considered unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
The mean number of females with living pups at the first litter check appeared slightly lower at 2000 ppm (this correlated with the apparent slightly lower number of implantation sites at 2000 ppm). No toxicological relevance was ascribed to this variation, since the difference from the concurrent control group was not statistically significant and values remained well within the range considered normal for rats of this age and strain.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
42 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
diet 700 ppm
Basis for effect level:
histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
One pup at 250 ppm was found dead at first litter check. During lactation, one pup of the control group, six pups at 250 ppm, one pup at 700 ppm and two pups at 2000 ppm were found dead or went missing. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
No developmental toxicity was observed up to the highest dose level tested (2000 ppm) equivalent to 168 mg/kg bw based on intake of females.
Executive summary:

The summary is the same as in the repeated and fertility section of the source substance Galbascone

Introduction and method: The substance was administered via the diet to male and female Wistar Han rats at dietary concentrations of 250, 700 and 2000 ppm (10 rats/sex/dose level) according to OECD 422 Guideline (nominal ca: 133, 47 and 17 mg/kg bw). Concurrent controls (10 rats/sex) received basal diet without test item. Males were exposed for 10 weeks prior to mating, during mating, and up to termination (for 91 days). The females were exposed for 10 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 103 - 114 days). Analysis of diet preparations showed that the diets were prepared accurately and homogenously, and were stable during storage at room temperature under normal laboratory light conditions in an open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

The food and test substance intake of 250, 700 and 2000 ppm resulted in 16, 42 and 137 mg/kg bw, using the lower and thus most conservative values of males and females.

Results:Parental effects: There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

Heamatology and clinical biochemistry parameters: No adverse effects were observed.

Organ effects: liver: Relative liver weight was increased in males and females with 13% and macroscopic liver enlargement was seen at the high dose in some males. Microscopically a treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

Kidney: In males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

At 2000 ppm (ca 137 mg/kg bw) an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats and not in higher mammals, including man (Ref. 6, see end of section). In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. The granular casts and the increase in tubular basophilia are considered adverse for rats.

Microscopic examination in female urinary bladder revealed minimal to slight hypertrophy of the urothelium treated at 2000 ppm (ca 137 mg/kg bw). This test item-related change was considered to be adverse in this 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

Conclusion

Repeated dose systemic effects: The effects on the bladder in the females were considered adverse and therefore the overall NOAEL is set at 42 mg/kg bw.

Fertility: No fertility effects were observed up to and including 2000 ppm, the highest dose level tested. Based on the absence of adverse effects on fertility parameter, the NOAEL on fertility is >=2000 ppm (>=137 mg/kg bw) were seen.

Developmental toxicity: Developmental toxicity parameters were not affected up and including 2000 ppm (equivalent to 168 mg/kg bw, based on females only).Based on the absence of adverse effects on developmental toxicity parameters, the NOAEL developmental toxicity in this study is at least >=2000 ppm (>=168 mg/kg bw).

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