Registration Dossier

Administrative data

Description of key information

In a GLP-compliant OECD guideline 407 study, adverse effects on kidneys (tubular necrosis, degeneration and interstitial fibrosis) were observed starting from the lowest dose level of 50 mg/kg bw/day in males. Based on this, this dose level is considered to be a LOAEL. No NOAEL could be derived in the study.

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:
4 July 2017 - 7 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
yes
Remarks:
The study was originally designed as a OECD 422 study. However, as by mistake the strain of the male rats were Wistar instead of Wistar Han and the female rats were Wistar Han, it was decided not to continue the study with the mating period.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
yes
Remarks:
The study was originally designed as a OECD 422 study. However, as by mistake the strain of the male rats were Wistar instead of Wistar Han and the female rats were Wistar Han, it was decided not to continue the study with the mating period.
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
Version / remarks:
2000
Deviations:
yes
Remarks:
The study was originally designed as a OECD 422 study. However, as by mistake the strain of the male rats were Wistar instead of Wistar Han and the female rats were Wistar Han, it was decided not to continue the study with the mating period.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: substance change colour slowly especially in melted aggregate condition under air.
- Solubility and stability of the test substance in the solvent/vehicle: stability in propylene glycol for at least 5 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL (solutions); stability for at least 6 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (solutions). Stability for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions).

OTHER SPECIFICS:
pH 8-9 at concentration of 1% w/w
Specific gravity / density: 1.115 kg/L
Species:
rat
Strain:
other: Wistar Han (females) and Wistar (males)
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. By mistake, male Wistar rats were used in this study. This model is also 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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-13 weeks
- Weight at study initiation: 197-411 g
- Fasting period before study: no
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV) containing appropriate bedding and paper as cage enrichment, except during locomotor activities were they were individually housed in a Hi-temp polycarbonate cages without cage-enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: municipal water, ad libitum, except during locomotor activity measurements.
- Acclimation period: 6 and 7 days for females and males, respectively.

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed at the test facility. It is considered that there are no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 52-68
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 August 2017 To: 15 September 2017
Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because oral gavage is the preferred route for human risk assessment.
Vehicle:
propylene glycol
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 hours after completion of the preparation of the test item.
Test item was heated to a maximum of 87.6°C for maximally 3 hours and 21 minutes before use. Formulations were heated to a maximum temperature of 82.3°C for maximally 16 minutes to obtain visual homogeneity after which the formulations were allowed to cool down. Formulations were released for dosing when they obtained a temperature of 40°C or lower.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

- VEHICLE
- Justification for use and choice of vehicle: based on trial preparations performed at the Test Facility
- Amount of vehicle (if gavage): 5 mL/kg bw
- Specific gravity: 1.036
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples were analysed for homogeneity and accuracy of preparation using a validated method (LC-DAD). The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
Duration of treatment / exposure:
At least 28 days
Frequency of treatment:
Once daily 7 days/week, approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Concurrent vehicle controls (Group 1)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 14-day dose range finding study with oral exposure of Ethanol, 2,2’-[(3-methylphenyl)imino]bis- in rats and in an attempt to produce graded responses to the test item.
Positive control:
Not applicable.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for general health/mortality and moribundity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily beginning during the first administration of the test item and lasting throughout the dosing and recovery periods. During the dosing period, these observations were performed directly after dosing from Day 1 and also approximately 1 hour after dosing from Day 4, based on the peak effect of occurrence of clinical signs in a dose-range finding study.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: subjective appraisal was maintained during the study, but no quantitative investigation introduced.

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: WBC, absolute neutrophils, lymphocytes, monocytes, eosinophils and basophils counts, RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets; PT and APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at terminal necropsy
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined: ALAT, ASAP, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholestero, sodium, potassium, chloride, calcium, inorganic phosphate.

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 of treatment
- How many animals: selected 5 males and 5 females/group
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), fore- and hind-limb grip strength, motor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER:
Estrous cycle: Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (Pretest period), the first 14 days of treatment and on the day of scheduled necropsy. Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, on selected 5 animals/sex/group; examinations included 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.
The following organ weights were determined: brain, epididymis, adrenal glands, prostate glands, seminal vesicles, thyroid, heart, kidneys, liver, ovary, spleen, testes, thymus, uterus.

HISTOPATHOLOGY: Yes, on selected 5 animals/sex/group. Full histopathology was performed on selected controls and high-dose animals; for low- and mid-dose groups only gross lesions and target tissues (kidneys in males and skeletal musles in males and females) were examined.
The full histopathology included examination of the following tissues: artery, aorta, nasopharynx cavity, femur, bone marrow, sternum, brain (cerebellum, mid-brain, cortex), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, thyroid including parathyroid, pituitary gland, preputial gland, prostate, salivary gland, seminal vesicles, gross lesions, gut-associated lymphoid tissues, heart, kidney, cecum, colom, rectum, larynx, liver, lung, mandibular and mesenteric lymph nodes, skeletal muscles, optic and sciatic nerves, ovary, pancreas, skin, duodenum, jejunum, ileum, spinal cord, spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.
Statistics:
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. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The pairwise comparisons with the controls were conducted using Fisher’s exact test whenever the overall test is significant.
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% and or 5% levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A treatment related effect was observed in both males and females at 500 mg/kg bw/day. In week 1 and 2 most animals had a hunched posture and piloerection of the fur. Rales and laboured respiration up to a moderate degree was observed in the females that were euthanized for humane reasons at dose 500 mg/kg bw/day. During the last two days of treatment, red discoloration of the urine was observed in all animals dosed with the test item.
Salivation seen after dosing among all animals dosed with the test item was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other findings observed were considered to be within normal range.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females of 500 mg/kg bw/day group were euthanized for humane reasons at Day 8 and Day 13 of treatment, respectively. Clinical symptoms observed for these animals were rales, piloerection of the fur, hunched posture and gasping for air. Severe body weight loss of 19% was observed for female euthanized on day 8, and squaking was also observed for female euthanized on day 13. For both animals main macroscopic findings were in the gastrointestinal tract as distended with gas. In addition, female euthanized on day 8 had a reddish focus of 5x3 mm on the thymus and another female had a gelatinous thymus and swollen lungs. The microscopic correlate for the reddish focus on the thymus was moderate focal hemorrhage.
For the other findings there was no microscopic correlate.
For female euthanized on day 8 no definite cause of moribundity could be assessed from the slides examined. The minimal muscular necrosis/degeneration in the esophagus suggests this might be gavage related.
Main moribundity-related microscopic findings for female euthanized on day 13 were marked erosion/ulceration of the trachea epithelium with moderate lymphogranulocytic infiltrate. These findings were considered secondary to gavage trauma and therefore this death was considered primarily gavage related.
One male in the high-dose group died during blood sampling, which was considered to be an accidental death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In five males treated at 500 mg/kg bw/day a body weight loss of 2-10% was observed at Day 8 of treatment. Body weights recovered to normal levels during the following days. A statistical reduction in body weights was reached at Day 8 of treatment and a reduction in body weight gain at Day 8 and Day 15 of treatment for males treated at 500 mg/kg bw/day.
In two females, treated at 500 mg/kg bw/day, body weight loss of 10% and 19% (female killed on day 8 in extremis) was observed at Day 8 of treatment. For the surviving female, body weights recovered to normal levels during the following days.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males at 500 mg/kg bw/day, absolute and relative food consumption was lower over Days 1-8 compared to controls. Normal values for absolute and relative food consumption were noted for the remainder of the study period. Food consumption values were similar to control levels for males up to 150 mg/kg bw/day and females up to 500 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
An increased water consumption with an accompanying increase in urine production was observed for all males (including controls). This was investigated by the veterinarian. No issues with the health of the male animals were found that could explain the increase in water consumption. However, later it became apparent that the higher water consumption was caused by the difference in strain (i.e. Wistar versus Wistar Han).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in hematology parameters, all at 500 mg/kg, distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values compared to the control group were indicated between parentheses.
• Lower mean corpuscular haemoglobin concentration (MCHC) in males (4%). As this change was minimal and no corroborative findings were noted, this was not considered toxicologically relevant.
• Higher white blood cell (WBC) count in females (72%). This increase was the result of the observed (non-significant) increase in lymphocytes (68%).
• Higher number of reticulocytes in females (48%; not statistically significant).
• Higher red blood cell distribution width (RDW) in females (13%).
Coagulation parameters of treated rats were considered not to have been affected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in clinical chemistry parameters distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values or fold-change compared to the control group were indicated between parentheses.
• Higher aspartate aminotransferase (ASAT) levels in males at 500 mg/kg bw/day (66%; not statistically significant).
• Higher bilirubin levels in males at 50, 150 and 500 mg/kg bw/day (15%, 23% and 58%, respectively) and in females at 500 mg/kg bw/day (70%). This was not statistically significant for males at 50 mg/kg bw/day.
• Higher bile acids levels in males at 500 mg/kg bw/day (x2; without statistical significance; due to one animal) and in females at 500 mg/kg bw/day (x3).
• Lower glucose levels in males at 500 mg/kg bw/day (17%) without statistical significance. As the control group included one outlier and no statistical significance was reached, this finding was not considered toxicologically relevant.
• Higher chloride levels in males at 50, 150 and 500 mg/kg bw/day (2%, 3% and 3%, respectively) and in females at 50 and 150 mg/kg bw/day (4% and 3%, respectively). Since the increases were small and there was no dose response relationship, this finding was not considered toxicologically relevant.
The statistical significant change noted for sodium concentration for males at 150 mg/kg bw/day was not regarded toxicologically relevant as there was no dose response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity were similar between control and treated animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following changes in organ weights distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values compared to the control group were indicated between parentheses.
• Test item-related lower thymus weights (absolute and relative to body weights) were noted in the 150 and 500 mg/kg bw/day group males (29% decrease at both dose levels compared to controls).
• Test item-related higher kidney weights (relative to body weights) were noted in the 500 mg/kg bw/day treated males (13% increase compared to controls)
• Relative increase in liver weights for females treated with 150 mg/kg bw/day (17%). Since there was no dose response relationship, this finding was not considered toxicologically relevant.
• An increase in absolute kidney weights in females (11%) and a non-statistical increase in relative kidney weights (7%). The increase in absolute kidney weight was considered the result of variation in terminal body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. All of the remaining macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in three females treated with 0, 50 and 500 mg/kg bw/day and in one female treated with 150 mg/kg bw/day, is related to a stage in the estrous cycle and is a normal
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Ethanol, 2,2’-[(3-methylphenyl)imino]bis- were noted in the kidneys of males starting at 50 mg/kg bw/day and the skeletal muscle of the 500 mg/kg bw/day group males and females.
In kidneys, tubular necrosis, single cell in the outer medulla, was present in males starting at 50 mg/kg bw/day up to moderate degree (no clear dose-response). Tubular degeneration in the outer medulla, was present in males starting at 50 mg/kg bw/day up to moderate degree (dose-response increase in severity). Interstitial fibrosis, peritubular in the outer medulla was present in males starting at 50 mg/kg bw/day up to moderate degree (no clear dose-response). Tubular brown pigment, outer medulla was present in males treated at 500 mg/kg bw/day up to slight degree.
Multifocal myofiber degeneration of skeletal muscle was present at increased incidence and severity in males and females treated at 500 mg/kg bw/day up to slight (males) or moderate (females) degree. Inflammatory cell infiltrate was present at increased incidence and severity in males and females treated at 500 mg/kg/day up to slight degree.
The remainder 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to be affected by treatment. All females of the control and treated groups had regular cycles of four to five days during the pre-test and treatment periods.
Details on results:
the following interpretation of the results was provided by the study director:
Observed mortality was not considered test-item related, since the deaths occured occurred early during the study period and no microscopic findings related to treatment with the test-item could be found. Considering the magnitude of the observed body weight loss, the effects on body weights were considered adverse at dose 500 mg/kg bw/day. Red discoloration of the urine observed during the last two days of treatment may be related to the microscopic findings noted for the kidneys.
The observed increases in white blood cell count, reticulocytes and red blood cell distribution in females at 500 mg/kg bw/day were considered non-adverse by the study director in the absence of morphological correlates. Also clinical biochemistry changes (higher ASAT in males at 500 mg/kg bw/day, higher bilirubin levels in males starting at 50 mg/kg bw/day and in females at 500 mg/kg bw/day and higher bile acids levels for both sexes at 500 mg/kg bw/day) were considered non-adverse in the absence of morphologic correlates.A test item-related decrease in thymus weight starting from 150 mg/kg bw/day was considered non-adverse based on the lack of histopatological changes.
In kidneys, tubular necrosis, degeneration and interstitial fibrosis were observed starting from the lowest dose level in males. At the low and mid dose the tubular epithelium was basophilic and columnar compared to basophilic and low cuboidal at the highest dose. This indicates that the tubular epithelium is actively regenerating at the low and mid dose, but at the high dose the regenerating capacity of the tubular cells is decreased. The increased incidence and severity of multifocal myofiber degeneration in skeletal muscle in males and females at 500 mg/kg bw/day was considered to be adverse based on the degenerative nature of the finding.
Key result
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
myofibres
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Formulation analysis:

The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies 110.4 ( n = 6), 108.2 (n = 2) and 103.3 (n = 6), respectively).

The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation 1.3% and 1.5%).

Conclusions:
In a GLP-compliant OECD guideline 407 study, adverse effects on kidneys (tubular necrosis, degeneration and interstitial fibrosis) were observed starting from the lowest dose level of 50 mg/kg bw/day in males. Based on this, this dose level is considered to be a LOAEL. No NOAEL could be derived in the study.
Executive summary:

In a GLP-compliant OECD guideline 407 study, male Wistar and female Wistar Han rats were treated with solutions of the test item at dose levels of 0 (vehicle controls), 50, 150 and 500 mg/kg bw/day in propylene glycol for 28 days. Mortality occurred in two females treated with 500 mg/kg bw/day; however, the deaths were considered unrelated to treatment. The observed clinical signs inluded hunched posture and piloerection at 500 mg/kg bw/day. Severely reduced body weight gain in males, accompanied by reduced food consumption, occurred in males and two females of 500 mg/kg bw/day; this finding was considered adverse. Observed haematological and clinical chemistry changes (increases in white blood cell counts, reticulocytes and red blood cell distribution in females at 500 mg/kg bw/day, increased ASAT in males at 500 mg/kg bw/day, higher bilirubin in males at 50 mg/kg bw/day and females at 500 mg/kg bw/day and higher bile acids in both sexes at 500 mg/kg bw/day) were considered non-adverse in the absence of morphological correlates. A tubular cell injury in kidneys of males, consisting of tubular necrosis, degeneration and interstitial fibrosis in the outer medulla was observed in males starting from 50 mg/kg bw/day.

At the highest dose these findings were accompanied by brown tubular pigment. At the low and mid dose the tubular epithelium was basophilic and columnar compared to basophilic and low cuboidal at the highest dose. This indicates that the tubular epithelium is actively regenerating at the low and mid dose, but at the high dose the regenerating capacity of the tubular cells is decreased. At 500 mg/kg bw/day, the increased incidence and severity of multifocal myofiber degeneration in skeletal muscle were observed in males and females. Based on the results of the study, the NOAEL could not be established; based on the adverse effects in male kidneys at the lowest dose level the LOAEL is considered to be 50 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 (data derived in a GLP-compliant guideline study)
System:
urinary
Organ:
kidney

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

Mode of Action Analysis / Human Relevance Framework

In the available OECD guideline 407 study with rats, a tubular cell injury in kidneys of males, consisting of tubular necrosis, degeneration and interstitial fibrosis in the outer medulla, was observed starting from the lowest tested dose of 50 mg/kg bw/day. In females, only an increase in absolute kidney weights and a non-statistical increase in relative kidney weights was seen at the highest tested dose level of 500 mg/kg bw/day. Adverse effects in kidneys of male rats are often related to the accumulation of alpha-2 -microglobulin, a protein which is not found in female rats and in higher mammals. Alpha-2 -microglobulin nephropathy is characterized by the accumulation of hyaline droples in the proximal tubules, subsequent single cell necrosis, the formation of granular casts at the junction of the proximal tubule and the thin loop of Henle and the presence of regenerative tubules. As alpha-2 -microglobulin is not present in higher mammals, including humans, adverse effects in male rat kidneys caused by alpha-2 -microglobulin accumulation, are conisdered to be irrelevant for human toxicological risk assessment. In the present case, however, no evidence of hyaline droplet accumulation was seen at histology. Furthermore, in the available OECD guideline 421 study with rats, tubular degeneration at a slight degree was also present in one female at the dose level of 300 mg/kg bw/day, and tubular basophilia was present in 3 females up to a moderate degree at the same dose level. It should also be noted that in the OECD guideline 407 study different strains (Wistar male and Wistar Hand female rats) were used in the study, making a direct comparison of the effects observed in two sexes difficult. Observed adverse histopathological changes in female rats in the OECD 421 study suggest that the adverse tubular cell injury seen in male rats in the OECD guideline 407 study are not related to the alpha-2 -microglobulin accumulation. Therefore they should be regarded as potentially relevant to humans.

In addition, the increased incidence and severity of multifocal myofiber degeneration in skeletal muscle were observed in males and females in the OECD guideline 407 at 500 mg/kg bw/day. Based on degenerative nature of this effect it is considered to be adverse. As no species-specific mechanism is indicated, this effect should be regarded as potentially relevant to humans.

Additional information

In a GLP-compliant OECD guideline 407 study, male Wistar and female Wistar Han rats were treated with solutions of the test item at dose levels of 0 (vehicle controls), 50, 150 and 500 mg/kg bw/day in propylene glycol for 28 days. Mortality occurred in two females treated with 500 mg/kg bw/day; however, the deaths were considered unrelated to treatment. The observed clinical signs inluded hunched posture and piloerection at 500 mg/kg bw/day. Severely reduced body weight gain in males, accompanied by reduced food consumption, occurred in males and two females of 500 mg/kg bw/day; this finding was considered adverse. Observed haematological and clinical chemistry changes (increases in white blood cell counts, reticulocytes and red blood cell distribution in females at 500 mg/kg bw/day, increased ASAT in males at 500 mg/kg bw/day, higher bilirubin in males at 50 mg/kg bw/day and females at 500 mg/kg bw/day and higher bile acids in both sexes at 500 mg/kg bw/day) were considered non-adverse in the absence of morphological correlates. A tubular cell injury in kidneys of males, consisting of tubular necrosis, degeneration and interstitial fibrosis in the outer medulla was observed in males starting from 50 mg/kg bw/day.

At the highest dose these findings were accompanied by brown tubular pigment. At the low and mid dose the tubular epithelium was basophilic and columnar compared to basophilic and low cuboidal at the highest dose. This indicates that the tubular epithelium is actively regenerating at the low and mid dose, but at the high dose the regenerating capacity of the tubular cells is decreased. At 500 mg/kg bw/day, the increased incidence and severity of multifocal myofiber degeneration in skeletal muscle were observed in males and females. Based on the results of the study, the NOAEL could not be established; based on the adverse effects in male kidneys at the lowest dose level the LOAEL is considered to be 50 mg/kg bw/day.

Justification for classification or non-classification

In the available OECD guideline 407 study with rats, adverse effects on kidneys, manifested as tubular necrosis, degeneration and interstitial fibrosis, were observed in male rats starting at the lowest dose level of 50 mg/kg bw/day. In accordance with Regulation (EC) 1272/2008 (CLP), adverse toxicological effects which indicate significant organ damage and appreciable cell death in vital organs incapable of regeneration support classification for specific target organ toxicity following repeated exposure. According to Regulation (EC) 1272/2008 (CLP), classification in Category 2 is appropriate when adverse effects in a 90 -day repeated dose toxicity study with rats are seen at the dose levels within the cut-off values of 10-100 mg/kg bw/day. In the present case, the LOAEL of 50 mg/kg bw/day is derived in a 28 -day study with rats. Using a default factor 3 for the extrapolation from subacute to subchronic exposure duration, the extrapolated LOAEL for a 90 -day study would correspond to 16.7 mg/kg bw/day. Therefore classification of the substance as STOT RE Cat. 2, H372 is appropriate in accordance with Regulation (EC) 1272/2008.