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Description of key information

A 90-day study was performed with URALAC P 1920C according to OECD/EC guidelines and GLP principles. It is noted that the study was performed as a data requirement for a registration in China.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2015 - 15 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The sub-chronic study was performed to fill the data requirements of a China registration dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
dd 03 November 2015
Limit test:
no
Specific details on test material used for the study:
Purity/composition correction factor: no correction factor required
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
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 142-173 g (males), 116-143 g (females)
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex in Macrolon cages. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum, except during locomotor activity monitoring and overnight before blood collection
- Water: tap water ad libitum, except during locomotor activity monitoring
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

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

IN-LIFE DATES: 14 April to 14 July 2015 (males); 14 April to 15 July 2015 (females)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing (stored at room temperature) and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (factor 1.036). The formulations were heated to a maximum of 83°C for a maximum of 1 hour under nitrogen to obtain visual homogeneity. (only on Day 1: 1 hour and 20 minutes and for 3 days without nitrogen, see deviation). Formulations were allowed to cool down below 40°C before dosing. All test item preparations appeared as white suspensions.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): The choice was based on trial formulations performed at WIL Research Europe and on information provided by the Sponsor.
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations), in Weeks 1, 4 and 13. Accuracy of the highest concentration was also analyzed in Week 6. Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions were analyzed in Week 1 (highest and lowest concentration) and Week 4 and 6 (highest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In order to set the dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing, a dose range finding study was performed.
Three female rats (6-8 weeks old) per group were exposed to 500 and 1000 mg/kg body weight for 14 days (10 mL/kg body weight/day). The animals were observed daily, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing for clinical signs and twice daily for mortality. Body weight and food consumption was monitored weekly. All animals were subjected to an external, thoracic and abdominal examination on Day 15 after the last observation of clinical signs (scheduled necropsy). No organs were fixed. Animals were not deprived of food prior to necropsy.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations (clinical signs and arena) were conducted and functional observations were started between 1-2 hours after dosing. At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of
treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Following instillation of tropicamide solution both eyes were examined by means of an ophthalmoscope.
- Time schedule for examinations: at pretest: All animals; at Week 13: controls and high dose group animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study, before sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available
- How many animals: all
- All parameters according to guideline were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study, before sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available
- How many animals: all
- All parameters according to guideline were examined.

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: During Week 12-13 of treatment
- Dose groups that were examined: All groups, 5 animals /sex/group (starting 1-2 hours after dosing)
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength; motor activity test (total movements and ambulations were reported)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to the guidelines

ORGAN WEIGHTS: Yes (all major organs according to the guidelines)

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all control and high dose animals;
- all gross lesions.
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. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were noted during the observation period that were considered to be related to treatment. No abnormalities during weekly arena observations were noted during the observation period. Any clinical signs noted during the treatment period, including hunched posture and piloerection, occurred among all dose groups at low incidence and severity with no apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
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):
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight remained similar to the control level over the study period.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment. The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicological relevant changes were noted in haematological parameters. A statistically significant change in relative neutrophil counts was noted in males (decrease of 14%, 20% and 21 for the males dosed at 100, 300 and 1000 mg/kg bw/day compared to control males). As the average concentration in control males was highly influenced by a single male (which had a neutrophil concentration of 33.2%) and this effects was not seen in females, this was considered to be not toxicologically relevant. In addition, a reduction in red blood cells was found in males dosed at 300 mg/kg bw/day (-5% compared to controls), and reduced prothrombin time was seen in males dosed at 100 and 300 mg/kg bw/day (respectively -6% and -4% compared to the control males). Both these effects were small, not dose-related and not seen in females, therefore it was considered to be an incidental finding and not related to exposure to the test item. In females, an increase was seen in platelets at the two highest dose groups (both groups +15% compared to control females). This observation was influenced by a single control female with a low platelet count. Taking this into account and based on the fact that no other effects were seen in females, the increased number of platelets was not considered to be toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher alanine phosphatase activity was measured in males and females at 1000 mg/kg bw day (+45% for males and +12% for females compared to controls). Furthermore higher glucose level in exposed males (+6%, +8% and +34% in the males exposed to 100, 500 and 1000 mg/kg bw, respectively). In females, glucose levels were only statistically significantly increased in the group exposed to 500 mg/kg bw/day compared to controls (+30%; increase at low and high dose +5% and +6%, respectively). In absence of any other adverse findings these effects were not considered to be toxicologically relevant.
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):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. The motor activity measurements showed a similar motor activity habituation profile for all dose groups with a decreasing trend in activity over the duration of the test period. An apparent trend towards higher motor activity was noted in males at 1000 mg/kg bw/day. This finding was highly influenced by one male. Since this animal showed no corroborative findings in the parameters determined in this study and the change was only slight in nature, this apparent trend was considered not toxicologically relevant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related findings in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. Any changes in macroscopic examination were considered to be unrelated to treatment as these
occurred in the absence of a dose-related trend.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations. All histologic changes were considered to be incidental findings and 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:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including highest dose (1000 mg/kg bw/day) tested
Key result
Critical effects observed:
no

Results dose range finding study:

No mortality occurred. At 500 mg/kg bw/day, hunched posture was noted mostly at 1 and/or 3 hours after dosing in all three animals. At 1000 mg/kg bw/day, hunched posture was noted mostly at 3 hours after dosing (and/or sometimes after 1 hour) in all three animals. Salivation was noted on one day immediately after dosing in all three animals. No unexpected effects were seen on body weight and at necropsy.

Analytics:

The concentrations analysed in the test item formulations of all exposed groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 112%).

A small response at the retention time of the test substance was observed in one of the chromatograms of the control group formulation prepared for use in Week 1. It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. In all other formulations of the control group, no test substance was detected.

The formulations of the low dose group were homogeneous (i.e. coefficient of variation ≤ 7.3%). For the formulation of the high dose group prepared for use in Week 1, the coefficient of variation was above the criterion for homogeneity of 10% (i.e. coefficient of variation 15%). Measurements in Week 4 and Week 13 showed that formulations of the high dose group were homogeneous (i.e. coefficient of variation ≤ 5.5%).

Analysis of the low and high dose group formulations prepared in Week 1 yielded a relative difference of ≤ 8.4% after storage. Based on this, formulations at the entire range were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours. Analysis of the high dose group formulation prepared in Week 4 showed a significant decrease in concentration (relative difference of up to -17%). Additional analysis of the high dose group formulation prepared in Week 6 however confirmed stability of the high dose group formulations (relative difference -7.0%).

Conclusions:
Based on the results of a 90-day study performed according to OECD/EC guidelines and GLP principles, the NOAEL for repeated sub-chronic expsoure is found to exceed 1000 mg/kg bw/day, as no adverse effects were seen at any dose level in this study.
Executive summary:

A 90-day study was performed with URALAC P 1920C according to OECD/EC guidelines and GLP principles. Male and female rats were exposed for 90 consecutive days by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. Chemical analysis of the test item formulations and the control formulations showed that the rats were dosed correctly.

No mortality occurred during the study. No toxicologically significant changes were noted in any of the parameters determnined in this study, i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination. Clinical biochemistry revealed a higher alanine phosphatase activity and a higher glucose level in males at 1000 mg/kg bw/day. These changes were not supported by any corroborative changes in parameters determined in this study, including microscopic examination and therefore considered not toxicologically significant.

Based on these results it is concluded that the NOAEL for repeated sub-chronic exposure exceeds 1000 mg/kg bw/day, as no adverse effects were seen up to and including the highest dose used in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A reliable study is used (Klimisch 1 study)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 90-day study was performed with URALAC P 1920C according to OECD/EC guidelines and GLP principles. Male and female rats were exposed for 90 consecutive days by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. Chemical analysis of the test item formulations and the control formulations showed that the rats were dosed correctly.

No mortality occurred during the study. No toxicologically significant changes were noted in any of the parameters determined in this study, i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination. Clinical biochemistry revealed a higher alanine phosphatase activity and a higher glucose level in males at 1000 mg/kg bw/day. These changes were not supported by any corroborative changes in parameters determined in this study, including microscopic examination and therefore considered not toxicologically significant.

Based on these results it is concluded that the NOAEL for repeated sub-chronic exposure exceeds 1000 mg/kg bw/day, as no adverse effects were seen up to and including the highest dose used in this study.

Justification for classification or non-classification

The registered substance is not classified for repeated oral dose toxicity according to Regulation (EC) No 1272/2008.

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