Registration Dossier
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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 231-335-2 | CAS number: 7493-74-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 Feb - 08 Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- combined repeated dose and reproduction / developmental screening
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 Feb - 08 Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- July 29, 2016
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD (SD), SPF
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.
VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL, respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
- Duration of treatment / exposure:
- Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until the day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13
Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration. - Frequency of treatment:
- once daily, 7 days/week
- Dose / conc.:
- 15 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 12 (main groups)
6 (recovery groups; for control and high dose groups) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of the vehicle control were administered with the vehicle alone (corn oil).
- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition
BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No
HEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: not specified
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: total leukocyte count (WBC), mean corpuscular hemoglobin (MCH), total red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC), hemoglobin (HGB), platelet count (PLT), hematocrit (HCT), reticulocyte count (absolute (RETA) and relative (RET%) counts), mean corpuscular volume (MCV), WBC differential count (absolute and relative (%) differential counts: including neutrophils (NEU), eosinophils (EOS), basophils (BAS), monocytes (MON), lymphocytes (LYM) and other cells as appropriate (i.e.; large unstained cells (LUC)), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: 16 h (overnight) prior to scheduled sacrifice
- How many animals: all scheduled sacrifice animals in the main and the recovery groups
- Parameters checked: glucose (GLU), alanine aminotransferase (ALT), blood urea nitrogen (BUN), total bilirubin (TBIL), creatinine (CREA), alkaline phosphatase (ALP), total protein (TP), gamma glutamyl transpeptidase (GGT), albumin (ALB), creatine phosphokinase (CK), albumin/globulin ratio (A/G), calcium (Ca), total cholesterol (TCHO), inorganic phosphorus (IP), triglyceride (TG), sodium (Na), phospholipid (PL), potassium (K), aspartate aminotransferase (AST), chloride (Cl)
URINALYSIS:No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before scheduled sacrifice
- Dose groups that were examined: first six animals per sex in main group (if possible) and in all animals of the recovery group
- Battery of functions tested: sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity
IMMUNOLOGY: No
OTHER:
Thyroid Hormone Analysis:
- Time schedule for examinations: at termination
- Dose groups that were examined: all adult animals in main and recovery group
- Parameters checked: thyroid hormone (T4) and the thyroid stimulating hormone (TSH) - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
The animals were carefully examined for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.
ORGAN WEIGHTS: Yes (see table 1)
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.
HISTOPATHOLOGY: Yes (see table 2)
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except reproductive organs collected from first six animals per sex in main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically. - Statistics:
- Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the control and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.
In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.
Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no changes observed in the reproductive system in this animal. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency. - 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):
- In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(see table 3) - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results due to a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group.
(see table 4) - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased absolute and relative weights of liver (absolute weights: up to 1.27-folds over control, relative weights: up to 1.24-folds over control) and spleen (absolute weights: up to 1.42-folds over control, relative weights: up to 1.37-folds over control) were noted in both sexes at 150 mg/kg bw/day. (see table 4) Also, decreased absolute weights of the left and right testis (88% of control, respectively), and the left and right epididymides (91% of control, respectively) were observed. These changes did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related changes were observed in liver, stomach, spleen and hepatic lymph node of males and/or females at 150 mg/kg bw/day.
In the liver, yellow or dark-red discoloration, yellow focus/foci, enlarged, small, abnormal shape and/or irregular surface of all lobe or specific lobes (caudate, left or median lobe) were observed in both sexes at 150 mg/kg bw/day. After recovery period, a yellow discoloration and/or irregular liver surface were still observed in both sexes at 150 mg/kg bw/day.
In the stomach, adhesion of the stomach (stomach to liver) was observed in one male at the recovery at 150 mg/kg bw/day.
In the spleen, enlargement was observed in one male at 150 mg/kg bw/day. After the recovery period, enlargement was also observed in one female at 150 mg/kg bw/day and white discoloration was observed in one male and female at 150 mg/kg bw/day.
Enlarged hepatic lymph nodes were observed in both sexes at 150 mg/kg bw/day, but they were not observed any more at the end of the recovery period.
Other macroscopic findings noted in the main and the recovery groups were considered to be incidental or spontaneous changes that are commonly seen in SD rats with a given low incidence or severity. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.
Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.
Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period, lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia and slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.
Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.
Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed at 50 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical biochemistry
- gross pathology
- haematology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The NOAEL for systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day.
Table 3. Haematological findings
Group |
|
WBC (x10³/µL)
|
LYMA (x10³/µL)
|
MONA (x10³/µL) |
LUCA (x10³/µL)
|
FIB (mg/dL)
|
PLT (10³/µL)
|
males |
|||||||
Control |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
10.45 |
8.01 |
0.35 |
0.12 |
182.6 |
994.3 |
|
SD |
2.165 |
1.414 |
0.134 |
0.047 |
9.06 |
86.03 |
|
15 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
12.33 |
9.75 |
0.44 |
0.14 |
186.0 |
974.8 |
|
SD |
2.937 |
2.488 |
0.221 |
0.052 |
7.16 |
111.10 |
|
50 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
11.52 |
9.07 |
0.4 |
0.14 |
191.8 |
1118.4*R |
|
SD |
3.175 |
2.476 |
0.148 |
0.075 |
11.87 |
82.91 |
|
150 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
14.64+D |
11.76+D |
0.50 |
0.16 |
223.2+R |
1200.6+R |
|
SD |
3.732 |
3.182 |
0.265 |
0.079 |
30.48 |
227.41 |
|
Recovery group - Control |
n |
6 |
6 |
6 |
6 |
6 |
6 |
mean |
10.17 |
7.42 |
0.39 |
0.10 |
199.9 |
1095.7 |
|
SD |
2.447 |
1.609 |
0.127 |
0.041 |
10.97 |
88.91 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
6 |
6 |
6 |
6 |
mean |
10.93 |
8.90 |
0.29 |
0.12 |
202.4 |
1105.0 |
|
SD |
1.803 |
1.311 |
0.092 |
0.038 |
7.12 |
166.78 |
|
females |
|||||||
Control |
n |
9 |
9 |
9 |
9 |
9 |
9 |
mean |
9.37 |
6.22 |
0.47 |
0.12 |
216.3 |
1190.2 |
|
SD |
1.673 |
1.994 |
0.12 |
0.049 |
22.14 |
236.35 |
|
15 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
8.66 |
5.81 |
0.42 |
0.11 |
14.7 |
1194.0 |
|
SD |
2.041 |
1.726 |
0.152 |
0.035 |
0.46 |
221.87 |
|
50 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
12 |
12 |
mean |
9.42 |
6.08 |
0.38 |
0.12 |
15.2*R |
1237.7 |
|
SD |
2.808 |
2.307 |
0.123 |
0.050 |
0.60 |
139.20 |
|
150 mg/kg bw/day |
n |
11 |
11 |
11 |
11 |
11 |
11 |
mean |
12.14*D |
8.55 |
0.76+D |
0.19*R |
15.1 |
1289.4 |
|
SD |
2.691 |
2.644 |
0.186 |
0.086 |
1.03 |
329.31 |
|
Recovery group - Control |
n |
6 |
6 |
6 |
6 |
6 |
6 |
mean |
6.86 |
5.42 |
0.17 |
0.08 |
172.1 |
1093.2 |
|
SD |
0.903 |
0.646 |
0.071 |
0.025 |
6.48 |
118.97 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
6 |
6 |
6 |
6 |
mean |
6.23 |
4.92 |
0.20 |
0.08 |
181.1*D |
1219.7 |
|
SD |
1.172 |
1.042 |
0.106 |
0.033 |
3.84 |
270.14 |
R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)
D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)
WBC = total leukocyte count
LYMA = absolute lymphocyte count
MONA = absolute monocyte count
LUCA = absolute large unstained cells counts
FIB = fibrinogen
PLT = platelet count
Table 3. Clinical biochemistry findings
Group |
|
ALT (IU/L) |
AST (IU/L) |
GGT (IU/L) |
TBIL (mg/dL) |
males |
|||||
Control |
n |
12 |
12 |
12 |
12 |
mean |
44.3 |
172.6 |
0.49 |
0.183 |
|
SD |
42.60 |
83.42 |
0.497 |
0.1209 |
|
15 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
mean |
34.4 |
149.4 |
0.41 |
0.152 |
|
SD |
11.40 |
35.09 |
0.293 |
0.0270 |
|
50 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
mean |
34.9 |
152.0 |
0.73 |
0.150 |
|
SD |
6.52 |
16.69 |
0.308 |
0.0234 |
|
150 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
mean |
124.0*R |
251.3 |
4.15+R |
0.195*R |
|
SD |
181.65 |
216.19 |
6.111 |
0.0341 |
|
Recovery group - Control |
n |
6 |
6 |
6 |
6 |
mean |
47.4 |
168.5 |
0.44 |
0.131 |
|
SD |
26.76 |
40.47 |
0.148 |
0.0105 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
6 |
6 |
mean |
53.8 |
162.5 |
0.80+D |
0.140 |
|
SD |
12.46 |
36.32 |
0.207 |
0.0290 |
|
females |
|||||
Control |
n |
9 |
9 |
9 |
9 |
mean |
65.5 |
181.8 |
1.18 |
0.118 |
|
SD |
22.29 |
46.30 |
0.747 |
0.0227 |
|
15 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
mean |
59.8 |
164.5 |
0.78 |
0.122 |
|
SD |
15.82 |
32.37 |
0.355 |
0.0178 |
|
50 mg/kg bw/day |
n |
12 |
12 |
12 |
12 |
mean |
70.1 |
162.1 |
1.45 |
0.135 |
|
SD |
16.19 |
27.38 |
0.564 |
0.0237 |
|
150 mg/kg bw/day |
n |
11 |
11 |
11 |
11 |
mean |
134.0 |
259.5 |
10.01+R |
0.143 |
|
SD |
176.91 |
210.57 |
8.246 |
0.0336 |
|
Recovery group - Control |
n |
6 |
6 |
6 |
6 |
mean |
44.1 |
142.8 |
1.08 |
0.197 |
|
SD |
10.60 |
25.76 |
0.252 |
0.0289 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
6 |
6 |
mean |
43.2 |
190.2+D |
1.15 |
0.175 |
|
SD |
13.61 |
25.34 |
0.425 |
0.0341 |
R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)
D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)
ALT = alanine aminotransferase
AST = aspartate aminotransferase
GGT = gamma glutamyl transpeptidase
TBIL = total bilirubin
Table 4. Findings in organ weight
Group |
|
Liver absolut (g) |
Liver relative (g/kg bw) |
males |
|||
Control |
n |
12 |
12 |
mean |
13.623 |
2.7296 |
|
SD |
1.8269 |
0.17755 |
|
15 mg/kg bw/day |
n |
12 |
12 |
mean |
13.787 |
2.7626 |
|
SD |
1.2970 |
0.21793 |
|
50 mg/kg bw/day |
n |
12 |
12 |
mean |
14.437 |
2.8353 |
|
SD |
1.7329 |
0.19210 |
|
150 mg/kg bw/day |
n |
12 |
12 |
mean |
16.385+D |
3.3854+D |
|
SD |
1.5829 |
0.23912 |
|
Recovery group - Control |
n |
6 |
6 |
mean |
14.781 |
2.7965 |
|
SD |
1.1645 |
0.19102 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
mean |
15.223 |
2.8543 |
|
SD |
1.2307 |
0.20696 |
|
females |
|||
Control |
n |
9 |
9 |
mean |
12.366 |
3.7479 |
|
SD |
1.3052 |
0.33675 |
|
15 mg/kg bw/day |
n |
12 |
12 |
mean |
12.924 |
3.8280 |
|
SD |
0.8599 |
0.21893 |
|
50 mg/kg bw/day |
n |
12 |
12 |
mean |
12.632 |
3.7100 |
|
SD |
1.0564 |
0.18965 |
|
150 mg/kg bw/day |
n |
11 |
11 |
mean |
15.757+R |
4.5949+R |
|
SD |
2.2078 |
0.52216 |
|
Recovery group - Control |
n |
6 |
6 |
mean |
8.684 |
2.8012 |
|
SD |
0.5044 |
0.06238 |
|
Recovery group 150 mg/kg bw/day |
n |
6 |
6 |
mean |
9.687*D |
3.1183+R |
|
SD |
0.7912 |
0.18695 |
R = Dunn Rank Sum Test Significant at the 0.01 level (+) or at 0.05 level (*)
D = Dunnett LSD Test Significant at the 0.01 level (+) or at 0.05 level (*)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- July 29, 2016
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Allyl phenoxyacetate
- EC Number:
- 231-335-2
- EC Name:
- Allyl phenoxyacetate
- Cas Number:
- 7493-74-5
- Molecular formula:
- C11H12O3
- IUPAC Name:
- allyl phenoxyacetate
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD (SD), SPF
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10 weeks (males); 12 weeks (females)
- Weight at study initiation: 355.0 - 439.4 g (males); 242.3 - 295.3 g (females)
- Housing: During acclimation, pre-treatment, treatment, postmating and recovery period: 2 animals per cage; Mating period: 1 male and 1 female; Gestation period: 1 mated female; Lactation period: neonates were kept with the dam; animals were kept in stainless-steel cages (255W×465L×200H mm) and dams with pups in polycarbonate cages (260W x 420D x 180H mm) on aspen animal bedding
- Diet: Lab Diet® #5053 (PMI Nutrition International, USA) irradiated by gamma-ray, ad libitum
- Water: filtered, ultraviolet light-irradiated municipal tap water, ad libitum
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: Microbial monitoring for diet was performed and a certificate of analysis for the diet was provided by the supplier. The drinking water was analyzed every 6 months for specified contaminants.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 - 22.8
- Humidity (%): 40.9 - 53.8
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil. Dose formulations were prepared at least one time per week and stored at room temperature. Dose formulations were transferred to the animal room at room temperature.
VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
- Amount of vehicle: 2 mL/kg
- Lot/batch no.: MKBZ9899V - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The verification of the dose level concentration was determined by gaschromatography prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of dose formulations were 106.84, 102.04, and 101.53% at the dose levels of 7.5, 25, and 75 mg/mL respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
- Duration of treatment / exposure:
- Main groups:
males: at least 50 days, starting 2 weeks before mating, during mating until day prior to sacrifice
females: for 2 weeks prior to mating and continued until lactation day 13
Recovery groups:
at least 50 days; Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration. - Frequency of treatment:
- once daily, 7 days/week
- Details on study schedule:
- not applicable
Doses / concentrationsopen allclose all
- Dose / conc.:
- 15 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 12 (main groups)
6 (recovery groups; for control and high dose groups) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose level was selected based on the results of a repeated 2-week dose range-finding study in Sprague-Dawley rats in which doses of 15, 60 and 240/150 mg/kg bw/day have been tested. In that study, one found dead female was observed on treatment day 5 at dose level of 240 mg/kg bw/day. The dose level of the high dose group was changed from 240 to 150 mg/kg bw/day on treatment day 6. Increased AST, ALT and yellow discoloration of liver were observed in males at 60 mg/kg bw/day. At 240/150 mg/kg bw/day, general toxicity including soft feces, lacrimation and soiled fur in males, and decreased body weight gain and food consumption in both sexes were observed. In addition, an increased AST and ALT were observed in males. Absolute and relative spleen weights were increased in both sexes. Based on the result of this dose range-finding study, 150 mg/kg bw/day was selected as the high dose, and 50 and 15 mg/kg bw/day were selected as the middle and the low dose, respectively. Animals of vehicle control were administered with the vehicle alone (corn oil).
- Rationale for animal assignment: Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and of group assignment), estrus cycle and for being free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality and morbidity observations were conducted twice daily except for the acclimation and recovery period where animals were observed once daily. In addition, it was conducted once on necropsy day.
- Cage side observations included: mortality/viability, clinical signs and general condition; during the gestation period: signs of abortion or premature delivery
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week
- Observations included: evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition
BODY WEIGHT: Yes
- Time schedule for examinations: first dosing day and then once per week; additionally on gestation days 0, 7, 14 and on lactation days 0, 4 and 13
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption per animal [g food/animal/day] was calculated by subtracting the amount of residual feed from the amount presented
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OTHER:
For haematology, clinical chemistry, neurobehavioural examination refer to IUCLID section 7.5.1 - Oestrous cyclicity (parental animals):
- A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle was examined during the treatment period until mating.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and to allow a correlation with the histopathology of the female reproductive organs. - Sperm parameters (parental animals):
- Parameters examined in P male parental generations:
testis weight, epididymis weight - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: thyroid hormone (T4) and the thyroid stimulating hormone (TSH) in blood on postnatal days 4 and 13
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals on treatment day 50
- Maternal/female animals: on lactation day 14; non-parturition females after 27 days from final mating day
- Recovery group: at least 14 days after first scheduled sacrifice of main group males and females
GROSS PATHOLOGY: Yes
The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined. Special attention was paid to the organs of the reproductive system. For lactating dams, the number of implantation sites (right and left) was counted. For non-parturition females, uterus implantation sites were examined after staining with ammonium sulfide according to KIT SOPs. Pregnancy was confirmed by implantation sites in the uterus.
ORGAN WEIGHTS: Yes
Paired organs were weighed together unless gross abnormalities were present. However, paired reproductive organs were weighed separately. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ/body weight ratios were calculated.
(for details see 7.5.1)
HISTOPATHOLOGY: Yes
Tissues shown in table 2 from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative, and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximately 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.
All tissues except for the reproductive organs collected from the first six animals per sex in the main group were further processed to slides, stained with hematoxylin and eosin (H&E), and examined microscopically. All reproductive organs collected from the main groups were further processed to slides, stained with H&E, and examined microscopically. Target organs from the recovery group animals were additionally evaluated microscopically.
(for details see 7.5.1) - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at postnatal day 13.
- All animals were subjected to external postmortem examinations
GROSS NECROPSY
- Gross necropsy consisted of external examinations - Statistics:
- Mean values and standard deviations were calculated. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using the Pristima System (Version 7.X. Xybion Medical Systems Corporation, USA) or Statistical Analysis Systems (SAS/STAT Version 9.2 and 9.4, USA). Data were considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data were analyzed using the Analysis of Variance (ANOVA), and the significance of inter-group differences were analyzed using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test, and the significance of inter-group differences was assessed between the VC and treated groups using the Dunn’s Rank Sum Test.
For comparing the control group with the recovery group, the data were analyzed for homogeneity for variance using the F-test. Homogeneous data were analyzed using the T-test and the significant difference was assessed between control and recovery group using the Dunnett’s Test. Heterogeneous data were analyzed using the Kruskal-Wallis Test and significant difference was assessed between control and recovery group using the Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weights. The litter size was used as the covariate. Litter data were statistically evaluated using the statistical unit as a litter.
Data presented as frequencies were analyzed by χ2-test followed by the Fisher's exact test where necessary. - Reproductive indices:
- - Precoital Time = No. of days taken to mate
- Mating index (%) = (No. of animals with evidence of mating / No. of animals paired) × 100
- Fertility index (%) = (No. of animals with impregnation or pregnancy / No. of animals paired) × 100
- Fecundity/Pregnancy Index (%) = (No. of animals with impregnation or pregnancy / No. of animals with evidence of mating) x 100
- Unaccounted-for sites (%) = (No. of implantation sites/litter) – (No. of pups born/litter) / No. of implantation sites/litter x 100
- Delivery Index = No. of dams with live pups on Day 0 of lactation / No. of pregnant dams x 100 - Offspring viability indices:
- - Viability Index = No. of live pups on Day 4 of lactation / No. of live pups at birth x 100
- Weaning Index = No. of live pups on Day 13 of lactation / No. of pups on Day 4 of lactation after culling x 100
- Sex ratio = (No. of male pups on PND 0/litter) / (No. of total pups on PND 0/litter) x 100
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related moribund animals were observed in any group throughout the study.
In males at 15, 50 and 150 mg/kg bw/day, salivation was observed in 11, 12, and 18 animals, respectively. In females at 15, 50, and 150 mg/kg bw/day, salivation was observed in 6, 12, and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have any dose-response.
In one female of control group found dead on gestation day 21 one early resorption, one late resorption and 7 dead fetuses were observed in the uterine examination. In the result of microscopic findings, moderate hemorrhage, slight alveolar edema and moderate perivascular congestion were observed in the lung. Moderate centrilobular necrosis in liver, right atrium dilatation in heart, slight atrophy in spleen and minimal mononuclear cell infiltration and hemorrhage in uterus were also observed. These were considered to be incidental since it was observed in the control group and there were no correlated microscopic changes in other animals in the same control group.
Non-parturition was observed in two females of control group and in one female at 150 mg/kg bw/day. Three females were subjected to unscheduled sacrifice on gestation day 27 and all of them were non-pregnant. In the results of microscopic findings, minimal squamous cell hyperplasia in stomach was observed in one animal at 150 mg/kg bw/day, but there were no other changes observed in the reproductive system in this animal. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No test item-related deaths occurred in any group throughout the study.
One female of control group was found dead on gestation day (GD) 21. Two females of control group and one female at 150 mg/kg bw/day were subjected to unscheduled sacrifice on gestation day 27 since they were non-pregnant. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No test item-related changes in body weight and in body weight gain were observed in both sexes during the study. Statistically significant changes in body weight gain during the study were not considered test item-related since they were transient.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No test item-related changes in food consumption were observed in both sexes during the study.
A statistically significant change in food consumption during the study was not considered test item-related since there was no correlation with body weight changes and there was no dose-dependency. - 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):
- In males, total leukocyte count (WBC, 1.40-fold over control), absolute lymphocyte count (LYMA, 1.47-fold over control), absolute monocyte count (MONA, 1.43-fold over control), absolute large unstained cells counts (LUCA, 1.33-fold over control), fibrinogen (FIB, 1.22-fold over control) were increased at 150 mg/kg bw/day and platelet count (PLT, up to 1.21-folds over control) was increased at 50 and 150 mg/kg bw/day. The increases of WBC, LYMA, FIB and PLT were statistically significant. In females, WBC (1.30-fold over control), LYMA (1.37-fold over control), MONA (1.62-fold over control) and LUCA (1.58-fold over control) were increased at 150 mg/kg bw/day. The increases of WBC, MONA and LUCA were statistically significant. These changes recovered or showed a tendency of recovery at the end of the recovery period. Increased leukocyte counts, fibrinogen and platelet count in males and/or females at 150 mg/kg bw/day were correlated with inflammatory changes in the liver.
Other statistically significant changes were not considered test item-related, because these changes were minimal, there were no dose-relationships, and no correlations with microscopic changes and/or observed only in the recovery group.
(for result tables refer to 7.5.1) - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, alanine aminotransferase (ALT, 2.80-fold over control), aspartate aminotransferase (AST, 1.46-fold over control), gamma glutamyl transpeptidase (GGT, 8.47-fold over control) and total bilirubin (TBIL, 1.07-fold over control) were increased at 150 mg/kg bw/day. The increases of ALT, GGT and TBIL were statistically significant. In females, ALT (2.05-fold over control), AST (1.43-fold over control), TBIL (1.21-fold over control) and GGT (8.48-fold over control) were also increased at 150 mg/kg bw/day. The increase of GGT was statistically significant. These changes recovered or showed a tendency to recover at the end of the recovery period. Increases of alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase and total bilirubin in males and/or females at 150 mg/kg bw/day were considered to be results of a massive damage of hepatocytes.
Other statistically significant changes were not considered test item-related, because changes were minimal, there were no dose-relationships, no correlated microscopic changes and/or observed only in recovery group. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related changes in functional behavior examinations were observed in both sexes during the study.
A statistically significant decrease in grip strength of forelimb in males at 50 and 150 mg/kg bw/day was not considered test item-related since there was no dose-dependency and no change in other parameters during the functional behavioral examinations. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Test item-related changes were observed in liver, spleen, hepatic lymph node, and stomach of both sexes at 150 mg/kg bw/day.
Liver:
Slight to marked cholangiofibrosis, minimal to marked hydropic degeneration, minimal to marked necrosis, minimal to marked bile duct hyperplasia, minimal to slight hepatocyte hypertrophy and/or minimal to slight pigmented macrophages in both sexes and slight fibrosis in capsule in one male were observed at 150 mg/kg bw/day. These changes were observed in the periportal region except a fibrosis in capsule. After recovery period, cholangiofibrosis, hydropic degeneration and necrosis were fully recovered and bile duct hyperplasia and hepatocyte hypertrophy showed a tendency of recovery in both sexes at 150 mg/kg bw/day. In the 150 mg/kg bw/day recovery group, minimal to moderate periportal fibrosis in both sexes were also observed and pigmented macrophage was similar to the main groups in both sexes.
Cholangiofibrosis, hydropic degeneration, and necrosis were considered to be an adverse effect of the test-item and are well known and often observed as hepatocellular toxicity caused by various xenobiotics and chemicals. Periportal fibrosis was observed and shown to be a recovery reaction.
Bile duct hyperplasia, hepatocyte hypertrophy, pigmented macrophages and/or fibrosis in the capsule accompanied the above named findings were not considered as an adverse effect at the severity as seen here. These changes were considered to be adaptive changes to a drug-induced increase in a metabolic workload or were considered as secondary findings related to the aforementioned adverse effects in the liver.
Spleen and hepatic lymph node:
Minimal to slight lymphoid hyperplasia in both sexes at 150 mg/kg bw/day and slight chronic inflammation in the capsule in one female at 150 mg/kg bw/day were observed. At the end of the recovery period and lymphoid hyperplasia showed a tendency of recovery in both sexes at 150 mg/kg bw/day and a capsular fibrosis in both sexes and chronic inflammation in capsule were observed in females at 150 mg/kg bw/day. In the hepatic lymph node, moderate lymphoid hyperplasia, slight congestion were observed in both sexes at 150 mg/kg bw/day.
These changes in spleen and hepatic lymph nodes were considered to be associated with a secondary response to the inflammatory changes in the liver.
Stomach:
Minimal to slight squamous cell hyperplasia in the non-glandular region was observed in both sexes at 150 mg/kg bw/day and fully recovered after the recovery period. This change was considered to be non-adverse but an adaptive response following a local irritation caused by the test item since it fully subsided after the recovery period.
Other changes observed in microscopic findings were considered as incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among the control and the treated groups. - Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in the adult male animals.
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- A decreased absolute weight of testes and epididymides in males at 150 mg/kg bw/day did not represent a meaningful toxicologic finding as there were no correlated microscopic findings and/or it fully recovered during the recovery period.
- Reproductive performance:
- no effects observed
Effect levels (P0)
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- fertility
- Effect level:
- >= 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects observed up to the highest dose tested
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- haematology
- clinical biochemistry
- organ weights and organ / body weight ratios
- gross pathology
- histopathology: non-neoplastic
Target system / organ toxicity (P0)
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significant decrease in body weight of the F1 pups was observed on PND 13 (male pups: 92% of VC, female pups: 91% of VC) at 150 mg/kg bw/day.
(see table 1) - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormone anylysis:
No test item-related changes in the thyroid hormone (T4) and the thyroid stimulating hormone (TSH) were observed in pups.
Effect levels (F1)
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No adverse effects observed at 50 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Generation:
- F1
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
Target system / organ toxicity (F1)
- Key result
- Critical effects observed:
- no
Overall reproductive toxicity
- Key result
- Reproductive effects observed:
- no
Any other information on results incl. tables
Table 1. Pups body weight (males)
Group |
|
Control |
15 mg/kg bw/day |
50 mg/kg bw/day |
150 mg/kg bw/day |
PND 0 |
Mean |
7.04 |
6.75 |
6.96 |
7.05 |
SD |
0.44 |
0.65 |
0.35 |
0.50 |
|
Covariate adjusted mean |
6.97 |
6.79 |
7.01 |
7.01 |
|
n |
9 |
12 |
12 |
11 |
|
PND 4 (preculling) |
Mean |
11.52 |
11.11 |
11.08 |
10.71 |
SD |
1.25 |
1.69 |
0.97 |
0.85 |
|
Covariate adjusted mean |
11.35 |
11.08 |
11.28 |
10.65 |
|
n |
9 |
12 |
12 |
11 |
|
PND 4 (postculling) |
Mean |
11.82 |
11.38 |
11.51 |
11.02 |
SD |
1.32 |
1.63 |
0.99 |
0.83 |
|
n |
9 |
12 |
12 |
11 |
|
PND 13 |
Mean |
35.78 |
35.13 |
35.05 |
32.32+D |
SD |
1.46 |
2.93 |
1.83 |
2.68 |
|
Covariate adjusted mean |
35.51 |
34.98 |
35.24 |
32.51*D |
|
n |
9 |
12 |
12 |
11 |
+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)
Table 2. Pups body weight (females)
Group |
|
Control |
15 mg/kg bw/day |
50 mg/kg bw/day |
150 mg/kg bw/day |
PND 0 |
Mean |
6.58 |
6.32 |
6.646.96 |
6.637.05 |
SD |
0.41 |
0.63 |
0.38 |
0.53 |
|
Covariate adjusted mean |
6.51 |
6.36 |
6.69 |
6.59 |
|
n |
9 |
12 |
12 |
11 |
|
PND 4 (preculling) |
Mean |
10.82 |
10.46 |
10.66 |
10.26 |
SD |
1.27 |
1.67 |
0.97 |
0.98 |
|
Covariate adjusted mean |
10.64 |
10.44 |
10.88 |
10.20 |
|
n |
9 |
12 |
12 |
11 |
|
PND 4 (postculling) |
Mean |
10.99 |
10.72 |
11.02 |
10.48 |
SD |
1.19 |
1.67 |
1.11 |
1.10 |
|
n |
9 |
12 |
12 |
11 |
|
PND 13 |
Mean |
34.09 |
33.60 |
34.19 |
30.64*R |
SD |
1.46 |
3.27 |
1.71 |
3.64 |
|
Covariate adjusted mean |
33.86 |
33.47 |
34.36 |
30.80*D |
|
n |
9 |
12 |
12 |
11 |
* R = Dunn Rank Sum Test Significant at the 0.05 level+D = Dunnett LSD Test Significant at the 0.01 level or at 0.05 level (*)
Applicant's summary and conclusion
- Conclusions:
- The NOAEL for parental systemic toxicity was considered to be 50 mg/kg bw/day in both sexes, based on cholangiofibrosis, hydropic degeneration and necrosis of liver in both sexes at 150 mg/kg bw/day. The NOAEL for parental fertility was considered to be 150 mg/kg bw/day, since no adverse effects were observed on parental reproductive function.
The NOAEL for developmental toxicity was considered to be 50 mg/kg bw/day based on decreased pup body weights on postnatal day 13 at 150 mg/kg bw/day. This effect was considered to be a secondary effect of parental systemic toxicity. No further effect on development was observed.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.