Registration Dossier

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Administrative data

Description of key information

Oral (OECD 407), 28 days, rat (m/f): NOAEL systemic = 100 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
2013-09-04 to 2014-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 03 Oct 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2001/59/EC
Version / remarks:
adopted 06 Aug 2001
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 – 8 weeks (males) and 8 – 9 weeks (females)
- Weight at study initiation: 200 - 257 g (mean 215.13 g) males and 141 - 166 g (mean 151.75 g) females
- Housing: individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 030713)
- Diet: Altromin 1324 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder with the help of a mortar and the ground test item was weighed into a tared glass bottle on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was mixed thoroughly using a homogenizer. The test item formulation was prepared once a week and stored at ambient temperature.


VEHICLE
- Concentration in vehicle: 4, 10, 20 and 25* mg/mL (*treatment with 25 mg/mL for the first three days, afterwards treatment with 20 mg/mL)
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 1101401034
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For determination of the concentration of test item in dosing formulations, samples of 5 mL were retained from low and mid dose group in study weeks 2 and 3 and from the high dose group in study weeks 1 and 4. Stability of the test item in the vehicle was assessed from one of the dose preparations after 8 days of storage.
Concentration analysis revealed a mean achieved concentration of 100.0%, 103.5% and 100.1% in the low, middle and high dose group, respectively. Homogeneity was determined for the low and high dose group only and revealed a mean recovery between 103.5 to 109.2% at the low dose group and between 102.4 and 106.1% at the high dose group, respectively. The stability of the control and high dose group after 7 days of storage was between 97.7 and 95.7%.
Duration of treatment / exposure:
28 days with 14 day recovery period
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
5 animals were treated for the first three days with 125 mg/kg bw/day and afterwards with 100 mg/kg bw/day for the remaining treatment period and 2 animals were treated with 100 mg/kg bw/day for the whole treatment period.
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Animals were treated for the first three days with 125 mg/ kg bw/day and afterwards with 100 mg/kg bw/day for the remaining treatment period.
No. of animals per sex per dose:
20 and 50 mg/kg bw/day: 5 (main group)
100 mg/kg bw/day: 6 (main group) and 6 (recovery group); each 5/6 males and 5/6 females were treated at 125 mg/kg bw/day for the first 3 days of treatment and. Due to mortality observed at 125 mg/kg bw/day, the high dose level was reduced to 100 mg/kg bw/day from Day 4 onward. To compensate for the animals that died in the period that the high-dose group was administered 125 mg/kg bw/day, 1 male and 1 female rat per group were added to the high-dose group and administered 100 mg/kg bw/day for the whole treatment period of 28 days.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dosing was based on a range finding study
- Rationale for animal assignment: 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
- Post-exposure recovery period in satellite groups: 14 days

The highest dose level was chosen with the aim of inducing toxic effects without deaths or severe suffering. Animals from high dose group were treated with 125 mg/kg bw/day from Day 1 - 3 . After 2 spontaneous deaths, from Day 4 the dose was reduced to 100 mg/kg bw and 4 reserve animals were added to the high dose group.
Positive control:
no
Observations and examinations performed and frequency:
CAGES SIDE OBSERVATIONS: Yes
- Time schedule for examinations: The animals were checked once daily for clinical signs and twice daily for morbidity and mortality, except for weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first administration and at least once a weak thereafter.
- Detailed clinical examinations included: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION: Yes
- Time schedule for food consumption: Food consumption was measured weekly during the treatment and recovery period in the recovery animals.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 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.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Haematocrit, haemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, platelet count, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells. Coagulation parameters examined included prothrombin time and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine, total protein, albumin, urea, total bilirubin, total bile acids, total cholesterol, glucose, sodium and potassium.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed with samples collected prior to or as part of the sacrifice of the animals.
- Metabolism cages used for collection of urine: The samples were drawn from the bladdder during necropsy.
- Animals fasted: Yes
- Parameters checked: Specific gravity, nitrite, pH value, protein, glucose, ketones, urobilinogen, bilirubin, blood and leukocytes.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before the first exposure and once during the four weeks of exposure, as well as in the last week of the recovery period multiple detailed behavioural observations were made using a functional observational battery of tests.
- Dose groups that were examined: All animals in all groups


IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. One day after the last administration (study Day 29) all surviving animals of the main group and 2 weeks after the last administration all surviving animals of the recovery group (study Day 43) were sacrificed using anesthesia and were 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 following organ weights were determined for all animals: Liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland and heart.

HISTOPATHOLOGY: Yes. Samples of the following tissues from each animal were preserved and examined microscopically: Brain (cerebrum, cerebellum and pons), spinal cord, eye, liver, kidney, adrenal glands, stomach, small and large intestines (including Peyer's patches), thymus, thyroid glands, spleen, lung and trachea, mammary glands, skin, heart, ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating glands, urinary bladder, lymph nodes (mesenteric and axillary), peripheral nerve (e.g. sciatic nerve) with skeletal muscle, sternum with bone marrow, pituitary gland, oesophagus and head with nasal cavity.
Statistics:
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy).
The toxicological and pathological data were evaluated via the validated computerized system E-Workbook. 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 test item-treated with control animals 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. The statistics were performed with E-Workbook software (p<0.05 is considered as statistically significant).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Moderate to severe salivation, moving the bedding and piloerection were noted in all treatment-groups and considered to be treatment-related. Slight indications of diarrhea, piloerection, slight salivation and moving the bedding were also noted among control animals, but the clinical signs were more prominent and consistently observed in test item-treated animals. Moving the bedding, salivation and piloerection were only seen immediately after dose administration, therefore the findings were considered to be transient and non-adverse.
In addition, high dose group males showed transient occurences of respiratory sounds, eyes half closed, eschar and bloated belly. In females of the high dose group, nasal discharge, alopecia and bloated belly was noted in the main and recovery group. Transient appearances of respiratory sounds (slight to severe) were observed in females of all dose groups, but more frequently seen in animals of the high dose group. The findings were considered to be treatment-related and toxicologically relevant. For details please refer to tables 1 and 2 under “Any other information on results incl. tables”.
Mortality:
mortality observed, treatment-related
Description (incidence):
50 mg/kg bw/day: 1/5 females died (study Day 22), treatment-related
100/125 mg/kg bw/day: After two spontaneous deaths at 125 mg/kg bw/day the dose level was adjusted to 100 mg/kg bw/day from study Day 4 onward. Within the whole study period, 3/6 males (study Days 4, 9 and 17) and 1/6 females (study Day 17) of the main group and 2/6 males (study Days 3 and 4) and 1/6 females (study Day 3) of the recovery group died or were killed for humane reasons during the treatment preriod, treatment-related. All deaths/unscheduled sacrifices were attributed to local lesions in the forestomach, treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
20 mg/kg bw/day: reduced body weight gain in both sexes during the first week of treatment, not treatment-related
50 mg/kg bw/day: slightly decreased body weight gain in females at the beginning of treatment, not treatment-related
100/125 mg/kg bw/day: considerably attenuated body weight increase in males during the first and second week of treatment (approx. 15 and 70% of controls, respectively, statistically not significant), during this time mortality occured in 4/10 males in this group, treatment-related findings consistent with changes in food intake; increased body weight gain in females during the first 2 weeks (118 and 112% of control) followed by a slight decrease in the third week (approx. 3 g), both increase and decrease statistically not significant, findings in females were attributed to the marked decrease in body weight of a single animal and accompanied by mortality of a second female in this group; consistent with changes in food intake
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
100/125 mg/kg bw/day: Decreased in males between Days 1-28 due to lower food consumption in week 1 and 2 (-39.4% and -16.9%, statistically not significant), attributed to two isolated males which were found dead or prematurely killed for ethical reasons. A slight decrease within the first two study weeks (-15% and -18.9%) was also observed in the male recovery group, which were attributed to individual animals with poor health condition. In females there was a considerable decrease in food consumption in first study week in animals of the main group (-41.6%, statistically not significant) and a slight decrease (-14.8%) in animals of the recovery group. All findings in males and females were considered to be secondary due to histopathological changes in the forestomach and attributed to treatment. All observations were found to be fully reversible in recovery animals after two weeks of recovery.
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
Description (incidence and severity):
20 mg/kg bw/day: A statistically significant decrease in haemoglobin was observed in males, the data were within the historical control range and considered to be not treatment-related.
50 mg/kg bw/day: A statistically significant decrease in haemoglobin and in red blood cell count was observed in males at the end of the treatment period. The data were within the historical control range and fully reversible after 2 weeks of recovery and therefore considered to be not treatment-related. A statistically significant increase in mean corpuscular volume (MCV) and a statistically non-significant increase in reticulocytes (Ret) was noted in females at the end of treatment, as well as a statistically significant increase in basophils. The increases in MCV and Ret were without dose relation and all findings were within the historical control range and fully reversible after 2 weeks recovery and therefore considered to be not treatment-related.
100/125 mg/kg bw/day: A statistically significant decrease in haematocrit and haemoglobin was noted in males. The changes remained within the historical control range and were fully reversible after two weeks of recovery and therefore considered to be not treatment-related. Males of the recovery group showed a statistically significant decrease in mean Ret and a statistically significant decrease in white blood cells. The findings were not consistent with animals of the main group and therefore not attributed to treatment. In females, there was a statistically significant increase in MCV and Ret at the end of treatment. The effects were fully reversible after 2 weeks of recovery in recovery animals and remained within the historical control range. Therefore all findings were not related to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
20 and 50 mg/kg bw/day: A statistically non-significant increase in alanine aminotransferase and aspartate aminotransferase and a statistically non-significant increase in cholesterol was noted in both sexes at the end of treatment. The findings occurred without dose-relation and were within the historical control range and thus considered to be without treatment-relation.
100/125 mg/kg bw/day: A statistically significant increase in total bile acids was noted in males at the end of treatment, which was fully reversible after two weeks of recovery. In addition, a statistically significant decrease in total protein was observed in male recovery animals at the end of recovery. The observations remained within the historical control range and were without histopathological abnormalities, therefore the findings were considered to be not treatment-related. In both sexes, there was a statistically non-significant increase in alanine and aspartate aminotransferase as well as cholesterol. Without a clear dose-relation the findings were considered to be not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
High levels of erythrocytes were found in 2/6 males and 2/6 females at the end of the recovery period and a high level of glucose was found in 1/6 females at the end of treatment. In addition, an increased urine protein concentration was observed in 1/6 female at the end of the recovery period. All observations were slight, in the normal range of normal variation and without dose relation and therefore considered to be not treatment-related.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
20 mg/kg bw/day: increased relative liver weight in males, not dose-related, reversible, in the absence of histopathological findings the changes were considered to be related to metabolic adaption but not treatment-related
50 mg/kg bw/day: decreased absolute weight of epididymides, not dose-related, considered to be incidental; increased relative liver weight in males, not dose-related, reversible, in the absence of histopathological findings the changes were considered to be related to metabolic adaption but not treatment-related; increased mean absolute + relative spleen weight and increased relative thyroid weight in males at the end of treatment, sex specific, without histopathological findings, considered to be possibly related to compensation of local irritative effects in the forestomach; increased absolute liver weight in females, no dose relation, considered to be a change of metabolic adaption.
100/125 mg/kg bw/day: increased relative liver weight in males, not dose-related, reversible, in the absence of histopathological findings the changes were considered to be related to metabolic adaption but not treatment-related; increased mean absolute + relative spleen weight and increased relative thyroid weight in males at the end of treatment, sex specific, without histopathological findings, considered by the study author to be possibly related to compensation of local irritative effects in the forestomach; the cause of the effects is unclear.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings being indicative of necrotizing inflammation in the forestomach were observed in all test item groups of both sexes at 20, 50 and 100/125 mg/kg bw/day. Findings at 100/125 mg/kg bw were found to be non-reversible after two weeks of recovery. Thickened and/or dark red mucosa and several foci were observed at macroscopical examination and the findings were accompanied by forestomach epithelial (squamous) hyperplasia and hyperkeratosis, parakeratosis and/or dyskratosis at histopathological examination. The incidence and group mean severity of the lesions was higher at 50 and 100/125 mg/kg bw/day. The findings were considered to be treatment-related but explained to be a result of local irritating effects and thus not attributed to systemic toxicity. The necrotic and inflammatory lesions showed a tendency to recover after 14 days of recovery. For details please refer to table no. 3 under "any other information on results incl. tables". In addition, gross necropsy findings in the lungs, spleen and seminal vesicles were observed in single cases in individual animals, but the observations were explained by accidental aspiration or the poor condition of the animals and due to treatment. For details please refer to table no. 4 under "Any other information on results incl. tables".
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrotizing inflammation in the forestomach was observed in all test item groups of both sexes at 20, 50 and 100/125 mg/kg bw/day, which was accompanied by forestomach epithelial (squamous) hyperplasia and hyperkeratosis, parakeratosis and/or dyskratosis. The incidence and group mean severity of the lesions was higher at 50 and 100/125 mg/kg bw/day. The findings were considered treatment-related but to be a result of local irritating effects and thus not attributed to systemic toxicity. After 14 days of recovery, the necrotic and inflammatory lesions were still present but to a lesser extent and not all animals were affected. For details please refer to table no. 5 under "any other information on results incl. tables".
In addition, several necrotic/inflammatory lesions and/or reactive alterations to the local injuries were recorded in the trachea, lung and/or oesophagus of decedent animals (please refer to table no. 6 under "any other information on results incl. tables"). The findings were attributed to the animal’s morbidity. Lymphoid atrophy of spleen and/or atrophy of thymus were recorded in some decedents and considered to be secondary responses to peracute stressful condition. 3/6 males of the high dose group showed reduced/low secretion of seminal vesicles and coagulating glands, but as histomorphological alterations were lacking the findings were considered to be secondary events due to poor conditions. In addition, 1/6 females of the high dose group had focal decidual-like alterations of the uterus and vaginal mucification. The causes of the alterations were unclear, but as no other histomorphological alterations were noted the findings were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Details on results:
There was necrotizing inflammation in the forestomach in all test item-treated groups of both sexes with a dose dependently increasing incidence, which was accompanied with forestomach epithelial (squamous) hyperplasia and hyperkeratosis, parakeratosis and/or dyskeratosis as reactive changes to the forestomach injuries.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effect observed
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Primary or secondary results of local irritative effects in the forestomach. As the forestomach is not a human tissue, there is no relevance for human exposure.
Critical effects observed:
no

Table 1: Clinical findings in males

Clinical sign Occurence / Total number of animals Recovery animals
0 mg/kg bw/day 20 mg/kg bw/day 50 mg/kg bw/day 100/125 mg/kg bw/day 0 mg/kg bw/day 100/125 mg/kg bw/day
Diarrhea, slight 3/5 3/5 2/5 4/6 1/5 2/6
Piloerection, slight 4/5 4/5 5/5 6/6 / 5/6
Salivation, slight 1/5 5/5 5/5 4/6 2/5 4/6
Moving the bedding 3/5 5/5 5/5 6/6 1/5 6/6
Salivation, moderate 1/5 5/5 5/5 4/6 / 5/6
Salivation, severe / 5/5 5/5 6/6 / 5/6
Piloerection, moderate / 1/5 1/5 5/6 / 4/6
Piloerection, severe / / 4/5 2/6 / 5/6
Respiratory sounds, moderate / / / 2/6 / /
Eyes half closed / / / 2/6 / /
Respiratory sounds, strong / / / 2/6 / 2/6
Nasal discharge, slight / / / 2/6 / /
Diarrhea, severe / / / 4/6 / 3/6
Diarrhea, moderate / / / 1/6 / 1/6
Eschar / / / / / 1/6
Respiratory sounds, slight / / / 1/6 / /
Bloated belly / / / / / 1/6

Table 2: Clinical findings in females

Clinical sign Occurence / Total number of animals Recovery animals
0 mg/kg bw/day 20 mg/kg bw/day 50 mg/kg bw/day 100/125 mg/kg bw/day 0 mg/kg bw/day 100/125 mg/kg bw/day
Diarrhea, slight / / / 6/6 3/5 2/6
Piloerection, slight / 5/5 5/5 6/6 / 6/6
Salivation, slight 5/5 4/5 6/6 1/5 5/6
Moving the bedding / 5/5 5/5 6/6 / 6/6
Salivation, moderate 5/5 5/5 6/6 / 5/6
Salivation, severe / 5/5 5/5 6/6 / 5/6
Piloerection, moderate / 1/5 1/5 6/6 / 5/6
Piloerection, severe / / / 6/6 / 5/6
Respiratory sounds, moderate / / 1/5 3/6 / 1/6
Eyes half closed / / / / / 1/6
Respiratory sounds, strong / / / 2/6 / 1/6
Nasal discharge,red / / / 1/6 / 2/6
Nasal discharge, slight / / / 1/6 / 2/6
Diarrhea, severe / / / 1/6 / 2/6
Diarrhea, moderate / / 2/6 / 2/6
Respiratory sounds, slight / 1/5 2/5 / / 3/6
Nasal discharge, moderate / / / 1/6 1/5 /
Alopecia, flanks / / / 1/6 / /
Bloated belly / / / 1/6 / /
Alopecia / / / 1/6 / /

Table No. 3: Macroscopical findings in the stomach observed at gross necropsy

Sex Macroscopic finding Necropsy finding occurence/total no. of animals at dose [mg/kg bw/day]
At the end of treatment After recovery
0 20 50 100/125 0 100/125
Male Thickened mucosa 1/5 / 1/5 2/6 / /
Focus depressed / / 1/5 1/6 / /
Abnormal content, foam / / / 1/6 / /
Gaseous distention, slimy fluid / / / 1/6 / /
Focus(es) (dark), mucosa fundus, foci, dark red 5x1 mm / / 1/5 / / 1/6
Focus(es) (dark), several, black 2 mm / / 1/5 1/6 / /
Focus(es) (dark), mucosa fundus, foci, dark red 4x1 mm / / / 1/6 / /
Focus(es) (dark), several, black 4x1 mm / / / / / 1/6
Discoloured (dark), mucosa fundus, discoloration red / / / / / 1/6
Gaseous distention, red / / / / / 1/6
Discloured (dark), granular mucosa / / / / / 1/6
Female Gaseous distention / / 1/5 1/6 / /
Abnormal content, slimy / / / 1/6 / /
Discoloured (dark), mucosa red / / / / / 1/6

Table No. 4: Macroscopic findings at gross necropsy without treatment relation

Sex Macroscopic finding Necropsy finding occurence/total no. of animals at dose [mg/kg bw/day]
At the end of treatment After recovery
0 20 50 100/125 0 100/125
Male Seminal vesicles, small in size / / / 2/6 / 1/6
Seminal vesicles, small in size, both vesicles / / 1/5 / / /
Duodenum, gaseous distention / / / 3/6 / 1/6
Jejunum, discoloured (dark) / / / / / 1/6
Jejunum, gaseous distention / / / 3/6 / 2/6
Ileum with Peyer's patches, gaseous distention / / / 2/6 / 1/6
Caecum, gaseous distention / / 1/5 3/6 / /
Colon/rectum/anus, gaseous distention / / / 3/6 / /
Kidneys, discoloured (pale) / / / 1/6 / /
Kidneys, dilated pelvis, right / 1/5 1/5 / 1/5 /
Kidneys, dilated pelvis, left / / / / / 1/6
Kidneys, dilated pelvis / 1/5 / / / /
Kidneys, discoloured (pale), right, both, beige / 1/5 / / / /
Kidneys, discoloured (pale), both, light brown / 1/5 / / / /
Kidneys, large in size / / / / / 1/6
Thymus, discoloured (dark) / / / 1/6 / 1/6
Brain, cut / / / 1/6 / /
Caecum, fluid content / / / / / 1/6
Spleen, cut in two pieces / / / / / 1/6
Lungs, not collapsed / / / / / 1/6
Female Axillary lymph nodes, discoloured, dark / / 1/5 / / /
Mesenteric lymph nodes, discoloured, dark / / 1/5 / / /
Ovaries, discoloured, both / 1/5 / / / /
Ovaries, discoloured, both, dark red / / / 1/6 / /
Ovaries, discoloured (dark), left, dark red / / / / 1/5
Ovaries, discoloured (dark), right, red / / / / / 1/6
Uterus/cervix, fluid distension / 1/5 1/5 / / 1/6
Uterus/cervix, nodules / / / 1/6 / /
Pancreas, discoloured (dark) reddish 1/5 / / / 1/5 2/6
Duodenum, gaseous distention / / 1/5 1/6 / 1/6
Jejunum, gaseous distention / / 1/5 1/6 / 1/6
Ileum with Peyer's patches, gaseous distention / / / 1/6 / 1/6
Ileum with Peyer's patches, discoloured, dark / / 1/5 / / /
Caecum, gaseous distention / / 1/5 1/6 / /
Caecum, fluid content / / / / / 1/6
Colon/rectum/anus, gaseous distention / / 1/5 1/6 / /
Spleen, small in size / / / 1/6 / /
Kidneys, dilated pelvis, both / 1/5 / / 1/5 /
Thyroid, cut in two pieces / / / / / 1/6
Lungs, discoloured (dark), red / / / / / 1/6
Thymus, discoloured (dark), red / / / / / 1/6

Table No. 5: Histopathological findings in the forestomach of surviving animals

Histopathological finding
incidence/severity
At the end of treatment After recovery
0 mg/kg bw/day 20 mg/kg bw/day 50 mg/kg bw/day 100/125 mg/kg bw/day 0 mg/kg bw/day 100/125 mg/kg bw/day
5 males 5 females 5 males 5 females 5 males 4 females 3 males 5 females 5 males 5 females 4 males 5 females
Inflammation, necrotizing 0 0 3/1.3 2/1.0 5/1.4 4/1.8 3/2.0 5/1.2 0 0 0 0
Epithelial hyperplasia, reactive 0 0 3/1.3 2/1.0 5/2.0 4/1.8 3/2.3 5/2.0 0 0 0 0
Dyskeratosis 0 0 2/1.5 2/1.0 3/1.3 2/2.5 3/1.7 4/1.0 0 0 0 0
Hyperkeratosis and parakeratosis 0 0 3/1.3 2/1.0 5/1.8 4/1.8 3/2.0 5/1.8 0 0 4/1.0 4/1.0
Granulation 0 0 0 0 0 0 0 0 0 0 2/1.0 3/1.3

Table No. 6: Histopathological findings in decedent animals

Histopathological finding
incidence/severity
At the end of treatment After recovery
50 mg/kg bw/day 100/125 mg/kg bw/day 100/125 mg/kg bw/day
Trachea
Inflammation, necrotising 0 1/1.0 1/1.0 0 1/4.0 0
Hypertrophy, mucosal epithelium 0 0 0 1/1.0 0 0
Flocculent content in the lumen 0 0 0 0 1/2.0 0
Lung
Aspiration, pneumonia 0 0 1/1.0 1/1.0 0 0
Bronchial mucous content, with celluluar debris and foreign material 0 0 1/1.0 0 0 0
Increased macrophages, at bronchiolar alveolar duct area, with flocculent material 0 0 0 0 0 1/1.0
Haemorrhage, at bronchiolar alveolar duct area, with flocculent material 0 0 0 0 1/1.0 0
Oesophagus
Inflammation, necrotising (recorded as grade 3)
focal, submucosal (recorded as grade 1)
0 0 2/1.0 1/3.0 0 0
Epithelial hyperplasia, reactive 0 0 2/1.0 1/2.0 0 0
Dyskeratosis 0 0 0 1/1.0 0 0
Hyperkeratosis and parakeratosis 0 0 1/2.0 0 0 0
Forestomach
Inflammation, necrotizing 0 1/1.0 3/2.7 1/2.0 2/3.5 1/4.0
Epithelial hyperplasia, reactive 0 0 3/2.0 1/2.0 2/1.5 1/1.0
Dyskeratosis 0 0 2/1.5 1/2.0 2/1.0 1/1.0
Hyperkeratosis and parakeratosis 0 1/1.0 3/2.0 1/2.0 2/2.0 1/1.0
Spleen
Lymphoid atrophy 0 1/2.0 0 1/4.0 2/1.0 1/1.0
Thymus
Atrophy 0 1/1.0 2/1.0 1/3.0 2/1.5 1/1.0
Conclusions:
Necrotic inflammation in the forestomach was observed in all test item treated groups of both sexes, showing a dose-dependent increase in the severity and in the number of animals affected. Based on the data changes recorded in the study were not considered to be due to the systemic exposure to the test item, and were rather considered to be the primary or secondary results of local irritative effects of the test item in the forestomach, caused by the corrosive nature of the test item. As the forestomach is not a human tissue there is no relevance for human exposure. Based on the results of this study, the NOAEL for systemic toxicity was 100 mg/kg bw/day in male and female rats, which was the highest doses tested. The NOAEL for local toxicity was 50 mg/kg bw/day in male and female rats. As the forestomach is not a human tissue, the exposure is not relevant for humans.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study with the registered substance. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Item 8.6, of Regulation (EC) No. 1907/2006 (REACH).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item was investigated for repeated dose oral toxicity in a 28-day study conducted according to OECD guideline 407 and in compliance with GLP (2013-0392-DGT). Groups of 5 rats/sex were exposed daily to the test item by oral gavage at initial doses of 20, 50 and 125 mg/kg bw/day for a treatment period of 28 days. Due to mortality observed at 125 mg/kg bw/day, the high dose level was reduced to 100 mg/kg bw/day from Day 4 onward. To compensate for the animals that died in the period that the high-dose group was administered 125 mg/kg bw/day, 1 male and 1 female rat per group were added to the high-dose group and administered 100 mg/kg bw/day for the whole treatment period of 28 days. A similar constituted group received the vehicle (PEG 400) and served as control. For the control and high-dose group, recovery (satellite) groups were included. The animals (5/sex for control, 1/sex at 100 mg/kg bw/day for 28 days of treatment and 5/sex at 125 mg/kg bw/day for the first 3 days of treatment and reduced to 100 mg/kg bw/day for the remaining 25 days of treatment) were treated in an identical manner to the animals of the main group, but observed for a 14-day recovery period after treatment before study termination.

During treatment and during recovery, clinical signs and mortality was recorded daily, while body weight and food consumption was recorded weekly. Ophthalmological examinations were performed prior to administration and within the last week of treatment or recovery. In addition, haematological and clinical chemistry parameters were investigated and urinalysis was conducted prior to the sacrifice of the animals. At scheduled necropsy, selected organs were weighed and the animals (that survived to schedule sacrifice and those that died or were terminated prior to schedule sacrifice) were analysed for macroscopic and histopathological findings.

Stability, homogeneity and the concentration of the test item in the vehicle were confirmed by analytical methods. Mortality was mainly observed in animals of high dose group. After two spontaneous deaths at 125 mg/kg bw within the first three days of treatment, the high dose level was set to 100 mg/kg bw from study Day 4 onward. During the treatment period, 5/12 males and 2/12 females died. The deaths were observed after 3-4 days of treatment in 1/6 males of the main group and in 2/6 males and 1/6 females of the recovery group, and after 9 and 17 days of treatment in 2/6 males and in 1/6 females of the main group. The deaths were attributed to local lesions in the forestomach and considered to be treatment-related, due to the corrosive effect of the test substance.

Clinical signs of toxicity were observed during the treatment period only. High dose group males showed transient occurences of respiratory sounds, eyes half closed, eschar and bloated belly. In females of the high dose group, nasal discharge, alopecia and bloated belly was noted in the main and recovery group. Transient appearances of respiratory sounds (slight to severe) were observed in females of all dose groups, but more frequently seen in animals of the high dose group. These findings were considered to be treatment-related and toxicologically relevant. In addition, moderate to severe salivation, moving the bedding and piloerection were noted in all treatment-groups and considered to be treatment-related. Slight indications of diarrhea, piloerection, slight salivation and moving the bedding were also noted among control animals, but the clinical signs were more prominent and consistently observed in test item-treated animals. The observations were made immediately after dose administration, therefore the findings were considered to be transient and non-adverse.

Animals of the lower dose groups showed a slightly reduced body weight gain at the beginning of treatment, which was not considered to be treatment related. In contrast, there was a considerably attenuated body weight gain in males of the high dose group during the first and second week of treatment (approx. 15 and 70% of controls, respectively). In females, an increased body weight gain was noted during this the first two weeks of treatment, followed by a slight decrease during the third week of treatment. The changes in body weight development in animals of the high dose group were attributed to the poor health condition of individual animals and therefore attributed to treatment and considered to be toxicologically relevant. Consistent with this finding, animals of the high dose group showed a reduced food consumption over the whole treatment period. Decreased food consumption in males during the first two study weeks (-39.4% and -16.9%) were attributed to two isolated males which were found dead or prematurely killed for ethical reasons. A slight decrease within the first two study weeks (-15% and -18.9%) was also observed in the male recovery group, which were attributed to individual animals with poor health condition. In females there was a considerable decrease in food consumption in first study week in animals of the main group (-41.6%, statistically not significant) and a slight decrease (-14.8%) in animals of the recovery group. All findings in males and females were considered to be secondary due to histopathological changes in the forestomach and attributed to treatment. All observations were found to be fully reversible in recovery animals after two weeks of recovery.

Haematology revealed a statistically significant decrease in haemoglobin in males of all test item-treated groups. The values remained all within the range of historical control data, therefore the changes on haemoglobin were not attributed to treatment and considered to be incidental. At 50 mg/kg bw/day, a statistically significant decrease in red blood cell count was observed in males at the end of the treatment period, as well as a statistically significant increase in basophils in females. All findings were fully reversible after two weeks of recovery, without dose-relation and remained within the laboratory’s historical control data and were therefore considered to be unrelated to treatment. In the high dose group, a statistically significant decrease in haematocrit was noted in males. The changes remained within the historical control range and were fully reversible after two weeks of recovery and therefore considered to be not treatment-related. High dose group males of the recovery group showed a statistically significant decrease in mean reticulocytes and a statistically significant decrease in white blood cells. The findings were not consistent with animals of the main group and therefore not attributed to treatment. In females, there was a statistically significant increase in mean corpuscular volume and in reticulocytes at the end of treatment at 50 and 100 mg/kg bw/day, but a dose-response relationship was not evident. The effects were fully reversible after 2 weeks of recovery in recovery animals and remained within the historical control range. Thus, all changes on haematology were not attributed to treatment and considered to be incidental.

In clinical biochemistry at the end of treatment, there was a (statistically non-significant) increase in alanine and aspartate aminotransferase and in cholesterol in males and females of all test item-treated groups. The findings occurred without dose-relation and were within the laboratory’s historical control range and thus considered to be without treatment-relation. In the high dose group, a statistically significant increase in total bile acids was noted in males at the end of treatment, which was fully reversible after two weeks of recovery. In addition, a statistically significant decrease in total protein was observed in male recovery animals at the end of recovery. The observations remained within the historical control range and were without histopathological abnormalities, therefore the findings were considered to be not treatment-related.

Urinalysis revealed a number of findings in animals of the high dose group only, comprising high levels of erythrocytes at the end of the recovery period in both sexes and high levels of glucose and high urinary protein content at the end of treatment or at the end of the recovery period in individual animals. All observations were slight, in the normal range of normal variation and without dose relation and therefore not attributed to treatment.

At gross necropsy, thickened and/or dark red mucosa of the stomach and several foci was observed in the majority of animals that died or were sacrificed prior to scheduled termination, but also in surviving animals of the control (1/5 males and 0/5 females), mid (2/5 males and 0/4 females) and high dose group (3/3 males and 0/5 females). /. The findings were indicative of necrotising inflammation and in line with histopathological abnormalities found at microscopical examination. The observations were considered to be related to treatment, and a result of local irritating effects of the test substance. In the recovery group, the necrotic and inflammatory lesions showed a tendency to disappear after 14 days of recovery, but the mentioned lesions were still present in 2/4 surviving males and in and 0/5 Surviving females of the recovery group.

Organ weight analysis revealed a statistically significant increase in relative liver weight in males of all dose groups and in females at 50 mg/kg bw/day, which was not dose-related, reversible in recovery animals and, in the absence of histopathological findings, related to metabolic adaption. Increases in absolute and relative spleen weight and in relative thyroid weight were noted at 50 and 100/125 mg/kg bw/day in males at the end of the treatment period only. Because the observations were sex specific and without histopathological abnormalities, the relevance is unclear. However, these effects are not considered to be toxicologically relevant as they were not observed at the end of the recovery period.

Histopathological examination confirmed the findings of necrotising inflammation observed during gross necropsy. Forestomach epithelial (squamous) hyperplasia and hyperkeratosis, parakeratosis and/or dyskratosis were noted in animals of all treatment groups and showed a dose dependentincrease in the incidence and severity. In high-dose recovery animals, necrotic and inflammatory lesions, dyskeratosis and reactive hyperplasia were no longer present. Instead, submucosal granulation indicating the healing process of necrotizing inflammation was observed in the forestomach. Hyperkeratosis without parakeratosis was still observed in recovery animals, but the severity regressed after 14 days recovery period.The observations were considered to be treatment-related and to be the primary or secondary result of local irritating effects, rather than to be a result of systemic toxicity. As the forestomach is not a human tissue, there is no relevance for human exposure.

Based on the experimental findings and under the conditions of the test, the NOAEL for local toxicity was 50 mg/kg bw/day for male and female rats. The local effects were observed in the forestomach and are not considered to be relevant to humans. The NOAEL for systemic toxicity was derived to be 100 mg/kg bw/day for male and female rats.

Justification for classification or non-classification

Oral repeated exposure with the test item induced necrotising inflammation in the forestomach of both sexes with a dose dependently increasing incidence and corresponding histopathological findings. The observations were considered to be the primary or secondary result of local irritating effect of the test item in the forestomach rather than to be caused by systemic toxicity. As the forestomach is not present in humans, there is no relevance for human exposure. The NOAEL for systemic toxicity in male and female rats was derived to be 100 mg/kg bw/day.

The available data on repeated dose toxicity following oral administration do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.

No classification for repeated dose toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.