Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 03, 2016 to May, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Minor study plan deviations that did not impact the study integrity
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation:
Males: approximately10 weeks.
Females: approximately 11 weeks.
- Housing: Animals were housed in groups of 5 animals of the same sex in Macrolon plastic cages (MIV type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): tap water
- Acclimation period: At least 5 days prior to start treatment.


ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities.
Route of administration:
oral: gavage
Details on route of administration:
Using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
corn oil
Details on oral exposure:
Dose volume: 5 mL/kg body weight.
Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature protected from light was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
- Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day prior to scheduled necropsy.
- Females that delivered were exposed for 42-47 days (most females) or 54 days (one female), i.e. two weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42 days.
Routinely, females that are littering are left undisturbed.The omission of one day of dosing over a period of several weeks is considered not to affect the toxicological evaluation
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on a preliminary dose range-finder
- Rationale for animal assignment (if not random): random
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortalitty/viability checked at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION :
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes (F0 generation only)
- Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION (FUNCTIONAL OBSERVATIONS): Yes
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested during the lactation period from lactation Day 4 onwards (all before blood sampling). These tests were be performed after observation for clinical signs (incl. arena observation, if applicable).

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test (Ref. 4) was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Salivation was noted after dosing among animals treated at 100 mg/kg (one male), 300 mg/kg (two males, all females) or 1000 mg/kg (all animals), mostly at a slight degree, with a dose-dependent time of onset (starting at Day 6 at 1000 mg/kg and at Day 18 at the lower dose levels). This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption before or after allowance for body weight were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were not affected by treatment.
Statistically significant differences noted in the number of platelets in females (lower at 300 and 1000 mg/kg) were considered to be unrelated to treatment due to the absence of a dose-related response and all values were within normal limits.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean alanine aminotransferase (ALAT) activity was slightly higher at 1000 mg/kg in both sexes (statistically significant in females only), particularly due to moderately higher values in a few individual animals.
Slightly decreased levels of total bilirubin were observed in all treatment groups (statistically significant for females treated at 300 and 1000 mg/kg). All values were within the range of the historical control data and were therefore considered not toxicologically relevant.
Other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment as these changes occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.
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 was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar 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):
There were test item-related organ weight changes in the liver (both sexes), kidneys (males only) and possible test item-related organ weight changes in the thymus (males only). For the thymus weights, all values of the treated males were within the range of the historical control data. There were no other test item-related organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the kidneys of males treated at 300 or 1000 mg/kg, and in the thyroid gland and liver of males and females treated at 1000 mg/kg.
An increased incidence and severity of hyaline droplet accumulation was present in males at 300 and 1000 mg/kg up to moderate degree.
An increased incidence and severity of tubular basophilia was present in males at 1000 mg/kg up to moderate degree.
Granular casts were present in males at 1000 mg/kg up to marked degree. In the liver, hepatocellular hypertrophy was present in males and females at 1000 mg/kg at minimal degree.
In the thyroid gland, follicular cell hypertrophy was present at slightly increased incidence and severity in both sexes at 1000 mg/kg up to slight (males) or minimal (females) 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:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 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.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the study conditions, the NOAEL was considered to be 300 mg/kg bw/day based on histopathological renal changes at 1000 mg/kg bw/day in males.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance in rat according to OECD Guideline 422, in compliance with GLP. The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy (for 29 d). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation (mostly for 42-47 d). Females that failed to deliver offspring were treated for 42 d. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 h at room temperature protected from light.

No toxicologically relevant changes were noted in the in-life parameters examined in this study up to 1000 mg/kg bw/day (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption). Treatment-related findings were limited to slight salivation after dosing in a single male at 100 mg/kg bw/day and in up to all animals at the higher dose levels. This was considered to be a physiological response rather than a sign of systemic toxicity. At microscopic examination, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted in the kidneys of male rats treated at 300 or 1000 mg/kg bw/day. The hyaline droplets were considered to represent alpha2u globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. At 1000 mg/kg bw/day, the increased hyaline droplet accumulation was accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia. This combination of findings was considered to be adverse. The higher kidney weights of males at 1000 mg/kg bw/day were considered to be related to the microscopic renal changes. At 300 mg/kg bw/day, the increased hyaline droplet accumulation occurred in the absence of indicators of renal tubular damage and was, therefore, considered to be non-adverse. Males and females treated at 1000 mg/kg bw/day showed several changes indicative of an effect on the liver, namely an increase in liver weight (liver to body weight ratios were about 20% higher), and, in a few animals, minimal hepatocellular hypertrophy and moderately higher plasma activity of alanine aminotransferase (ALAT). These findings were considered to be non-adverse as they occurred in the absence of histological evidence of liver damage and corroborative clinical pathology changes. The test substance related increase in incidence and severity (up to slight) of follicular cell hypertrophy observed in the thyroid of males and females treated at 1000 mg/kg bw/day was regarded to be an adaptive response to the induction of hepatic enzymes and considered non-adverse at the incidences and severities recorded. A test substance related effect on the thymus weights could not be excluded, however, the statistically significantly reduced thymus weights in males treated at 300 and 1000 mg/kg bw/day were likely the result of relatively high values in the vehicle control group. All values of the treated males were within the range of the historical control data. Furthermore, these organ weight changes were neither corroborated by microscopic changes in the thymus nor by any other indicator of toxicity. No treatment-related or toxicologically relevant changes were noted in haematology parameters or findings at macroscopic examination.

Under the study conditions, the NOAEL was considered to be 300 mg/kg bw/day based on histopathological renal changes at 1000 mg/kg bw/day in males (Hartman-Van Dycke, 2017).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Good quality database
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

Additional information

A study was conducted to determine the repeated dose toxicity of the test substance in rat according to OECD Guideline 422, in compliance with GLP. The substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy (for 29 d). The females that delivered were treated for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 d of lactation (mostly for 42-47 d). Females that failed to deliveroffspringwere treated for 42 d. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 h at room temperature protected from light.

No toxicologically relevant changes were noted in the in-life parameters examined in this study up to 1000 mg/kg bw/day (i.e. mortality, clinical appearance, functional observations, body weight and body weight gain, food consumption). Treatment-related findings were limited to slight salivation after dosing in a single male at 100 mg/kg bw/day and in up to all animals at the higher dose levels. This was considered to be aphysiological response rather than a sign of systemic toxicity.At microscopic examination, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted in the kidneys of male rats treated at 300 or 1000 mg/kg bw/day. The hyalinedroplets were consideredto represent alpha2u globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. At 1000 mg/kg bw/day, the increased hyaline droplet accumulation was accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia. This combination of findings was considered to be adverse. The higher kidney weights of males at 1000 mg/kg bw/day were considered to be related to the microscopic renal changes. At 300 mg/kg bw/day, the increased hyaline droplet accumulation occurred in the absence of indicators of renal tubular damage and was, therefore, considered to be non-adverse. Males and females treated at 1000 mg/kg bw/day showed several changes indicative of an effect on the liver, namely an increase in liver weight (liver to body weight ratios were about 20% higher), and, in a few animals, minimal hepatocellular hypertrophy and moderately higher plasma activity of alanine aminotransferase (ALAT). These findings were considered to be non-adverse as they occurred in the absence of histological evidence of liver damage and corroborative clinical pathology changes. The test substance related increasein incidence and severity (up to slight) of follicular cell hypertrophy observed in the thyroid of males and females treated at 1000 mg/kg bw/day was regarded to be an adaptive response to the induction of hepatic enzymes and considered non-adverse at the incidences and severities recorded. A test substance related effect on the thymus weights could not be excluded, however, the statistically significantly reduced thymus weights in males treated at 300 and 1000 mg/kg bw/day were likely the result of relatively high values in the vehicle control group. All values of the treated males were within the range of the historical control data. Furthermore,these organ weight changes were neither corroborated bymicroscopic changes in the thymus nor by any other indicator of toxicity. No treatment-related or toxicologically relevant changes were noted in haematology parameters or findings at macroscopic examination.

Under the study conditions, the NOAEL was considered to be 300 mg/kg bw/day based on histopathological renal changes at 1000 mg/kg bw/day in males (Hartman-Van Dycke, 2017).

Justification for classification or non-classification

Based on the results of a 28 d oral toxicity study conducted in rats, the test substance does not warrant classification for repeated dose toxicity according to the EU CLP criteria (EC 1272/2008).