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Description of key information

The Repeated dose 28-day oral toxicity study with GR-88 -0778 was performed by daily gavage in the rat, followed by a 14 -day recovery period according to the OECD Guideline No.: 407.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2015 to 13 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.
Qualifier:
according to guideline
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name (as stated in the report): GR-88-0778
Lot No: Batch 6
Aspect: Colourless liquid
Expiration date: March 29, 2017
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han) (outbred, SPF-Quality).
Details on species / strain selection:
- Rationale: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Total number of animals: 30 males and 30 females (females were nulliparous and non-pregnant).
- Age at start of treatment: Approximately 6 weeks.
- Identification: Earmark and tattoo.
- Acclimatization period: At least 5 days before the start of treatment under laboratory conditions
- Health inspection: Upon receipt of the animals
- Room conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tubes.
Vehicle:
corn oil
Details on oral exposure:
1 one dose volume 5 mL/kg body weight per animal
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
At least 28 days. Animals were dosed up to the day prior to necropsy of Main group animals.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Based on the results of an 8-day range finding study
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Based on the results of an 8-day range finding study
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Based on the results of an 8-day range finding study
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Based on the results of an 8-day range finding study
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
14d recovery group
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
14d recovery group
No. of animals per sex per dose:
30 males and 30 females (females were nulliparous and non-pregnant), 1 one dose volume 5 mL/kg body weight per animals.
Control animals:
yes, concurrent vehicle
Positive control:
none
Observations and examinations performed and frequency:
Mortality / Viability:
At least twice daily.

Clinical signs:
At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study (project 508338)). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

Functional Observations:
During Week 4 of treatment, the following tests were performed on all Recovery Group 1 and 4 animals and all Main Group 2 and 3 animals after dosing at no specific time point, but within a similar time period after dosing for the respective animals (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (project 508338)) (abbreviations mentioned in the respective tables indicated between brackets):
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.Based on (potential) treatment-related findings noted in motor activity at the end of treatment, the motor activity test was conducted for all Recovery Group 1 and 4 animals during the last week of the recovery period.

Body weights: Weekly.

Food consumption: Weekly.

Water consumption:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Sacrifice and pathology:
On the scheduled day of necropsy, animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem
examination.
Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy.
All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Other examinations:
see details below.
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 (Ref. 1; manyto-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 (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
− The Fisher Exact-test (Ref. 3) was applied to frequency data.
− The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. No findings were noted during the arena observations in this study.
Salivation seen after dosing among all animals dosed at 150 and 500 mg/kg was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test substance rather than a sign of systemic toxicity.
Scabs noted in one male at 500 mg/kg was only observed during the recovery period and therefore considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
At 500 mg/kg, males had lower body weight and reduced body weight gain during the treatment and recovery period (statistically significant on most occasions during the treatment period only), and appeared to show a slight dose-related trend across male dose groups. Weight gain compared to Day 1 of the recovery phase was similar between the control group and males at 500 mg/kg.
Body weights and body weight gain of females were considered to have been unaffected by treatment.
Since the body weight changes were slight in nature, these changes were not considered to be adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicological relevant changes in food consumption before or after correction for body weight were recorded.
The slightly higher relative food consumption in males at 500 mg/kg is considered to result from the lower body weights at this dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Slight changes in haematology parameters consisted of lower red blood cell count, haemoglobin and haematocrit levels, higher prothrombin time and/or lower platelet count in males and females at 500mg/kgbw/d. These changes suggest an effect on red blood cell metabolism. However these changes were slight in nature and not supported by clear morphological findings and therefore not considered to be adverse.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters were observed in animals at 500 mg/kg compared to control animals at the end of the treatment phase:
− Higher alanine aminotransferase activity (ALAT) in males and females
− Higher alkaline phosphatase activity (ALP) in males and females (not statistically significant for males)
− Higher total bilirubin in females (also statistically significant at 150 mg/kg)
− Higher albumin in males
− Higher bile acids in females
− Higher creatinine in males (also statistically significant at 150 mg/kg)
− Lower cholesterol in males (non-significant at 150 mg/kg)
− Higher potassium in males
− Lower chloride in males and females
At the end of a 14-day recovery period, higher potassium was recorded for males and females at 500 mg/kg.
A slight change in alanine aminotransferase activity (ALAT) of females at the end of recovery, is considered not toxicologically relevant since it was only a slight change and in revised direction compared to treatment period.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
For males and females at 500 mg/kg a lower motor activity was recorded. Since this lower motor activity was absent after a 14-day recovery period and was not supported by any other clinical signs or abnormalities during functional observations or morphological changes in neuronal tissues, the changes in motor activity were not considered to represent an adverse effect on neurobehaviour.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the 28-day treatment period, statistically significant higher liver weights were noted in males and females at 500 mg/kg (absolute: +27% and +37% and relative to body weights +33% and +34%, respectively) and in males at 150 mg/kg (relative: 10%). In females significant higher liver weights were noted after the 14-day recovery period (absolute: +12% and relative to body weights +16%). There were no statistically significant organ weight changes after the 14-day recovery period in males.
Any other differences, including those that reached statistical significance were considered not to be GR-88-0778-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related gross observations.
All findings were considered to be unrelated to treatment as these occurred in the absence of a doserelated trend and were in the expected range for these rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings after treatment with GR-88-0778 were noted in the thyroid gland and liver of males and females and kidneys of males.
A slightly increased incidence and severity of hypertrophy of follicular cells was recorded in the thyroid gland at 500 mg/kg. In males this was recorded in 5/5 animals (2 minimal, 3 slight) and in 3/5 females (2 minimal, 1 slight). After a 14-day treatment free period there was complete recovery for this finding.
The minimal severities up to 3/5 animals per group recorded in males of the remaining main and recovery Groups including controls were considered to be within background.

Hypertrophy of the follicular epithelium of the thyroid glands is often a secondary effect associated with centrilobular hypertrophy in the liver and subsequent increased thyroid hormone elimination (Ref. 9).
The slightly increased incidence and severity of follicular cell hypertrophy recorded in the thyroid gland in both sexes at 500 mg/kg after treatment for 28 days with complete recovery after a 14-day treatment free period is regarded to be an adaptive change and considered non-adverse (Ref. 10).

In the liver centrilobular hepatocellular hypertrophy was recorded in both sexes at a minimal degree in 1/5 males at 150 mg/kg and in 3/5 males and 4/5 females at 500 mg/kg. After a 14-day treatment free period there was complete recovery for the hepatocellular hypertrophy in females and partial recovery for the hepatocellular hypertrophy in males; this was still present in 1/5 males at 500 mg/kg.

In the kidneys hyaline droplet accumulation was recorded at an increased incidence and severity in main males at 500 mg/kg (5/5 males, 2 slight, 3 moderate). After a 14-day treatment free period there was complete recovery for this finding. The minimal or slight severities recorded in males of the remaining main and recovery Groups including controls were considered to be within background.
At 500 mg/kg the hyaline droplet accumulation in the kidney was accompanied by tubular degeneration/regeneration in 3/5 males (1 minimal, 2 slight), which showed complete recovery after
the 14-day treatment free period.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
Histopathological examination revealed changes in the thyroid gland and liver of males and females and kidneys of males.
The increased incidence and severity of hyaline droplet accumulation as recorded in the kidneys of males at 500 mg/kg likely represents alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 6).
The increased hyaline droplet accumulation at 500 mg/kg was accompanied by minimal to slight tubular degeneration/regeneration and was therefore considered to be an adverse finding.

Hypertrophy of the follicular epithelium of the thyroid glands is often a secondary effect associated with centrilobular hypertrophy in the liver and subsequent increased thyroid hormone elimination (Ref. 9).
The slightly increased incidence and severity of follicular cell hypertrophy recorded in the thyroid gland in both sexes at 500 mg/kg after treatment for 28 days with complete recovery after a 14-day treatment free period is regarded to be an adaptive change and considered non-adverse (Ref. 10).
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the morphological changes in the liver of males and females.
Critical effects observed:
no
Conclusions:
Based on the morphological changes in the liver of males and females and in the kidney of males a No Observed Adverse Effect Level (NOAEL) for GR-88-0778 of 150 mg/kg was established.
Executive summary:

The Repeated dose 28-day oral toxicity study was with GR-88 -0778 was performed by daily gavage in the rat, followed by a 14 -day recovery period according to the OECD Guideline No.: 407.

Based on the results of an 8-day range finding study the dose levels for this 28-day oral gavage study were selected to be 0, 50, 150 and 500 mg/kg.

The test substance, formulated in corn oil, was administered daily for 28 days by oral gavage to SPFbred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test substance in corn oil were prepared accurately and sufficient homogeneity was shown (at the lowest and highest level), and were stable over at least 6 hour.

Dose levels up to 500 mg/kg of GR-88-0778 were well tolerated by males and females. No mortality occurred and no clinical signs of toxicity were noted.

For males and females at 500 mg/kg a lower motor activity was recorded. Since this lower motor activity was absent after a 14-day recovery period and was not supported by any other clinical signs or abnormalities during functional observations or morphological changes in neuronal tissues, the changes in motor activity were not considered to represent an adverse effect on neurobehaviour.

Slight reduced body weight gain and lower body weight were observed in males at 500 mg/kg, which was not accompanied by reduced food consumption. No effects were noted in body weight or food consumption of females. Since the body weight changes were slight in nature, these changes were not considered to be adverse.

Histopathological examination revealed changes in the thyroid gland and liver of males and females and kidneys of males. The increased incidence and severity of hyaline droplet accumulation as recorded in the kidneys of males at 500 mg/kg likely represents alpha2uglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. This male rat specific protein is not present in female rats nor in higher mammals, including man.

The increased hyaline droplet accumulation at 500 mg/kg was accompanied by minimal to slight tubular degeneration/regeneration and was therefore considered to be an adverse finding.

Although no clear correlation with histopathological findings was noted a slightly higher creatinine and potassium level and lower chloride level were noted (predominantly in males) for which a relation to treatment could not be excluded. Hepatocellular hypertrophy in the liver combined with a weight increase of over 25% as recorded in males and females at 500 mg/kg is considered an adverse finding. There was partial recovery for this finding in males at 500 mg/kg and complete recovery in females of this dose level. Slight changes noted in clinical biochemistry, that could be related to these morphological changes, are higher alanine aminotransferase and alkaline phosphatase activity, higher total bilirubin, albumin, bile acids and lower cholesterol in males and/or females. The minimal hepatocellular hypertrophy recorded in one male at 150 mg/kg in absence of significant liver weight changes and in absence of any degenerative changes is considered to be non-adverse.

Hypertrophy of the follicular epithelium of the thyroid glands is often a secondary effect associated with centrilobular hypertrophy in the liver and subsequent increased thyroid hormone elimination. The slightly increased incidence and severity of follicular cell hypertrophy recorded in the thyroid gland in both sexes at 500 mg/kg after treatment for 28 days with complete recovery after a 14-day treatment free period is regarded to be an adaptive change and considered non-adverse.

Slight changes in haematology parameters consisted of lower red blood cell count, haemoglobin and haematocrit levels, higher prothrombin time and/or lower platelet count in males and females at 500 mg/kg. These changes suggest an effect on red blood cell metabolism. However these changes were slight in nature and not supported by clear morphological findings and therefore not considered to be adverse.

In conclusion, based on the morphological changes in the liver of males and females and in the kidney of males a No Observed Adverse Effect Level (NOAEL) for GR-88-0778 of 150 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
Guideline GLP study
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the data available only reversible and adaptive responses were found following exposure to Nympheal, and as such no classification is necessary according to the (EC) No 1272/2008 Regulation (CLP).