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Description of key information

Based on the results of the 90-day repeated dose study, the NOAEL was determined to be 1000 mg/kg bw/d for male and female Wistar rats.

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:
2015-05-18 and 2016-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study according to guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: 47 – 50 days old (male/female)
- Weight at study initiation: males: 178-219 g; females: 123-165 g
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % aqueous methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in 0.5 % methycellulose in a frequency based on the stability features of the test item in the vehicle (stable at room temperature at least for one day, at 5 +/- 3°C for 3 days).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore 0.5 % methycellulose was used for preparing formulations appropriate for oral administration. 0.5 % methycellulose is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored at 5 ± 3°C until the analysis. Formulation samples were diluted with acetonitrile and then analysed by a HPLC method.
Measured concentrations varied between 99 and 114 % of the nominal concentrations and all formulations were considered to be homogeneous.

HPLC conditions:
Detector: 222 nm
Column: Phenomenex, Luna 3μ C18 (2) 100A,
150 x 4.6 mm, 3 μm, No.: 637921-4
Mobil Phase: Acetonitrile : Water = 95 : 5 (v/v)
Flow Rate: 1.2 mL/min
Injection volume: 10 μL
Temperature: 25 °C
Retention time: 9.5 min ±5 %
Run time: 12 minutes
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week basis, every day at a similar time (+/- 2 hours)
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
other: nominal concentrations
No. of animals per sex per dose:
10 animals/sex in the control and dose groups; 5 animals/ sex in the control and high dose groups for recovery observations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 1000, 300 and 100 mg/kg bw/day is based on findings obtained in previous repeated dose toxicity studies with the test item in the rat (Reproduction/Developmental Toxicity Screening Test, Study no. 552.421.4150).
- Rationale for selecting satellite groups: five animals per sex in the control and in the high dose group (according to guideline)
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighing was performed on day 0, then weekly. Fasted body weight was measured on day of necropsy (Days 90 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION:
- The food consumption was determined on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period
- Dose groups that were examined: all control and high dose test animals prior to test termination (day 86)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90) and on recovery animals at the end of recovery period (day 118)
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90) and on recovery animals at the end of recovery period (day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (day 84)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, including organ weights (see table No. 4); All animals were necropsied one day after the last treatment (main groups) or after four weeks recovery period (recovery groups).
HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups.
Statistics:
Statistical analysis was done for the following data: body weight, food consumption, hematology, blood coagulation, clinical chemistry and organ weight data.
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
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:
no effects observed
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
No animals died during the course of the study. Toxic signs related to the test item were not detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations and in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period.

BODY WEIGHT AND WEIGHT GAIN
The body weight development of male and female animals was undisturbed in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods.

FOOD CONSUMPTION
The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 mg/kg bw/day) during the entire observation period (treatment and recovery periods).

OPHTHALMOSCOPIC EXAMINATION
There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day).

HAEMATOLOGY
Hematology examinations did not reveal test item related changes in the evaluated hematological parameters (1000, 300 and 100 mg/kg bw/day).

CLINICAL CHEMISTRY
Pathological changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 1000, 300 and 100 mg/kg bw/day.

NEUROBEHAVIOUR
Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 1000, 300 or 100 mg/kg bw/day).
The behavior and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

ORGAN WEIGHTS
There were no test item related changes in weights of the examined organs at the end of the treatment or recovery periods at 1000, 300 and 100 mg/kg bw/day.

GROSS PATHOLOGY
Specific macroscopic alterations related to treatment with the test item were not observed in male or female animals at 1000, 300 or 100 mg/kg bw/day at the terminal necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examinations did not reveal any test item related changes at 1000 mg/kg bw/day dose level.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed in the highest dose tested
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day for male and female Wistar rats.
Executive summary:

The objective of this study was to obtain information on the possible health hazards after repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in the high dose and control dose animals in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day doses corresponding to concentrations of 0, 200, 60 and 20 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. Each five animals/sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle (0.5 % aqueous methylcellulose) for the test item and its sufficient stability in the vehicle was analytically verified up front. Recovery of the test item was 94 and 98 % of nominal concentrations at approximately 1 and 200 mg/mL in 0.5 % aqueous methylcellulose, respectively. Thus, it was proved to be stable in a refrigerator (5 ± 3°C) of at least 3 days. Concentrations of the test item in the dosing formulations varied between ranges of 99 % and 114 % of nominal concentrations at each analytical occasion confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups.

The results of study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. The body weight development of male and female animals was not affected in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). Hematology examinations did not reveal clear test item related adverse changes in the evaluated parameters (1000, 300 and 100 mg/kg bw/day). Adverse findings were not detected in any of the clinical chemistry parameters in male or female animals at 1000, 300 and 100 mg/kg bw/day. No specific macroscopic alterations or changes in weights of the examined organs at the end of the treatment or recovery periods related to treatment with the test item were observed in male or female animals at 1000, 300 or 100 mg/kg bw/day. Histopathological examinations did not reveal any test item related changes at the 1000 mg/kg bw/day dose level.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day for male and female Hsd.Han:Wistar rats.

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:
GLP and guideline 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

Key study

Repeated dose toxicity: oral (90 days)

A 90 -day repeated dose study with the test item was conducted according to OECD 408.

The test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day doses corresponding to concentrations of 0, 200, 60 and 20 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. Each five animals/sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle (0.5 % aqueous methylcellulose) for the test item and its sufficient stability in the vehicle was analytically verified up front. Recovery of the test item was 94 and 98 % of nominal concentrations at approximately 1 and 200 mg/mL in 0.5 % aqueous methylcellulose, respectively. Thus, it was proved to be stable in a refrigerator (5 ± 3°C) of at least 3 days. Concentrations of the test item in the dosing formulations varied between ranges of 99 % and 114 % of nominal concentrations at each analytical occasion confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups.

The results of study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (1000, 300 or 100 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. The body weight development of male and female animals was not affected in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The mean daily food consumption was comparable in animals of the control and test item treated groups (1000, 300 and 100 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). Hematology examinations did not reveal clear test item related adverse changes in the evaluated parameters (1000, 300 and 100 mg/kg bw/day). Adverse findings were not detected in any of the clinical chemistry parameters in male or female animals at 1000, 300 and 100 mg/kg bw/day. No specific macroscopic alterations or changes in weights of the examined organs at the end of the treatment or recovery periods related to treatment with the test item were observed in male or female animals at 1000, 300 or 100 mg/kg bw/day. Histopathological examinations did not reveal any test item related changes at the 1000 mg/kg bw/day dose level.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day for male and female Hsd.Han:Wistar rats.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
90-day repeated dose toxicity study is used for chemical safety assessment.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
According to REACH Regulation (EU) No 1907/2006, Annex IX, 8.6.1 the test item should be tested for repeated dose toxicity via the most appropriate route. Testing by inhalation route can be considered as an inappropriate route as exposure and/or systemic availability via inhalation route is not expected. The vapour pressure of the test item is very low (< 1.3 x 10E-4 Pa at 25°C), therefore exposure of the substance as vapour is very limited. Furthermore, when used in its solid scales/ flakes form, substance particles would be probably coughed or sneezed out of the respiratory tract and if inhaled will not dissolve into the mucus lining of the respiratory tract due to the poor water solubility and the molecular size of the substance, thus, absorption is expected to be negligible. Therefore, testing by inhalation route additional to the available oral toxicity study is not required.
However, for risk assessment requirements, the DNEL for inhalation systemic effects was determined to be 10 mg/m³ the general dust limit (as the DNEL based on extrapolation of the oral toxicity study result was much higher than the general dust limit. For more information please refer to section 7 “ “Toxiclogical information”).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
According to REACH Regulation (EU) No 1907/2006, Annex IX, 8.6.1 the test item should be tested for repeated dose toxicity via the most appropriate route. Testing by dermal route can be considered as an inappropriate route as exposure and/or systemic availability via dermal route is not expected. The physiochemical properties of the substance (high logPow, very low water solubility and molecular size) will hinder dermal absorption. Furthermore, results of the dermal toxicity study, sensitization test and no skin irritation properties, support the assumption that no dermal absorption is negligible (please refer to the toxicokinetic statement in IUCLID section 7.1). Therefore, testing by dermal route additional to the available oral toxicity study is not required and risk assessment is based on the oral study.

Justification for classification or non-classification

Based on the results of the repeated dose testing the test item was not classified and labelled for repeated dose toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).