Registration Dossier

Administrative data

Description of key information

Under the conditions of the present study, the 28-Day Repeated Dose Oral Toxicity study with the test item including a 14-Day Recovery Period in male and female rats, with dose levels of 1.875, 7.5 and 30 mg/kg day was not associated with adverse toxicological findings.Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the test itemis considered to be 30 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-05 to 2013-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
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
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: 8 - 9 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 157 - 183 g; females: 126 - 148 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0715)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fiber bedding (lot no. 261111).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Preparation of the animals:

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange. Before the first
administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation
in body weight throughout the groups of males and females.

Administration of doses:

The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analysis
Each dosing concentration was analysed for nominal concentration and homogeneity of the test item in the vehicle at various intervals.
Stability analysis was performed on samples collected from the top and bottom dose levels.
Samples for the nominal concentration verification were taken in study week 1, 2, 3 and 4.
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1.
Samples for stability analysis were taken from top and bottom dose level in study week 1 at 0 hr and 6 hrs from high dose and low dose preparation.
All concentration samples were stored frozen (approximately -20°C) until the analysis was performed. All samples of dose formulations were
shipped on dry ice and protected from light to:
Dr. Matthias Eichler
IBACON GmbH
Institut für Biologische Analytik und Consulting IBACON GmbH
Arheilger Weg 17
64380 Rossdorf
Germany
The results were reported in an analytical phase report which was attached in the report of this study.
Duration of treatment / exposure:
The animals were treated with the test item or vehicle on 7 days per week for a period of 28 days.
Frequency of treatment:
The animals were treated once daily.
Remarks:
Doses / Concentrations:
1.875 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
7.5 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
40 animals (20 males and 20 females) were included in the main study (5 male and 5 female animals per group). The main study included one
control (C) and three dose groups (Low Dose = LD, Medium Dose = MD, High Dose = HD).
In addition, 20 animals (5 male and 5 female animals per group) were included in the control and high dose groups to be observed for a period of 14 days following the last administration.
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: random
- Section schedule rationale : all male animals together and all female animals together
Observations and examinations performed and frequency:
Body weight and food consumption:

The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the
treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.

Clinical observations:

All animals were observed for clinical signs during the entire treatment period of 28 days. The recovery animals were observed for an additional
period of 14 days following the last administration. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter. Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.

Functional observations:

Once before the first exposure and once in the fourth week of exposure as well as in the last week of the recovery period multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests . These tests were conducted in all animals.


Sacrifice and pathology:
Haematology:

Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).

Blood coagulation:

Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical biochemistry :

Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (ALP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Evaluation of thyroid hormones:

On the day of terminal sacrifice, blood was sampled from all surviving animals for evaluation of hormones. After overnight fasting of the animals,
blood from the abdominal aorta was collected in serum separator tubes at terminal euthanasia. The following hormones were examined at
BSL BIOSERVICE by Enzyme-linked immunosorbent assay (ELISA): Thyroid stimulating hormone (TSH), Triiodothyronine (T3), Thryroxine (T4).

Urinalysis:

An urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals. The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Pathology:

Gross necropsy-

One day after the last administration (study day 29) all surviving animals of the treatment period and 2 weeks after the last administration
all surviving animals of the recovery period (study day 43) were sacrificed using anesthesia (ketamine, medistar Arzeneimittelvertrieb, lot no: 00212, expiry date: 03/2014 and xylazin, Serumwerk, lot no. 00311, expiry date: 03/2013 or 00711, expiry date: 08/2013 (recovery animals)) and
subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial,
thoracic and abdominal cavities and their contents. The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed separately. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.

Organs weighed at Necropsy:

liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus,
thyroid/ parathyroid glands, spleen, brain, pituitary gland, heart.

The following tissues from all animals were preserved in 10% neutral buffered formalin except eyes, testes and epididymides which were fixed in
Modified Davidson’s fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin. brain (cerebrum, cerebellum and pons), spinal cord, eye, liver, kidneys, adrenal glands, stomach, small and large intestines (including Peyer´s patches), thymus, thyroid glands, spleen, lung and trachea, mammary glands, skin, heart, ovaries (females), uterus with cervix (females), vagina (females), testes (males), epididymides (males), prostate and seminal vesicles with coagulating glands as a whole (males), urinary bladder, lymphnodes (mesentric and axillary), peripheral nerve (e.g. sciatic nerve) with skeletal muscle, sternum with bone marrow, pituitary gland, oesophagus The animal found dead was also subjected to a gross necropsy and the organs preserved for a histopathological examination.

Histopathology:

All organs and tissues listed were evaluated in all animals of the control and high dose group sacrificed at the end of the treatment period and in the decedent animal. In addition, trachea was also evaluated in all animals of the low and medium dose group sacrificed at the end of the treatment period and in all recovery animals. For paired organs, both sides were examined. Macroscopic findings were evaluated in all study animals.


Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical
biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were
performed with a Student’s t-Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 was considered as statistically
significant).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality One female animal of the HD group died on study day 21. Based on macroscopic and histological lung findings, its death was considered to be most probably due to gavaging error and not to be directly related to the test item. Clinical Observations During the study clinical signs were recorded in males and females of control and treated group animals. The clinical findings like alopecia and eschars recorded in males and females were not considered to have toxicological relevance. Salivation, abnormal breathing, piloerection and slight diarrhea were observed transiently in few animals and were not assumed to have toxicological relevance. However, slight to moderate piloerection were noted in few recovery group animals (animals 26, 28 and 30), which persisted during the recovery period. None of the toxicological parameters corroborate this finding. Hence, the finding was not assumed to be of toxicological relevance. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality One female animal of the HD group died on study day 21. Based on macroscopic and histological lung findings, its death was considered to be most probably due to gavaging error and not to be directly related to the test item. Clinical Observations During the study clinical signs were recorded in males and females of control and treated group animals. The clinical findings like alopecia and eschars recorded in males and females were not considered to have toxicological relevance. Salivation, abnormal breathing, piloerection and slight diarrhea were observed transiently in few animals and were not assumed to have toxicological relevance. However, slight to moderate piloerection were noted in few recovery group animals (animals 26, 28 and 30), which persisted during the recovery period. None of the toxicological parameters corroborate this finding. Hence, the finding was not assumed to be of toxicological relevance. During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on boody weight gain as well as on food consumption.
Food consumption and compound intake (if feeding study):
not examined
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:
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:
no effects observed
Details on results:
Mortality.

One female animal (animal no. 49) of the HD group died on study day 21. Based on macroscopic and histological lung findings, its death was considered to be most probably due to gavaging error and not to be directly related to the test item.

Clinical Observations:

During the study clinical signs were recorded in males and females of control and treated group animals. The clinical signs are listed as follows,
Males (main group): Control- no findings; LD group- eschar at right cheek (1/5 animals), eschar at cheek (1/5 animals); MD group- alopecia at
forepaws (1/5 animals), abnormal breathing (1/ 5 animals); HD group- Alopecia on ventral thorax (1/5 animals), Slight diarrhea (1/5 animals).
Males (recovery group): Control- slight piloerection (1/5 animals); HD group- group Slight piloerection (4/5 animals), moderate piloerection
(2/5 animals), moving the bedding (1/5 animals), abnormal breathing (1/5 Animals), severe salivation (1/5 animals). Females (main group): Control- Alopecia on neck (2/5 animals), alopecia on ventral thorax (1/5 animals), alopecia (2/5 animals), eschar (2/5 animals), eschar on neck (175 animals), alopecia on forelimb (1/5 animals), eschar behind left ear (1/5 animals); LD group- no findings; MD group- eschar behind right ear (1/5 animals), eschar behind ears (1/5 animals), eschar at left shoulder (1/5 animals); HD group- Slight salivation (1/5 animals), abnormal breathing (2/5 animals).
Females (recovery group): Control- no findings; HD group- slight salivation (3/5 animals).

The clinical findings like alopecia and eschars recorded in males and females were not considered to have toxicological relevance.
Salivation, abnormal breathing, piloerection and slight diarrhea were observed transiently in few animals and were not assumed to have
toxicological relevance.

However, slight to moderate piloerection were noted in few recovery group animals (animals 26, 28 and 30), which persisted during the recovery
period. None of the toxicological parameters corroborate this finding. Hence, the finding was not assumed to be of toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery:

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development:

In both males and females, there were no treatment related changes recorded during treatment and recovery period in all treated groups when
compared to the corresponding control group. Statistically no significant changes were recorded for treated group values when compared to the corresponding control.

Food Consumption:

In both males and females, no statistically significant changes were noted for food intake in treated groups when compared to the corresponding
controls. However, there was substantial decrease in mean daily food intake noted in male and female LD and MD groups. There was no dose response pattern noted. Therefore, the changes were not considered to be of toxicological relevance.

Haematology and Blood Coagulation:

In males and females (main and recovery groups), statistically no significant difference was noted between treated and control groups for the
measured haematological parameters. Any variations among the groups were not assumed to be biologically relevant. Blood coagulation was not affected due to treatment in male and female animals. However, there was considerable decrease in mean aPTT value in
male LD group, which was not of toxicological relevance.

Clinical Biochemistry:

At the end of the treatment period, the statistical analysis revealed no significant changes for measured clinical biochemistry parameters in male treated groups when compared to corresponding control. However, the mean TBA value in male LD group was highly elevated, which was due to considerable increase in TBA value in two animals (animal 6 and 7). The changes were considered to be incidental and had no toxicological relevance. In females, statistically significant increases were noted for mean TP, Alb and Gluc values in LD group. There was no dose response pattern observed for this type of findings and hence the changes had no toxicological relevance.
At the end of recovery period, there was statistically significant increase in mean TP, Alb and Na values in male HD group. Statistically significant
increase was also noted for mean TP and Chol values in female HD group. However, all individual and mean values of TP, Alb and Chol were within
historical control range. The individual and mean values of Na of male HD recovery group were slightly above the borderline of historical control
value. There were also no changes recorded histopathologically that could corroborate the findings. Hence, the above changes were assumed to
have no toxicological relevance.

Evaluation of Thyroid Hormones:

During treatment period measured T3 and T4 concentrations in male and female animals were not considerably changed in LD, MD and HD groups
when compared to control. This is also applicable for the measured T3 and T4 concentrations in male and female animals in HD recovery group
when compared to corresponding control. The measured TSH concentrations in all animals were below limit of quantification.

Urinalysis:

No treatment related changes were recorded for urinalysis parameters in male and female treated groups including recovery group animals when
compared to the corresponding control group.

Pathology:

Few specific gross pathological changes were recorded for the female animals and were not considered to be treatment-related. The changes
recorded are listed as follows,
Control: fluid distended uterus with oviduct and cervix (animal 33); LD group: fluid distended uterus with oviduct and cervix (animal 38);
MD group: fluid distended uterus with oviduct and cervix (animal 41); HD group: reddish discoloured mandibular/ axillary lymph nodes and foam
filled dark spotted lung (animal 49); Control recovery: fluid distended uterus with oviduct and cervix (animal 55); HD recovery: fluid distended uterus with oviduct and cervix (animals 56, 58 and 59).

Organ Weight:

In males, there was statistically significant increase in relative brain weight of epididymides in LD group. In male recovery group, there was statistically significant increase in absolute liver weight in HD group but not for relative weights (brain and terminal body weights) of liver. In females, statistically significant decrease in absolute weight of adrenal and pituitary gland were recorded in MD group. These changes in male or female animals were
not assumed to have toxicological relevance due to either lack of dose response pattern and/or lack of histological changes.
In addition to above, there were also changes recorded in absolute and relative brain or body weights for few organs (liver, heart, kidneys, thyroid,
pituitary, adrenal gland, thymus, ovary, uterus or kidney) of LD or MD or HD groups male- female animals, but without having statistical significance and histological changes. Hence, the findings were not associated with toxicity.

Histopathology:

One female rat in HD group was found dead during the treatment period. Based on macroscopic and histological lung findings, its death was considered to be most probably due to gavaging error and not to be directly related to the test item. No test item-related macroscopic and no toxicologically significant histopathological changes were observed in this study. Histopathological findings attributable to treatment were seen in the trachea, only, and comprised a minimal or mild epithelial alteration in single males of MD and HD groups, as well as mild epithelial alteration, associated with mild (sub)epithelial mixed cell infiltration in two females of HD group. They had completely resolved after the 14-day recovery period.

In view of the low incidence of these changes and the lack of a clear dose relationship in the males, these findings were considered to be most
probably indicative of a local irritant effect of the test item formulation when accidentally reaching the trachea, e.g. after partial misgavaging or
after partial regurgitation/aspiration. They were therefore not considered to be of toxicological significance.

Dose Formulation Analysis:

The % nominal of formulation samples meant for the nominal concentration, stability and homogeneity verification are as follows,

- Nominal concentration: 90 -124 % in LD group, 95-106% in MD group, 102-107% in HD group.

- Stability analysis: 92 and 94% in LD group and 97 and 105% in HD group.

- Homogeneity analysis: 90-100% in LD group and 100-103% in HD group.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
Under the conditions of the present study, the 28-Day Repeated Dose Oral Toxicity study with the test item
including a 14-Day Recovery Period in male and female rats, with dose levels of 1.875, 7.5 and 30 mg/kg day was not associated with
adverse toxicological findings. The NOAEL was established to be 30 mg/kg body weight per day.
Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the test item is considered to be 30 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.
Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test item via oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem (sterile water), the vehicle used in this study. The 4 groups comprised of 5 male and 5 female Wistar rats.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically. To detect possible delayed occurrence or persistence of, or recovery from toxic effects, animals in the recovery group were observed for a period of 14 days following the last administration.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. In addition, trachea was also evaluated in all animals of the low and medium groups sacrificed at the end of the treatment period and in all recovery animals.

The following doses were evaluated:

Control:                         0          mg/kg body weight

Low Dose:                   1.875      mg/kg body weight

Medium Dose:            7.5          mg/kg body weight

High Dose:               30       mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was suspended in aqua ad injectionem and administered daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.


 Summary results :

One female animal of the HD group died on study day 21.Based on macroscopic and histological lung findings, its death was considered to be most probably due to gavaging error and not to be directly related to the test item.

In males and females, there were no clinical signs considered to be of toxicological relevance observed during treatment and recovery period.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

There were no ophthalmoscopic findings in any of the animals of this study.

In both males and females, no treatment related changes were recorded for body weight, body weight change and food intake during treatment and recovery period in all treated groups when compared to the corresponding control group.

In males and females (main and recovery groups), statistically no significant difference was noted between treated and control groups for the measured haematological and blood coagulation parameters. 

Test-item related alterations were not found in any of the parameters of clinical biochemistry analyzed at the end of the treatment and recovery period.

No treatment related changes were noted for measured T3 and T4 concentrations in male and female animals in LD, MD and HD groups when compared to control.

There were no test item-related changes noted for any of the urinary parameters tested.

There was no toxicological relevance considered for male and female organ weight (absolute and relative brain/ terminal body weight) data in treated groups when compared to corresponding control.

 

No test item-related macroscopic and no toxicologically significant histopathological changes were observed in this study.

 

Histopathological findings attributable to treatment were seen in the trachea, only, and comprised a minimal or mild epithelial alteration in single males of MD and HD groups, as well as mild epithelial alteration, associated with mild (sub)epithelial mixed cell infiltration in two females of HD group. They had completely resolved after the 14-day recovery period.

In view of the low incidence of these changes and the lack of a clear dose relationship in the males, these findings were considered to be most probably indicative of a local irritant effect of the test item formulation when accidentally reaching the trachea, e.g. after partial misgavaging or after partial regurgitation/aspiration. They were therefore not considered to be of toxicological significance.

Formulation sample analysis revealed nominal concentration:

90 -124 % in LD group, 95-106% in MD group, 102-107% in HD group;

stability analysis: 92 and 94% in LD group and 97 and 105% in HD group;

homogeneity analysis: 90-100% in LD group and 100-103% in HD group.

Conclusion:

U nder the conditions of the present study, the 28-Day Repeated Dose Oral Toxicity study with the test item including a 14-Day Recovery Period in male and female rats, with dose levels of 1.875, 7.5 and 30 mg/kg day was not associated with adverse toxicological findings. Based on the data generated from this study, the NOAEL (No Observed Adverse Effect Level) of the test item is considered to be 30 mg/kg body weight/day for the 28-day repeated dose oral toxicity study in male and female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study according to GLP available. No derivations and/or confounders. Klimisch rating 1 representing reliability without restrictions. Information valid and meets data requirements with regard to hazard identification and classification.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Available data allows for route-to-route extrapolation.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Testing was waived for animal welfare reasons because the test item is corrosive to skin. The available data allows for expert judgement concerning this endpoint.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Available data allows for route-to-route extrapolation.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Testing was waived for animal welfare reasons because the test item is corrosive to skin. The available data allows for expert judgement concerning this endpoint.

Additional information

The test item was administered daily in graduated doses of 0 (control), 31.25, 125 and 500 mg/kg body weight per day to 4 groups of test animals, one dose level per group for a treatment period of 14 days. No mortality occurred in the control and low dose group during the treatment period of this study. One male animal of the mid dose group was euthanized for animal welfare reasons. All animals of the high dose group died or were euthanized for animal welfare reasons. Macroscopic findings in these animals during necropsy are indicative of strong local effects in the gastro-intestinal tract as a cause of a bad health condition. Clinical signs were observed during the treatment period in all dose groups of this study. A slight decreased body weight gain was found for male and female mid dose animals between treatment days 7 and 14. Slightly decreased food consumption could be evaluated during the whole treatment period for male and female mid dose animals. Some values of haematology (platelet count, whit blood cell count) of male animals were increased when compared to the control animals. Platelet values were also increased in female mid dose animals. However, all values were still in the range of our historical control data. No treatment related changes were noted for measured T3 and T4 concentrations in male and female animals indicating no effects on the thyroide. Based on the results from this screening study, 31.25 mg/kg body weight per day was considered to be a minimal effect level.

When tested according to OECD TG 407, daily administration of 1,875, 7.5 or 30 mg/kg body weight to male and female rats for 28 consecutive days revealed no test item related systemic effects of toxicological significance. No mortality and no clinical signs of toxicity occurred . Body weight gain was not affected. There were no ophthalmoscopic findings and all parameters of the functional observation battery were within normal range. Haematology, clinical biochemistry and urinalysis were unobtrusive. T3 and T4 values were not affected. Organ weights in both, male and female animals of all dose groups, revealed no changes compared to the control animals. No test item related macroscopic and no toxicologically significant histopathological changes were observed. The NOAEL was established to be 30 mg/kg body weight per day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study according to GLP with Klimisch rating 1. Study design allows for endpoint related classification of registration substance.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: stomach

Justification for classification or non-classification

The results from the available studies with repeated exposure of the registration substance indicate that the test material is systemically available following oral administration. Mortalities occurred in all male and female animals at the highest dose of 500 mg/kg body weight per day after approximately 3 - 6 days of dosing due to general bad health condition. Decrease in food intake was observed in male and female animals of the 500 mg/kg and 125 mg/kg dose groups in the first week of treatment. Haematological findings revealed increases in white blood cell count in male and females from these two higher dose groups as well as increased neutrophil counts in males and increased monocyte, eosinophil and basophil counts in females. These haematological findings are interpreted to be associated with inflammatory changes related to treatment. An explanation may be seen in the strong irritative / corrosive properties of the test material which may exert a cytotoxic potential at any site of contact, e.g. the mucosa of the gastrointestinal tract. The no-observed-adverse-effect level (NOAEL) from the 28 -day subacute oral toxicity study was established at 30 mg/kg body weight per day.

Based on ECHA guidance on the application of the CLP criteria (version 3.0), substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement on the basis of the weight of all evidence available, including the use of recommended guidance values. In this respect, the evidence from studies with repeated dose toxicity studies indicate that the submission substance has the potential to be harmful to human health following repeated exposure. Looking closely at the effects occurred after repeated exposure all findings indicate an unspecific effect resulting from the local irritation of the gastrointestinal tract including the esophagus. This finding is supported by the corrosive property of the test substance. Due to the corrosivity of the test substance a reduced food intake resulting in a reduce body weight gain and inflammatory changes in hematology is observed as secondary effect. These local effects were also find in the new performed OECD 414 study. Taking into account all these information a classification with STOR-RE 2 is not applicable since the local irritation/corrosion effect is already covered by the Skin Corrosion 1C classification.