Registration Dossier

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined 28-day repeated dose toxicity with reproduction/developmental screening test.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
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:
other: OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421
Version / remarks:
July 1995
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3550
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3050
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: yes
- Solubility and stability of the test substance in the vehicle: yes, the substance is not expected to disintegrate in the vehicle and under the test conditions used in the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogenized to a visual acceptable level
Species:
rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10-12 weeks
- Weight at study initiation: males 281-318 grams, females 195-230 grams
- Fasting period before study: no
- Housing: in Macrolon plastic cages; premating in groups of 5 animals/sex/cage; females were caged together with males on a one-to-one basis for mating; post-mating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed; pups were kept with the dam until termination. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: free acess to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiӓten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: at least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material 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 to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 January 2016 To: 01 March 2016
Route of administration:
oral: gavage
Details on route of administration:
plastic feeding tube
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/v) were prepared daily within 6 hours prior to dosing and were homogenized to a visual acceptable level.

VEHICLE:
Based on trial formulations performed at CR.
amount of vehicle (gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase. On the first occasion samples from dosing formulations were analyzed for homogeneity (low and high concentrations) and accuracy of preparation (all concentrations, including vehicle control). Samples from mid and high concentration dosing formulations prepared on the second occasion were additionally analysed for homogeneity and accuracy.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was equal or below 10%.
Analysis of stability of the test substance under test conditions was not performed. The substance is a compact mixed metal oxide produced in a high temperature calcination process and is not expected to disintegrate in the chosen vehicle and under the test conditions used in this study, based on the following reasons:
-the substance is considered insoluble in water
-experiences made during the development of an ICP-MS method for analysis of the test substance in test media, i.e. very harsh conditions (mixture of strong mineral acids in combination with microwave digestion) are needed to decompose the substance into its metal ions
-due to the inorganic nature of the substance, stability measurements, based on an element in the substance, are not meaningful.
Duration of treatment / exposure:
males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
females: 42-54 days (2 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)
Pups were not dosed directly.
Frequency of treatment:
once daily for 7 days per week
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:
Dose levels were based on results of a 10-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: daily

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

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 4 of treatment, females during days 3-6 of lactation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity/ hearing ability / grip strength / motor activity / pupillary reflex

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (according to guidelines)

HISTOPATHOLOGY: Yes (according to guidelines)
Other examinations:
organ weights (5 animals/sex/group) according to guidelines
Weights of epididymides and testes from all remaining males were recorded.
Statistics:
Statistical methods used to analyze the data:
-Dunnett-test (many-to-one t-test) based on a pooled variance estimatewas applied for the comparison of the treated groups and the control groups for each sex if the variables could be assumed to follow normal distribution.
-Steel-test (many-to-one rank test) if the data could not be assumed to follow a normal distribution.
-Fisher Exact-test was applied to frequency data.
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):
Hunched posture and/or piloerection were observed on two separate occasions in two control females. At the incidence observed and because of their short-term presence in controls only, these signs were considered of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occured during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of the treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 1000 mg/kg bw/day treated females (statistically significant) increase in number of red blood cells and increased values of Hb and Ht were observed when compared to controls. In these females MCH, MCV and MCHC values (red cell derived parameters) were similar in comparison with the other dose groups including controls. The statistically significant difference apparent for the number of red blood cells in 300 mg/kg bw/day treated females was considered to be the result of a relatively low variation in the individual values within the dose group and to have occurred by change.
In males treated at 300 mg/kg bw/day a lower Hb value was observed, and in males from the 300 and 1000 mg/kg bw/day groups (stat sign) higher APTT values were observed compared to controls.
In females, in dose group 100 mg/kg bw/day a (stat sign) higher relative lymphocyte number and lower number of neutrophilswere observed. Lower RDW values were observed in females of all treated groups; lower MCH value was observed at 100 mg/kg bw/day and increased PT at 1000 mg/kg bw/day (achieving stat sign in most cases when compared to controls).
These changes were considered to be of no toxicological relevance as they were the result of relatively high control values (RWD) or occurred in the absence of treatment-related distribution and remained within the range considered normal for rats of this age and strain.
In a single female in dose group 300 mg/kg bw/day a low number of white blood cells was observed together with an increased number of neutrophils and decreased number of lymphocytes. This observation is often related to a non-specific stress response, which may be supported by the observations in the thymus. Since this observation was limited to a single female only and showed no dose effect relationship these findings were considered of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean value for total bilirubin in females dosed 100 mg/kg bw/day (stat sign when compared to controls) was considered to have occurred by chance and to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The foreleg grip strength in females showed a relatively large difference between the treated groups and controls but in the absence of a dose-response relationship and absence of corroborative effect in the hindlegs this finding was considered of no toxicological relevance. Moreover, all fore leg grip strength values were within the normal limits.
The variation in motor activity did not indicate a relation with treatment, as all groups showed the a similar habituation profile.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Thymus weight (abs and rel) was slightly (stat sign) increased in high dose females compared to controls.
Slightly higher (stat sign) relative kidney weights were noted in mid and high dose males compared to controls.
The changes observed in thymus weights in females and kidney weights in males are within the the normal limits for rats of this age and strain and therefore not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
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 1000 mg/kg bw/day (highest dose tested)
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Analysis of dose preparation:

The mean recoveries of the QC samples (range 85 -104%) fell within the criterion of 85 -115%. It demonstrated that the analytical method was adequate for the determination of the test item in the study samples.

A small response of the test item was observed in the control formulation (maximum of 0.16% response ratio to group 100 mg/kg bw/day).

The concentrations analyzed in the formulations of group 2 (100 mg/kg bw/day) were in agreement with target concentrations (mean accuracy between 98%), and the formulation was homogeneous (coefficient of variation 3.4%).

The concentrations analyzed in the first formulation of group 3 (300 mg/kg bw/day) was not agreement with target concentration (mean accuracy 79%); in the second formulation of this group analyzed concentration was in agreement with target concentration (mean accuracy 97%), and the formulation was homogeneous (coefficient of variation 4.1%).

In the high dose group (1000 mg/kg bw/day) concentrations analyzed were in agreement with target concentration in the first end second formulation (mean accuracy 89% and 102%, respectively). The first formulation was not homogeneous (coefficient of variation 14%), whereas the second formulation was demonstrated to be homogeneous (coefficient of variation 2.3%).

In a 10 -Day range-finding study, 3 females/group were dosed with 500 or 1000 mg/kg bw/day. Mortality, clinical signs , body weights and food consumption,were recorded At necropsy all animals were subjected to an external, thoracic and abdominal examination, and organ weights of liver and kidney were measured.

In this range-finding study only clinical signs (temporary hunched posture and/or piloerection mainly between 1 to 3 hours after dosing) were observed. Based on these results, the dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.

Conclusions:
Treatment with L 125 PLUS by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in adverse effects up to and including the highest dose level tested. Based on these results, the NOAEL in this study is at least 1000 mg/kg bw/day.
Executive summary:

L 125 was tested in a 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422. L 125 PLUS was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for two weeks prior to mating, during mating, and up to termination (29 days). The females were exposed for two weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously.

No treatment related adverse effects were observed in the parental males and females up to and including the highest dose level tested (1000 mg/kg bw/day) as evidenced by the absence of signs of toxicity or adverse changes in clinical appearance, haematology, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination. Based on these results a NOAEL of at least 1000 mg/kg bw/day was derived.

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion