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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:
18 Oct 2018 - 18 Jan 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Testing was initiated as requested in ECHA decision TPE-D-2114359620-51-01/F. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed in such a way that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to
humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: yellow, viscous clear liquid
- Storage conditions: at 20 ±5 °C, in the dark
Specific details on test material used for the study:
Testing was performed with two batches of the test item. Both batches were aqueous solutions of the test item. Dosing was corrected for the solid content. The first batch, used until day 34, had a dry residue content of 47.2%, the purity correction factor applied was 2.10. The second batch, used from day 35 until the end of the study, had a dry residue content of 48%, the purity correction factor applied was 2.08.
Stability in water for at least 6 hours at room temperature under normal laboratory light conditions was previously confirmed over the concentration range 1.0 to 200 mg/mL, Charles River Project 518371. Stability in water for at least 7 days in the refrigerator was also previously confirmed over the concentration range 1.0 to 200 mg/mL, Charles River Project 518371.

Test animals

Species:
rat
Strain:
other: Crl: WI(Han) rats
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 146-179 g (males), 119-146 g (females)
- Fasting period before study: No; No food or water was supplied during locomotor activity monitoring (maximum of 2 hours) and before necropsy (with a maximum of 24 hours)
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV). During locomotor activity monitoring, animals were housed individually.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 47-68
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 Oct 2018 To:18 January 2019

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage (1.895 ml/kg/day each until Day 34 and 1.796 ml/kg/day from Day 35) 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. The dose volume for each animal was based on the most recent body weight measurement. The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were not stirred continuously during dose administration, but swirled shortly before dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed using a validated analytical procedure (Test Facility Study No. 518371). Duplicate sets of samples (approximately 500 mg) for each sampling time point were send to
the analytical laboratory for concentration and homogeneity analysis. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ±10%. Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 518371) demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Charles River Study 518371 (also summarized in Section 7.5.1).
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 28-day repeated dose OECD 422 study with oral exposure of Dehytron® DC in rats (Charles River Study No. 518366, also summarized in Section 7.5.1). In the OECD 422 study, it was observed that foam formation easily occurred with this test item in formulation. This foam formation caused regurgitation which was expected to have lead to premature deaths at 1000 mg/kg bw/day. Therefore, dose volumes were decreased compared to the OECD 422 study to minimize the chance of regurgitation.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality/ moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily (0 to 30 minutes after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing Period

WATER CONSUMPTION : Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottle

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during Pretreatment Period in all animals, and at the end of the Dosing Period in Week 13 in all controls and in all high dose group animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 10 controls (males and females), 10 females/ dose group and 9, 8 and 10 males dosed at 100, 300 and 1000 mg/kg bw/day, respectively. Missing data on males were related to technical errors.
- Parameters according to the guidelines were examined. Plasma was analyzed for Prothrombin Time and Activated Partial Thromboplastin Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters according to the guidelines were examined. Serum was analysed for thyroid hormone levels (Triiodothyronine (T3), Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH))

URINALYSIS: No

FUNCTIONAL TESTS: Yes
- Functional tests were performed on the first 5 animals/sex/group during Week 12. These tests were performed after completion of clinical observations. The following tests were performed: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity (total movements and ambulations).

IMMUNOLOGY: No

OTHER:
- Daily vaginal lavage was performed for all females from Week 11 (Day 71) up to and including Week 13 (Day 91). Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by
vaginal lavage.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all rats)
Complete necropsy examination of all animals was done, including evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Main organs were weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (all rats)
The tissues mentioned in the guideline were evaluated, including any gross lesions. For the testes of all males of control group and highest dose group detailed qualitative examination was made, taking into
account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate
or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.
Statistics:
Levene’s test were used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed according to the matrix below when possible, but exclude semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made: low dose group vs. control group, mid dose group vs. control group and high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100, 300 and 1000 mg/kg bw/day incidental abnormal breathing sounds, labored breathing, shallow breathing, increased and decreased respiratory rate were observed in one or two animals per group on several days between Days 35 and 90. Moreover, fur loss, hunched posture and/or erected fur were noted in one or two animals per group on several days between Days 35 and 90. As the observations occurred in one or two rats per dose group and/or the observations were transient and did not persist, the effects were not considered adverse. Salivation was seen among all animals at 300 and 1000 mg/kg bw/day between Days 3 and 92 and incidental ploughing was observed in all males and one female at 1000 mg/kg bw/day directly after dosing. These findings were not considered to be toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). These signs were considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain in females at 100 and 300 mg/kg bw/day and females up to 1000 mg/kg bw/day remained in the same range as controls over the study period. A lower body weight compared to control was observed for males at 1000 mg/kg bw/day from the second study week onwards (0.88x compared to controls, respectively, at end of treatment). A minimal lower body weight compared to control was observed in males at 100 and 300 mg/kg bw/day starting in the second study week onwards (0.93x and 0.95x compared to controls, respectively, at end of treatment).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in females of all test item-treated groups was similar to the control level over the study period. A slightly lower food consumption was seen in males at 100, 300 and 1000 mg/kg bw/day starting in the second week.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters in all test item treated males and females up to 1000 mg/kg bw/day. At 300 and 1000 mg/kg bw/day platelet numbers were increased in males, however these changes occurred within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance. In males, white blood cell count decreased dose-dependently (7.3, 6.5, 6.4 and 6.0 *10E9/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). This was related to a decrease in lymphocytes (5.8, 5.0, 4.7 and 4.5*10E9/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). In absence of pathological effects related to this observation, these effects were not considered adverse. No such effect was seen in females.

Coagulation parameters of treated rats were considered not to have been affected by treatment. The statistically significant changes in prothrombin time in males at 100 and 300 mg/kg bw/day were considered to be unrelated to the test item as these occurred in the absence of a dose-related trend (18.8, 17.1, 17.6 and 17.6 s for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). No such effects were seen in females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical chemistry parameters in all test item-treated females. At 1000 mg/kg bw/day, alkaline phosphatase activity was slightly increased in males, however these changes occurred within the range considered normal for rats of this age and strain and were therefore considered to be of no toxicological significance (115, 123, 138 and 166 U/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively). Moreover, cholesterol (HDL and LDL) levels were increased in males, however this slight increase was mainly caused by a single animal and was therefore not toxicologically relevant (1.05, 1.09, 1.13 and 1.22 mmol HDL/L, 0.31, 0.31, 0.31 and 0.45 mmol LDL/L for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively).
Lower TSH values in all test item-treated male groups (0.376, 0.075, 0.113 and 0.038 uIU/mL for controls and males treated at 100, 300 and 1000 mg/kg bw/day, respectively) and lower T4 values (4.97, and 4.01 ug/dL for controls and males treated at 1000 mg/kg bw/day, respectively) were observed achieving a level of statistical significance when compared to controls. In the absence of a treatment-related distribution, these values were considered to be of no toxicological significance.
While few other changes in males and females were statistically significant, the alterations in clinical biochemistry parameters were unrelated to administration of the test item due to the minimal magnitude of the change and the absence of a dose response.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights. Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of an effect on final body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings 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. Stage dependent qualitative evaluation of spermatogenesis in the testes was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 71 up to and including Day 91). One control female, and one female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular estrous cycle length
showed no relationship to the dose, and was therefore considered unrelated to treatment

Effect levels

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 seen up to and including the highest dose level tested (1000 mg/kg bw/day)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses

Small responses at the retention time of the test item were observed in the chromatograms of the control group formulations prepared for use in Week 1, Week 6 and Week 13. The maximum contribution to the formulation samples based on peak area was 0.011% taking the dilution factor into account. As the maximum contribution was only 0.011%, the control group formulations were still acceptable.

The concentrations analyzed in week 1 in the formulations of groups exposed to the test item were in agreement with target concentrations (i.e. mean accuracies 98% and 100%).

The concentrations analyzed in week 6 in the formulations of the low and the mid dose group were in agreement with target concentrations (i.e. mean accuracies 97%).

The mean accuracies of the low and the mid dose group formulations were above the target concentration (i.e. 191% and 175% of target). An ‘out of specification’ investigation did not reveal an error. Redilution of the formulation samples resulted in comparable mean accuracies (i.e. 199% and 179% of target). Based on the overall results in this study, these single out of spec results were considered not to have affected the overall conclusion of the study.

The formulations of the low, mid and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 6.0%).

Applicant's summary and conclusion

Conclusions:
An oral sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles. No adverse effects were observed after 90-day repeated dosing to Dehyton DC, therefore the NOAEL was established to be 1000 mg/kg bw/day (based on solid content).
Executive summary:

A sub-chronic toxicity study was performed according to OECD/EC guidelines and GLP principles.

Wistar Han rats were orally exposed to Dehyton®DC by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day for ninety days. Accuracy and homogeneity of formulations were confirmed by chemical analyses. No mortality occurred. A lower body weight compared to control was observed for males at 1000 mg/kg bw/day from the second week onwards with corresponding lower food consumption. In addition, slightly lower body weight compared to control with corresponding lower food consumption was also seen in males at 100 and 300 mg/kg bw/day starting in the second week. At the severity observed and in the absence of any histopathological effects, these effects were not considered to be adverse. At 100, 300 and 1000 mg/kg bw/day incidental effects related to breathing (abnormal breathing sounds, labored breathing, shallow breathing and increased and decreased respiratory rate) were noted in one or two animals per group on several days between Days 35 and 90. However, at the severity observed and in the absence of any histopathological effects, these effects were not considered to be adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, estrous cycle determination, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). No adverse effects were seen on reproduction parameters (estrous cycle length, spermatogenesis, weight, appearance and histopathology of reproduction organs).

Based on these results, the no-observed-adverse-effect level (NOAEL) for sub-chronic exposure was found to be 1000 mg/kg bw/day.