Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral: In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats up to a dose level of 500 mg/kg body weight/day (OECD 422; Van Otterdijk, 2016). The NOAEL is established as 50 mg/kg body weight/day. The substance is therefore classified as STOT RE 2, according to CLP Regulation.

Repeated dose toxicity - inhalation: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated dose toxicity - dermal: A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

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:
2015-10-05 to 2016-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other:
Version / remarks:
OECD 421, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other:
Version / remarks:
EPA, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other:
Version / remarks:
EPA, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
Deviations:
no
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M14IB5308
- Expiration date of the lot/batch: 2016-02-03 (retest date)
- Purity test date: 2015-04-23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/ mL) was confirmed as part of the analytical method development and validation study (Test Facility Study No. 509777).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light tan hazy formulation (Groups 2 and 3), tan turbid formulation (Group 4)

OTHER SPECIFICS: correction factor is 1
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
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: males approx. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 303-339 g (males) and 191-246 g (females)
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period:At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities

IN-LIFE DATES: From: 2015-10-05 To: 2016-02-02
Route of administration:
oral: gavage
Details on route of administration:
Method: Oral gavage, using a plastic feeding tube.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose
Vehicle:
propylene glycol
Remarks:
specific gravility 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity of the test item. A correction factor of 1.00 was used. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 1-200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (2015-12-09; day 6 of treatment), according to a validated method (Test Facility Study no. 509777). Three sets of duplicate samples were collected. Two set of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concetnration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of +/- 10% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was <=10%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
31 days (males) except for male nos. 6, 7 and 26: 33 days
52-60 days (females that delivered)
44 and 39 days (female nos. 63 and 77 with total litter loss, resp)
42 days (females which failed to deliver)
Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately 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 (nominal)
Remarks:
group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
group 4
No. of animals per sex per dose:
10 animals/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 509045) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no mortality occurred and there were no clear effects on body weight and food consumption. Clinical signs consisted of occasional hunched posture on 1-3 days during treatment at 500 and 1000 mg/kg and rales in one animal at 500 mg/kg on the last days of treatment. Kidney weights were within normal range, however a clear increase in liver weight and liver to body weight ratio was noted at both 500 and 1000 mg/kg. Based on the increased liver weights, dose levels of 50, 150 and 500 mg/kg were selected for the main study in consultation with the Sponsor
- Rationale for animal assignment: randomized
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least 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 treatment (prior to first treatment) 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, 4, 7 and 13. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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, 4, 7 and 13. Relative food consumption was calculated and reported
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane 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 with K3-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell ditribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscul haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane 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 with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- thyroid hormone analysis

FUNCTIONAL OBSERVATIONS
- Time schedule: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity
Sacrifice and pathology:
SACRIFICE
- Male animals: all surviving animals, following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: all surviving animals, on PND 14-16 (females which delivered), on days 25-27 (females which failed to deliver, with evidence of mating) or within 24 hours of litter loss (females with total litter loss)

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin:
Cervix (F), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY: Yes
- all organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Thyroid glands and liver of all selected 5 males and females of Groups 2 and 3, and the spleen, ovaries and sternal bone marrow of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
Other examinations:
ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported
- The following organ weights and terminal body weight were recorded from the selected 5 animals/ sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavern osus muscle complex (LABC), Testes, Thyroid
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 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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test 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:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment.
One female at 500 mg/kg (no. 72) showed piloerection during three consecutive days in post-coitum. This sign was absent among other animals of the same dose group, and resolved while treatment continued. This clinical sign was therefore not considered to be related to treatment with the test item. Salivation seen after dosing among all test item treated groups 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 clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated unde r the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. One female at 150 mg/kg (no. 63) and one female at 500 mg/kg (no. 77) were sacrificed due to total litter loss and/or difficult parturition.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, body weight and body weight gain of females was lower than control means from day 11 to 20, being statistically significant on most occasions. These effects were considered secondarily to the lower number of pups born at this dose level.
Body weights and body weight gain of other dose groups were considered unaffected by treatment; any other statistically significant changes in body weight gain occurred in the absence of a treatment related trent, and were therefore not considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, food consumption was lower throughout the lactation phase, being statistically significant over Days 4-13. This was attributed to two litters (nos. 78 and 80) that each had only one pup. Food consumption was also statistically significantly lower at 500 mg/kg over Days 17-20 of the post coitum phase.
Food consumption before and after correction for body weight of other dose groups remained in the same range as controld over the treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
One female at 500 mg/kg (no. 75) had lower red blood cell counts, haemoglobin and haematocrit, and higher reticulocyte counts and red cell distribution width. Only for haemoglobin, the lower mean was statistically significant (mean approximately 18% lower than control mean).
Any other statistically significant changes in haematology parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Lower aspartate aminotransferase activity (ASAT) in males at 50, 150 and 500 mg/kg (approximately 28%, 44% and 60% lower than control mean, respectively) and in females at 500 mg/kg (not statistically significant for females due to high standard deviation for the control mean)
- Higher creatinine in males at 500 mg/kg (approximately 9% higher than control mean).
- Higher total protein and albumin in females at 500 mg/kg (approximately 5% and 8% higher than control mean, respectively; not statistically significant).
- Lower urea in females at 500 mg/kg (approximately 24% lower than control mean).
- Higher sodium in females at 500 mg/kg (approximately 2% higher than control mean).
- Lower potassium in females at 500 mg/kg (approximately 18% lower than control mean).

One control female (no. 47) showed a very high aspartate aminotransferase activity and bile acid level. No cause could be found for this high value based on parameters assessed in this study. It was considered that this did not adversely compromise interpretation of the study results. Any other statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend.

Thyroid hormone analyses: at 50, 150 and 500 mg/kg, serum levels of total T4 were statistically significantly lower in males. Mean T4 levels were approx. 22, 29 and 50% lower than controls, resp.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Motor activity was considered unaffected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Grip strength was similar across the groups for both sexes.

The apparent higher mean motor activity of females at 500 mg/kg was due to a high value for one female (no. 74; both total movements and ambulations). Since individual values of other animals of the same group remained in the range of values for control animals, this was not considered to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following test item differences in organ weights were observed between treated and control animals: Higher liver weight of males at 500 mg/kg (both absolute and relative to body weight) and females at 150 mg/kg (both absolute and relative to body weight) and 500 mg/kg (relative to body weight). Relative weight differences to control means were 33 and 27% for males and females at 500 mg/kg respectively, and 23% for females at 150 mg/kg. The increase in female liver weight was also evident at 50 mg/kg (relative weight increased with 19%; not statistically significant); Higher ovary weight at 50 and 500 mg/kg (absolute). Absolute weight difference to control mean was 23 and 25%, respectively (relative weight difference was 19 and 33%, respectively); Higher kidney weight of males at 500 mg/kg (relative to body weight; 18% higher than control mean).
Any other differences in organ weights, including those that reached statistical significance were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, one female (no. 77) had pale discolouration of the liver.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in:
- Thyroid gland: increased incidence and/or severity of hypertrophy of follicular cells in males at 500 mg/kg up to moderate degree and in females at 50, 150 and 500 mg/kg up to slight degree.
- Liver: Hepatocellular centrilobular scattered vacuolation in males and females at 150 and 500 mg/kg up to moderate degree; Hepatocellular centrilobular hypertrophy in males at 150 and 500 mg/kg and in females at 50, 150 and 500 mg/kg up to slight degree.
- Ovaries: increased incidence and severity of interstitial cell hypertrophy in females at 500 mg/kg up to slight degree.
An increased severity of extramedullary hematopoiesis in the spleen was noted in two females at 500 mg/kg (nos. 75 and 77) at marked degree. This finding was considered to be secondary to the failed pregnancy in these two females.
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 of histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
- analysis of dose preparations: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the results, the following NOAEL was derived for parental animals: 50 mg/kg/day (based on hepatocellular centrilobular vacuolation at 150 and 500 mg/kg). With the LOAEL being 150 mg/kg, the substance is therefore classified as STOT RE 2 according to CLP regulation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
other: Hepatobiliary system
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated toxicity: oral

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 50, 150 and 500 mg/kg bw/day via gavage (OECD 422; Van Otterdijk, 2016). The vehicle used was propylene glycol and the test solutions were prepared daily and administered within 6 hours after preparation.

The test item did not cause treatment-related mortality during the study; however, one female at 150 mg/kg and one female at 500 mg/kg were sacrificed due to total litter loss and/or difficult parturition. No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment. Other clinical signs noted were within the range of background findings for rats of this age and strain, lacked a dose-related trend, were considered a physiological response to the taste of the test item (salivation) or resolved during treatment (piloerection).

At 500 mg/kg, body weight and body weight gain of females was lower than control means from day 11 to 20, being statistically significant on most occasions. Body weight and body weight gain at other dose levels were considered unaffected by treatment. Also, food consumption was lower throughout the lactation phase at the highest dose level, being statistically significant over Days 4-13. This was attributed to two litters that each had only one pup. There was no effect on food consumption in other dose groups. One female at 500 mg/kg had lower red blood cell counts, haemoglobin and haematocrit, higher reticulocyte counts and red cell distribution width. All other hematological findings were not considered test item related. There were statistically significant changes in clinical biochemistry parameters such as lower aspartate aminotransferase activity (ASAT) in males at 50, 150 and 500 mg/kg and in females at 500 mg/kg, higher creatinine in males at 500 mg/kg, higher total protein and albumin in females at 500 mg/kg (not statistically significant), lower urea and potassium in females at 500 mg/kg but higher sodium levels. At 50, 150 and 500 mg/kg, serum levels of total T4 were statistically significantly lower in males.

Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.

The following test item differences in organ weights were observed: higher liver weight of males at 500 mg/kg (both absolute and relative to body weight) and females at 150 mg/kg (both absolute and relative to body weight) and 500 mg/kg (relative to body weight), higher ovary weight at 50 and 500 mg/kg (absolute).

At 500 mg/kg, one female had pale discoloration of the liver. Test item-related microscopic findings were: increased incidence and/or severity of hypertrophy of follicular cells, hepatocellular centrilobular scattered vacuolation, hepatocellular centrilobular hypertrophy, increased incidence and severity of interstitial cell hypertrophy. An increased severity of extramedullary hematopoiesis in the spleen was noted in two females at 500 mg/kg at marked degree. This finding was secondary to the failed pregnancy in these two females.

Based on the abovementioned considerations, the NOAEL was established as 50 mg/kg bw/day.

Repeated toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1).

Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Repeated toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1).

Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure.

Justification for classification or non-classification

For T001159, the NOAEL was established at 50 mg/kg bw/day, with an LOAEL of 150 mg/kg/day. Therefore, the test item is to be classified as STOT RE 2 according to CLP Regulation.