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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 Aug 2012 to 15 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to OECD guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate
EC Number:
700-204-6
Cas Number:
1174627-68-9
Molecular formula:
C9H17NO3
IUPAC Name:
methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Rhodiasolv Polarclean

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France.
- Age at study initiation: approximately 6 weeks old.
- Weight at study initiation:mean body weight of 182 g for males and 149 g for females.
- Fasting period before study: no.
- Housing: The animals were pair-housed, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust (SICSA, Alfortville, France). Each cage contained an object for the environmental enrichment of the animals (rat hut). The cages were placed in numerical order on the racks. In week 6 all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%):50 ± 20%
- Air changes (per hr):approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 01 October 2012 To: 31 December 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item dose formulations were prepared for 6 to 8 days (10 days being the maximum authorized as demonstrated by stability results from Rhodia SAS project No. 41005298, 14-day Dose Range Finding toxicity study), stored at +4°C prior to use and protected from light and delivered in brown flasks.

ADMINISTRATION OF DOSING SOLUTIONS:
The dose formulations were administered by gavage using a plastic syringe fitted with a metal gavage tube, once a day, at approximately the same time. The quantity of dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 5 mL/kg/day was used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method consisted of sampling 1 mL of dose formulations and diluting it appropriately with diluent to reach the nominal concentration of injection. The diluted samples were analyzed by High Performance Liquid Chromatography with Ultra-Violet detection (HPLC-UV), bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
other: actual ingested without correction for active substance content
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of the OECD 422 study conducted in Wistar rats (Rhodia SAS project No. 41005299). In this OECD 422 study, there were no toxicologically significant effects up to 1000 mg/kg/day. Therefore, 1000 mg/kg/day was selected as the high dose-level for this study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals.
- Rationale for animal assignment (if not random): n/a
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): n/a
Positive control:
n/a

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays. Each animal was observed once a day, at approximately the same time, for the recording of
clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all the animals once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes and mucous membranes, occurence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) and bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- 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: n/a (gavage)

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: n/a

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n/a

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals on one occasion at the end of the treatment period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all animals at the end of the treatment period.
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 7.5.1.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all animals at the end of the treatment period.
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 7.5.1.2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional Observation Battery)
- Time schedule for examinations: once in week 12
- Dose groups that were examined: all animals
- Battery of functions tested:
1) Detailed observations: The following parameters were assessed and graded:
. in the cage: "touch escape",
. in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydria sis),
. in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsio ns, gait, arousal (hypo- and hyper-activity), posture, stereotypic behavior, breathing, ataxia and hypotonia.
2) Reactivity to manipulation and different stimuli:The following parameters measurements, reflexes and responses were recorded:
. touch response,
. forelimb grip strength,
. pupillary reflex,
. visual stimulus response,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot splay,
. rectal temperature (at the end of the observation).
3) For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.
Sacrifice and pathology:
GROSS PATHOLOGY: A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes (see table 7.5.1.3)
Statistics:
Citox software (version D.6) was used to perform the statistical analyses of body weight, food consumption, hematology and blood biochemistry data.
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths. Ptyalism was observed in animals given the test item at 300 or 1000 mg/kg/day. While test item treatment-related, this finding is commonly observed and was considered of minor toxicological significance.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths. Ptyalism was observed in animals given the test item at 300 or 1000 mg/kg/day. While test item treatment-related, this finding is commonly observed and was considered of minor toxicological significance.
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were slight increases in absolute and relative kidney weights in high-dose females. However, in the absence of correlating microscopic findings, these increases were considered to be of no toxicological significance.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
There were no unscheduled deaths. Ptyalism was observed in animals given the test item at 300 or 1000 mg/kg/day. While test item treatment-related, this finding is commonly observed and was considered of minor toxicological significance.

BODY WEIGHT AND WEIGHT GAIN:
There were no effects on mean body weight and mean body weight gain.

FOOD CONSUMPTION:
There were no effects on mean food consumption.

OPHTHALMOSCOPIC EXAMINATION:
There were no findings at the ophthalmology examination at the end of the treatment period.

HAEMATOLOGY:
There were no effects on mean hematology parameters.

CLINICAL CHEMISTRY:
There were no test item treatment-related effects.

NEUROBEHAVIOUR:
- Functional Observation Battery: There were no test item treatment-related effects.
- Motor activity: There were no relevant differences in rearing and horizontal movements in test item-treated groups in both males and females when compared with control group.

ORGAN WEIGHTS:
There were slight increases in absolute and relative kidney weights in high-dose females. However, in the absence of correlating microscopic findings, these increases were considered to be of no toxicological significance.

GROSS PATHOLOGY:
There were no test item treatment-related necropsy findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test item treatment-related microscopic findings.

Effect levels

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: - Ptyalism observed in animals given the test item at 300 or 1000 mg/kg/day. - Slight increases in absolute and relative kidney weights in high-dose females.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item Rhodiasolv Polarclean, was administered daily to Wistar rats, by oral gavage, at dose-levels of 100, 300 or 1000 mg/kg/day for 13 weeks in a GLP-compliant study in accordance with OECD Guideline 408.
Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day in the absence of test item treatment-related toxicologically significant findings at this dose-level.
Based on the results of this study, the test substance Rhodiasolv Polarclean is not classified for repeated dose toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.
Executive summary:

The systemic toxicity and potential adverse effects of Rhodiasolv Polarclean following subchronic exposure via oral administration (gavage) was investigated in rats in a GLP- compliant study in accordance with OECD Guideline 408.

Methods:

Three groups of ten male and ten female Wistar rats received the test item, Rhodiasolv Polarclean (batch No. CY-12018030), at dose-levels of 100, 300 or 1000 mg/kg/day. In addition, one group of ten males and ten females received the vehicle alone (drinking water treated by reverse osmosis) and acted as a control group. The test item or the vehicle was administered during a 13-week period by gavage under a constant dosage-volume of 5 mL/kg/day. The actual test item concentration in each administered dose formulation was determined in weeks 1, 4, 8 and 12 using a validated HPLC/UV analytical method. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed once before the beginning of the treatment period and then once a week. A Functional Observation Battery (FOB) was performed for all animals once in week 12. Body weight and food consumption were recorded once a week during the study. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and in control and high-dose groups on completion of the treatment period. Hematology and blood biochemistry were performed on all animals at the end of the treatment period. On completion of the treatment period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from all animals from groups 1 and 4 and on all macroscopic lesions.

Results:

The test item concentrations in the administered dose formulations analyzed in weeks 1, 4, 8 and 12 remained within an acceptable range of variations when compared to the nominal values.

There were no unscheduled deaths.

Ptyalism was observed in animals given the test item at 300 or 1000 mg/kg/day. While test item treatment-related, this finding is commonly observed and was considered of minor toxicological significance.

No test item treatment-related effects were noted duyring FOB. There were no relevant differences in rearing and horizontal movements in test item-treated groups in both males and females when compared with control group.

There were no effects on mean body weight, mean body weight gain and mean food consumption.

There were no findings at the ophthalmology examination at the end of the treatment period.

There were no test item-related effects on mean hematology or blood biochemistry parameters.

There were slight increases in absolute and relative kidney weights in high-dose females.

However, in the absence of correlating microscopic findings, these increases were considered to be of no toxicological significance.

There were no test item treatment-related necropsy findings.

There were no test item treatment-related microscopic findings.

For anatomic pathology, the NOAEL (No Observable Adverse Effect Level) in this study was considered to be 1000 mg/kg/day in male and female rats.

Conclusion:

Under the experimental conditions of this study, the No Observable Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day in the absence of test item treatment-related toxicologically significant findings at this dose-level.

Based on the results of this study, the test substance Rhodiasolv Polarclean is not classified for repeated dose toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.