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Toxicological information

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:
March 13, 2015 - Sep 06, 2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted October 03, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Crl: WI (Han)
Breeder: Charles River, Germany
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8w
- Weight at study initiation: 224 (m), 172 (f)
- Fasting period before study:
- Housing: grouped
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage, once daily, 7 times a week for 4 weeks.
The volume of administration was 10 mL/kg body weight. The volume of administration per
animal was calculated by means of the LIM-System.
Vehicle:
other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): lab standard vehicle
- Concentration in vehicle: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): -
- Purity: analytical grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The quantification of the test item in the
dosing formulations was performed using a HPLC method with
UV detection. During the course of the study dosing formulations
(including control) were sampled at two different time points.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily, 7 times a week for 4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
0 mf/kg: 20
50 mg/kg: 10
150 mg/kg: 10
450 mg/kg: 20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): random
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Mortality
Mortality of each animal was checked daily. The parameter was recorded with the LIM-System.

Clinical Signs
The behavior and appearance of each animal was checked daily. The parameter was recorded
with the LIM-System.

Body Weight
Body weight was recorded before treatment and thereafter weekly. The parameter was recorded
with the LIM-System.
Body weight gain is a calculated parameter (difference to baseline value) that was determined for
each animal. The parameter was calculated for different intervals, e.g. weekly or total, with the
LIM-System.

Food Consumption
Food consumption was recorded once a week by weighing the food per cage which had not been
consumed. The parameter was recorded with the LIM- System.

Water Consumption
Water consumption was recorded twice a week by weighing the water per cage which had not
been consumed. The parameter was recorded with the LIM- System.

Functional Observational Battery (FOB)
A functional observational battery (FOB) was performed in the numerical first 5 males and 5
females per group before the first exposure, a week thereafter (approximately 1 hour after
treatment), and in week 4 (after motor activity measurements).
In week 4, the FOB was performed after the motor activity measurement. Animal numbers and
micro transponder implants were not identified for the laboratory staff performing the functional
observational battery. Therefore, the observers did not know to which treatment group the rats
belonged.

Motor Activity
In week 4, one hour after administration, the rats (the numerical first 5 males and 5 females per
group) were removed from their home cages and their motor activity was recorded in special
motor activity cages over 60 minutes at 5 minutes intervals.
The number of movements was evaluated by counting the number of interruptions of photo
beams (7 beams on the x-axis, 4 beams on the y-axis, 7 beams on the z-axis). This parameter is
called “counts” and given in numbers.
The system was also calculating the “on-time” (minutes), “off-time” (minutes), the “total
distance” the animal was moving (meter), “rearing” (number), and “rearing time” (minutes). The
data were transferred into the LIM-System. Assignment of the rats to the individual
measurements followed a randomization schedule. On each measuring day, measurements were
conducted simultaneously in 10 rats.

Clinical Pathology
In weeks 5 and 7 approximately 2.5 mL blood were taken sublingually under inhalation
anesthesia (isoflurane) from the designated rats. The blood samples were divided for
hematological and clinico-chemical examinations. Before blood sampling the animals were kept
in metabolism cages for the collection of urine for approximately 18 hours without food.
Sacrifice and pathology:
The animals were necropsied, examined for gross pathological alterations, the weights of
selected organs were recorded and histotechnical procedures and histopathological examinations
were performed.
Statistics:
Body weight, body temperature, food and water consumption, organ weights, hematology,
clinical chemistry, motor activity, functional observational battery (numerical parameters):
To compare the treatment groups with the control group, the following statistical procedures
were applied separately for each sex and each measuring point. To take the number of dose
groups into account all the test procedures used maintain a multiple significance level of =
0.05.
Absolute body weight, body weight gain (differences to baseline values on day 1), body
temperature, food and water consumption, organ weights (relative and absolute), clinical
pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and
urine weight), motor activity (total time period of 60 minutes), the dose groups were compared
with those of the control, using the multiple two-sided Dunnett-Test (Dunnett 1955,1964).
For all numerical parameters of the FOB, dose groups and the control groups were compared,
using the non-parametric 2-sided Kruskal-Wallis test (Hollander, 1973) followed by the
Wilcoxon-test rank sum test (Hollander, 1973).
For all parameters (except urinalysis) mean values, standard deviation, and number of animals
(N) were calculated.
For each numerical parameter of the FOB also minimum, maximum, median, and the frequencies
of scores were calculated. The descriptive parameters were listed as incidences per treatment
group in a table.
Software: Body weight, body temperature, food and water consumption, organ weights,
hematological parameters, clinico-chemical parameters (except the urinalysis parameters), motor
activity and the FOB parameters (except the frequencies of scores) were evaluated within the
LIM-System. The urinalysis examinations will be recorded with the LIM-System.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males, no statistically significant differences of body weight between dose groups and control
were observed. On day 28, a slightly lower body weight of approximately -3% was seen in 450
mg/kg/d males. This very slight effect was not statistically significant and no clear dosecorrelation
of effects was seen. Therefore, the observation was not considered treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was slightly increased at 450 mg/kg/d in weeks 1 and 3 in males, and at 150
mg/kg/d in week 1 in females. A decrease of -9.8% was observed in 450 mg/kg/d females in
week 1. The changes were statistically significant.

In week 1, the food consumption decrease in 450 mg/kg/d females correlated with the body
weight gain decrease and is therefore considered treatment-related. However, in males no
correlation of food consumption to body weight gain was seen, therefore, the change was not
considered to be treatment-related.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain was slightly lower in all male treatment groups compared to control. The
decreases were between -1.2 and -13.6% during the treatment period and -1.5 to -7.4% during
the recovery period (control and high dose). The changes were not statistically significant and no
clear dose-correlation of effects was seen. Therefore, the observation was not considered
treatment-related.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In males, water consumption showed a slight decrease (statistically significant) at 50 mg/kg/d
from day 1-4 and day 14-17. However, no dose-correlation of effects could be observed.
Therefore, these effects are not considered treatment related.
During the recovery period (day 28 to 42) no differences between the 450 mg/kg/d group and
control were observed.

In females, at 450 mg/kg/d water consumption was slightly increased, mostly statistically
significantly. At 50 mg/kg/d a slightly increased water consumption (statistically significantly)
was seen from day 1-4. No correlation with changes in urinalysis, serum clinical chemistry
parameters, or histopathological changes in the urinary tract were observed. Therefore, the
finding was considered to be treatment-related but not toxicologically relevant.
During the recovery period (day 28 to 42) no differences between the 450 mg/kg/d group and
control were observed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
All designated animals were investigated in the FOB before treatment on day -1, then on days 7
and 28 of treatment.
No treatment-related changes in autonomous and sensomotoric domain were observed. Of the
neuromuscular system parameters, mobility was statistically significantly decreased at 150 mg/kg/d
in males on day 28, no other parameters were changed. Of the central nervous system
parameters arousal and number of raisings were statistically significantly decreased in 150
mg/kg/d males on day 28, no changes of other parameters were seen including internal body
temperature and additional observations.
Since no clear dose-correlation of effects was seen, the changes in single parameters at the mid
dose level in the FOB are not considered to be treatment-related.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Oral administration of 50, 150 or 450 mg/kg of test item for 4 weeks revealed
minimal treatment-related single cell necroses of tubular epithelial cells in the renal cortex of
single males dosed with 450 mg/kg. After two weeks without treatment, full recovery of the
minimal kidney lesions was shown.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
food consumption and compound intake
water consumption and compound intake
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study the NOAEL (no observed adverse effect level) was established at 450 mg/kg/d, the NOEL (no observed effect level) was considered to be 50 mg/kg/d.
Executive summary:

Objective

The test item is an industrial chemical which needs to be registered in the European Union according to Annex VIII of the REACH regulation (>10-100 tons per annum). Such registrations require data on repeat dose toxicity, i.e. a subacute toxicity study in rats with oral

administration.

Study Design

The test item was administered orally by gavage, once daily 7 times a week for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 50, 150 or 450 mg/kg body weight. A similarly constituted control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium).

The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period 10 (5 male and 5 female) rats per group were scheduled for necropsy. The remaining rats of groups 1

and 4 were scheduled for a 2-week recovery period.

At the end of the treatment or recovery periods, all animals were subjected to a detailed necropsy and selected organ weights were recorded. Histopathological examinations were performed on selected organs of control and high dose animals (end of treatment period) and kidneys of

intermediate and recovery groups.

Results

Formulation analysis revealed that the dose groups were sufficiently exposed to the test item: The animals received mostly the anticipated concentrations (85 – 115%), with the exception of 3 samples with concentrations that were slightly above the predefined acceptance limit. This was

not considered to affect the validity and integrity of the study. No test item was detected in the control formulations.

All rats survived the treatment or recovery period until their scheduled necropsy. No treatmentrelated clinical signs were observed in males and females.

No treatment-related relevant changes of body weight were observed during the dosing and recovery period in males and females between dose groups and control. Body weight gain was slightly reduced in 450 mg/kg/d females throughout the dosing period between -35.2% (week 1)

and -17.2% (week 4) with statistical significance in week 3 (-24.7%). In week 1 this correlated with a reduced food consumption by -9.8% (statistically significant) and is considered to be treatment-related.

Water consumption showed no treatment-related changes in males at any dose level compared to control. In 450 mg/kg/d females water consumption was slightly increased, mostly statistically significant, compared to control during the treatment period. At 50 mg/kg/d a slightly increased water consumption (statistically significantly) was seen from day 1-4. No correlation with changes in urinalysis, serum clinical chemistry parameters, or histopathological changes in the urinary tract were observed. Therefore, the observation is considered treatment-related but not toxicologically relevant.

In the functional observational battery (FOB) no clearly treatment-related changes in autonomous, sensomotoric, neuromuscular, and central nervous system including body temperature measurements were observed. Measurement of motor activity revealed no treatmentrelated

changes in males, and a slight decrease of total distance and rearing numbers in females at 150 mg/kg/d and 450 mg/kg/d. All other motor activity parameters in females – number of counts, on-time, off-time, rearing time, did not show clear treatment-related differences of treatment groups versus control. Therefore, the slight effect on total distance and rearing number in females was considered to be treatment-related but not toxicologically relevant.

Evaluation of hematology, coagulation, clinical chemistry and urinalysis parameters at the end of the treatment and recovery period did not reveal any treatment related findings.

Necropsy revealed no treatment-related organ lesions in male and female rats of all dose groups at the end of treatment period, and in high dose animals at the end of the recovery period.

Determination or body and organ weights revealed no treatment-related weight changes in rats of both genders of all dose groups at the end of treatment period, and in high dose animals at the end of the recovery period.

Histopathology showed minimal treatment-related lesions in the kidneys of single male rats dosed with 450 mg/kg/d. Two out of 5 males showed minimal degenerative lesions - single cell necroses of tubular epithelial cells in the cortex. At 150 and 50 mg/kg/d no treatment-related lesions were found. At the end of the recovery period the full recovery of the minimal kidney lesions in single males was shown.

Conclusions

Daily oral administration of 50, 150 or 450 mg/kg/d of the test item to rats for 4 weeks was well tolerated. The slight reduction of body weight gain together with reduced food consumption observed in 450 mg/kg/d females after 1 week of dosing, the slightly increased water consumption in 450 mg/kg/d females during the treatment period, and the slightly changed motor activity parameters in 150 and 450 mg/kg/d females were considered to be treatment related but not adverse.

In histopathology minimal treatment-related single cell necrosis of tubular epithelial cells in the renal cortex of single males dosed with 450 mg/kg/d were observed. Full recovery of the minimal kidney lesions in single males was shown after a two week treatment-free period. The minimal kidney lesions were therefore not considered adverse. Under the conditions of the present study the NOAEL (no observed adverse effect level) was established at 450 mg/kg/d, the NOEL (no observed effect level) was considered to be 50 mg/kg/d.