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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 452-330-3 | CAS number: -
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- sub-chronic toxicity: oral
- Remarks:
- The oral sub-chronic toxicity study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 October 2015 - 28 April 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- -
- EC Number:
- 452-330-3
- EC Name:
- -
- Cas Number:
- 314020-40-1
- Molecular formula:
- C14H20N2O2
- IUPAC Name:
- 2-(2,6-diethyl-4-methyl-phenyl)propanediamide
- Test material form:
- solid: particulate/powder
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:WI(Han)
- Details on species / strain selection:
- Commonly used rodent species
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 201-272 g (males), 136-180 g (females)
- Fasting period before study: No
- Housing: Groups of five, by sex, in grid-floor cages suspended over paperlined trays.
- Diet: Pelleted diet, LabDiet 5L0S EURodent Diet (manufactured by PMI Nutrition International), supplied by International Product Supplies, ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: 13 days
DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for diet and water were retained within the Sequani archives. It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objective from being achieved.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 October 2015 To: 28 April 2016
Administration / exposure
- Route of administration:
- oral: gavage
- Details on route of administration:
- Animals were dosed once daily for at least 90 days, by gavage using a rubber catheter and disposable syringe at a constant dose volume of 10 mL/kg body weight until necropsy. Individual doses were adjusted according to the most recent body weight.
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5 % (w/v) aqueous carboxymethylcellulose with 0.1 % (v/v) Tween 80
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at intervals within known stability for each group separately, as a suspension in the vehicle. The required amount of test item was weighed into a container and a small quantity of vehicle was initially added to create a smooth paste using a spatula. After further addition of vehicle and mixing, the resultant suspension was made up to final volume or weight with vehicle, rinsing the spatula in the process and mixed. Formulations were then placed in an ultrasonic bath for a minimum of 15 minutes (in short bursts, as necessary, to prevent heating of the formulation) to break up any remaining large particulate. Formulations were divided into daily aliquots and were stored refrigerated (2-8°C), before use. Test item formulations were stirred for at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Formulations of the test substance in 0.5 % w/v aqueous carboxymethylcellulose with 0.1 % v/v Tween 80 at concentrations between 1 and 150 mg/mL, spanning those used in this study (1.5 mg/mL to 100 mg/mL), have been shown to be stable for up to six days when stored at room temperature, up to 12 days when stored refrigerated and one month when stored frozen.
Samples were taken from each formulation. Formulations prepared for use on Day 1 were analysed to assess their homogeneity and achieved concentrations. Having satisfactorily confirmed homogeneity on Day 1, subsequent formulations for use during Weeks 4, 8 and 12 were analysed to determine achieved concentrations only. Samples from vehicle used to dose Controls on Day 1 and during Weeks 4, 8 and 12 were analysed to confirm absence of test item. All samples were analysed for test substance using a validated method.
Results: Mean achieved concentrations of the test item formulations used to dose animals on Day 1 and during Weeks 4, 8 and 12 of the study were within 3% of nominal with coefficients of variation no greater than 2.5%, meeting the acceptance criteria (± 10% and ≤ 5%, respectively). No test substance was detected in the vehicle used to dose Control animals. It is considered, therefore, that formulations were both homogeneous and accurately prepared. - Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 15 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dose levels were selected in consultation with the Sponsor on the basis of results from a previous 28 day study in the rat. In this study, groups of five male and five female rats were dosed with 0, 15, 150, or 1000 mg/kg/day test substance. There were no test item effects on mortality, body weight, body weight gain, food intake or FOB measurements at any dose level. The liver was identified as the target organ and there was a dose-related increase in liver weight at 150 and 1000 mg/kg/day in males and females and changes in blood chemistry parameters in the females. A similar outcome was expected after 90 days dosing: the low dose level of 15 mg/kg/day was intended to be the NOEL and the high dose level of 1000 mg/kg/day was selected as the maximum permissible dose level for studies of this design.
- Rationale for animal assignment : Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on arrival.
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes (Mortality and morbidity)
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and, in addition, for the first four weeks of treatment, animals were observed before, shortly after and about one hour after dosing.
BODY WEIGHT: Yes
- Time schedule for examinations: Daily for the first week and weekly thereafter
FOOD CONSUMPTION: Yes
- Time schedule for examinations: Daily for the first week and weekly thereafter
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment (all rats), week 13 (control and high dose rats)
FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: All animals were observed once weekly, starting pre-dose, for their behaviour both within their cage and then after placement in an open arena. On one occasion during Week 13 of the study, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for all animals.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All
- The following parameters were examined.:haemoglobin concentration (Hb), total leucocyte count (WBC), red blood cell count (RBC), neutrophils (Neut), packed cell volume (PCV), lymphocytes (Lymph), mean cell volume (MCV), monocytes (Mono), mean cell haemoglobin (MCH), eosinophils (Eosin), mean cell haemoglobin concentration (MCHC), basophils (Baso), red blood cell distribution width (RDW), large unstained cells (LUC), platelet count (Plate), reticulocytes (Retics), absolute reticulocyte count (A retics), prothrombin time (PT), activated partial thromboplastin time (APTT).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All
- The following parameters were examined: urea, globulin (Glob), creatinine (Cren), albumin/globulin ratio (A/G), glucose (Gluc), total bilirubin (BiliT), alkaline phosphatase (ALP), cholesterol (Chol), alanine aminotransferase (ALT), triglyceride (Trigs), aspartate aminotransferase (AST), sodium (Na), gamma glutamyl transpeptidase (GGT), chloride (Cl), bile acids (B.Acids), calcium (Ca), total protein (T.Prot), potassium (K), albumin (Alb), inorganic phosphorus (I. Phos).
URINALYSIS: No
IMMUNOLOGY:No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- At the end of the treatment period all animals were killed by exposure to carbon dioxide gas in a rising concentration. All animals were weighed and examined externally. The abdominal cavity was opened and the animals were exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened and a full internal examination was performed.
ORGAN WEIGHTS: The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together): adrenal glands, prostate & seminal vesicles (incl. coagulating gland), brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroids (including parathyroids), liver, uterus (including cervix and oviducts), ovaries.
HISTOPATHOLOGY: Yes
- Initially, all the following tissues from Control and high dose animals (in addition to gross lesions from all groups) were examined. Subsequently (due to microscopic findings in the liver, thyroids, adrenals and ovaries of high dose animals), the liver and thyroids from both sexes, adrenals from males and ovaries from females were also examined from the low and intermediate dose groups.
- tissues examined: adrenal glands, pancreas, aorta, pharynx (laryngeal), bone marrow smear, pituitary, brain (3 levels examined), prostate & seminal vesicles (incl. coagulating gland), caecum, colon, rectum, duodenum, salivary gland (submandibular), epididymides, sciatic nerve, eyes (including optic nerves), skeletal muscle, femur & joint (including marrow), skin (with mammary gland), Harderian glands, spinal cord (3 levels examined LS and TS), heart, spleen, ileum, sternum with bone marrow, Jejunum (including Peyer’s patch), stomach, kidneys (one LS and one TS), submandibular lymph nodes, lacrimal glands, testes, larynx, thymus, liver, thyroids (including parathyroids), lungs (including mainstem bronchi), tongue, mammary gland (with inguinal skin), trachea, mesenteric lymph nodes, urinary bladder, nasal cavity (one section - 3rd level), uterus (including uterine cervix and oviducts), oesophagus, vagina, ovaries, all gross lesions. - Statistics:
- Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. All statistical tests were two-sided with minimum significance levels of 5 % and 1 %. Non-parametric statistics were not routinely conducted. When used, Dunnett’s test was conducted regardless of the outcome of the analysis of variance (ANOVA) or analysis of covariance (ANCOVA). Body weight, cumulative body weight gain from the start of dosing, food consumption, haematology, coagulation and blood chemistry parameters, grip strength and motor activity data were analysed using a parametric ANOVA. Organ weights were analysed using ANOVA for the absolute weights and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. For all of the parameters evaluated initially by ANOVA or ANCOVA, Dunnett's test was used to compare the Control and treated groups, based on the error mean square in the ANOVA or ANCOVA. The Dunnett's test was performed for all continuous data parameters, regardless of whether the initial ANOVA or ANCOVA was statistically significant. Qualitative functional observational battery parameters or any other parameters not mentioned above were not analysed statistically.
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Salivation throughout the treatment period for both sexes given 1000, 150 or 15 mg/kg/day. The number and frequency of these occasions increased in a dose related manner.
Yellow stained bedding was observed in cages for both sexes given 1000 mg/kg/day, and for males given 150 mg/kg/day. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Throughout the study females given 1000 mg/kg/day consistently gained statistically significantly more weight than Controls (p<0.01 to p<0.05), such that their cumulative mean body weight gains and mean body weights at the end of the dosing period were 30% and 10% higher, respectively, than the Control values. There were no effects on body weight in males at 1000 mg/kg/day or in either sex at 15 or 150 mg/kg/day.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- A marginal increase in mean food intake was observed for one out of two cages of females given 1000 mg/kg/day during the study. There was no effect on food intake during the study for males given any dose level and females given 15 or 150 mg/kg/day.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg/day females had a statistically significant increase in total white blood cell count due to an increase in neutrophils and monocytes (p<0.01 to p<0.05). Males given 1000 or 150 mg/kg had a statistically significant increase in platelet count and reduced prothrombin time (p<0.01), compared with Controls. There were no other effects on haematology or coagulation parameters.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- There was a marked, test item-related, approximately four fold increase (p<0.01) in mean plasma bile acid concentration in females given 1000 mg/kg/day and a slight non-significant increase in males from this group, compared with Controls. Aspartate aminotransferase activity was lower for both sexes given 1000 mg/kg/day (p<0.01) and for females given 15 or 150 mg/kg/day (p<0.05). Males given 1000 mg/kg/day also had low alkaline phosphatase activity (p<0.01) when compared with Controls. Group mean total plasma protein was increased (with high levels of albumin and globulin for males and only globulin for females) and associated albumin/globulin ratio was low for both sexes given 1000 mg/kg/day compared with Controls. Cholesterol was increased in both sexes given 1000 mg/kg/day (p<0.01 to p<0.05), compared with Controls. Coupled with the microscopic findings in the liver of high dose animals, these changes in this group were considered to be test item-related. Females given 1000 mg/kg/day also showed increase in inorganic phosphorus and triglyceride concentration (p<0.01 to p<0.05). Plasma chloride was slightly lower for both sexes given 1000 mg/kg/day (p<0.01 to p<0.05). Where statistically significant differences were observed for other blood chemistry parameters these differences were minimal and were considered to reflect normal biological variation.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- All treated females were more active and travelled a greater distance than Controls for the first and second 10 minutes of motor activity monitoring, achieving statistical significance (p<0.01 to p<0.05) only in animals given 1000 mg/kg/day. There were no differences from Controls for subsequent intervals. There was no effect in the males at any dose level. Forelimb grip strength was significantly (p<0.05) reduced for males given 1000 mg/kg/day, although their hind limb grip strength was similar to Controls. There were no effects on grip strength for females given 1000 mg/kg/day or either sex at any other dose level. There were no test item-related effects on functional observations or on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Group mean absolute and adjusted liver weights were higher than Controls for both sexes given 1000 mg/kg/day (39% and 41% for males, and 45% and 37% for females, respectively) and for males given 150 mg/kg/day (11%) (p<0.01). However, all relative liver weights for males given 150 mg/kg/day were well within the laboratory background ranges. Group mean adjusted thyroids weights were statistically significantly higher than Controls for all treated males (21%, 24% and 35% in the groups given 15, 150 or 1000 mg/kg/day respectively (p<0.01 to p<0.05)); mean absolute thyroid weight was also higher than the Control mean in these animals. Group mean absolute and adjusted thyroids weights were also higher than Controls (13% and 4% respectively) for females given 1000 mg/kg/day, but the differences did not achieve statistical significance. However, most relative thyroid weights for females in this group were at the upper limit of the laboratory background ranges and that for one female exceeded the normal upper limit. Group mean absolute and adjusted adrenal weights were statistically significantly lower (-16%; p<0.01) than Controls in female rats given 150 mg/kg/day. For females given 1000 mg/kg/day the group mean adjusted adrenal weights were higher than Control mean (4%). Other statistically significant inter-group differences were considered to reflect normal biological variation rather than any effect of the test substance.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Enlarged adrenals were noted in five out of ten females given 1000 mg/kg/day compared with one affected Control female. There was no microscopic correlation of this macroscopic finding and therefore the macroscopic findings were considered to reflect the inter-animal variation of normal aging changes in the female rat. A variety of spontaneous macroscopic changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Liver: Test item-related centrilobular hepatocyte hypertrophy (minimal or slight) was found in males and females given 1000 mg/kg/day. There was no such change in animals given 15 or 150 mg/kg/day.
Thyroids: Follicular cell hypertrophy (minimal) was found in males and females given 1000 mg/kg/day but not in animals given 15 or 150 mg/kg/day.
Adrenals: Vacuolation of the zona fasciculata and zona glomerulosa was found only in males given 1000 mg/kg/day but not in males given 15 or 150 mg/kg/day.
Ovaries: Hypertrophy and vacuolation of the interstitial gland was found in most of the females given 1000 mg/kg/day but not in females given 15 or 150 mg/kg/day.
A variety of spontaneous microscopic changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings is generally consistent with changes encountered in rats of this age kept under laboratory conditions.
Effect levels
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Remarks on result:
- other: Ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. Significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level.
Target system / organ toxicity
open allclose all
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- female reproductive system
- Organ:
- ovary
- Treatment related:
- yes
- Dose response relationship:
- no
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- endocrine system
- Organ:
- adrenal glands
- Treatment related:
- yes
- Dose response relationship:
- no
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- System:
- endocrine system
- Organ:
- thyroid gland
- Treatment related:
- yes
- Dose response relationship:
- no
Any other information on results incl. tables
Table 1: Intergroup comparison of body weight gain (g)-selected timepoints
|
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
Day |
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
1-2 |
3.9 |
4.9 |
5.4 |
6.0 |
3.8 |
2.8 |
3.4 |
1.4 |
1-4 |
16.6 |
17.2 |
18.9 |
18.8 |
10.7 |
7.7 |
8.4 |
11.0 |
1-8 |
36.0 |
37.5 |
39.3 |
39.1 |
20.1 |
18.1 |
20.8 |
25.7 |
1-22 |
90.2 |
88.3 |
95.8 |
91.4 |
47.0 |
46.8 |
47.3 |
61.0** |
1-50 |
167.4 |
156.8 |
167.9 |
167.9 |
80.3 |
80.3 |
79.2 |
97.9** |
1-71 |
198.4 |
182.7 |
195.6 |
196.7 |
88.7 |
91.7 |
89.6 |
108.1** |
1-92 |
223.8 |
206.7 |
216.7 |
219.2 |
92.8 |
98.5 |
97.7 |
120.7** |
* Statistically significant difference from control group mean, p<0.05
** Statistically significant difference from control group mean, p<0.01
Table 2: Intergroup comparison of food consumption (g/animal/day) -selected timepoints
|
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
Day |
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
1-2 |
22.4 |
21.9 |
23.8 |
19.5 |
15.9 |
14.7 |
15.3 |
11.2 |
3-4 |
22.4 |
22.3 |
24.1 |
22.5 |
17.4 |
14.9 |
15.7 |
16.3 |
7-8 |
24.7 |
24.4 |
26.2 |
23.2 |
17.4 |
18.3 |
18.6 |
17.8 |
15-22 |
24.4 |
24.3 |
26.6 |
24.7 |
20.4 |
18.7 |
19.3 |
20.6 |
43-50 |
24.7 |
24.0 |
26.3 |
23.9 |
17.9 |
19.1 |
19.0 |
20.5 |
64-71 |
25.2 |
25.6 |
24.4 |
24.9 |
19.4 |
19.3 |
18.7 |
20.1 |
85-92 |
24.7 |
24.5 |
25.9 |
23.8 |
17.1 |
18.4 |
18.4 |
19.3 |
1-92 |
25.0 |
24.7 |
26.3 |
24.7 |
18.2 |
19.0 |
18.8 |
19.9 |
No statistically significant differences from control group mean
Table 3: Intergroup comparison of selected haematology parameters
Parameter |
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
WBC (10^3/µL) |
6.965 |
6.043 |
6.391 |
7.215 |
4.499 |
4.752 |
5.232 |
6.115* |
Neut (%) |
12.43 |
13.03 |
11.19 |
13.82 |
12.67 |
12.30 |
11.32 |
12.41 |
Mono (%) |
1.57 |
1.66 |
1.39 |
1.73 |
1.98 |
2.10 |
2.25 |
2.94* |
Plate (10^3/µL) |
836.7 |
844.0 |
910.2 |
996.8** |
840.6 |
867.5 |
817.5 |
891.1 |
PT (sec) |
23.38 |
23.50 |
21.69** |
20.61** |
21.81 |
22.23 |
22.21 |
20.93 |
* Statistically significant difference from control group mean, p<0.05
** Statistically significant difference from control group mean, p<0.01
Table 4: Intergroup comparison of selected clinical chemistry parameters
Parameter |
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
B. Acids (µmol/L) |
9.33 |
7.34 |
9.58 |
10.77 |
11.41 |
7.44 |
12.44 |
45.64** |
AST (U/L) |
56.8 |
56.2 |
55.1 |
49.1** |
85.3 |
56.4* |
52.8* |
50.8** |
T. prot (g/dL) |
6.40 |
6.50 |
6.65* |
7.21** |
7.11 |
7.16 |
7.01 |
7.20 |
Alb (g/dL) |
4.03 |
4.10 |
4.18 |
4.19** |
4.94 |
4.93 |
4.74 |
4.56** |
Glob (g/dL) |
2.37 |
2.40 |
2.47 |
3.02** |
2.17 |
2.23 |
2.27 |
2.64** |
A/G ratio |
1.71 |
1.71 |
1.70 |
1.39** |
2.28 |
2.22 |
2.09** |
1.73** |
Chol (mg/dL) |
55.5 |
53.1 |
62.3 |
69.6* |
48.7 |
59.4* |
58.4 |
81.8** |
I. Phos (mg/dL) |
5.00 |
4.89 |
5.11 |
4.70 |
3.90 |
4.11 |
4.47 |
4.90** |
Trigs (mg/dL) |
132.1 |
117.8 |
131.1 |
132.9 |
94.7 |
97.1 |
89.9 |
153.5* |
Cl (nmol/L) |
100.4 |
100.8 |
100.2 |
98.7* |
100.6 |
100.2 |
99.7 |
97.1** |
* Statistically significant difference from control group mean, p<0.05
** Statistically significant difference from control group mean, p<0.01
Table 5: Intergroup comparison of motor activity
Parameter |
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
Movement (a) |
|
|
|
|
|
|
|
|
1 |
899.2 |
1004.7 |
993.6 |
1272.5 |
1069.4 |
1509.3 |
1454.6 |
1642.8** |
2 |
638.6 |
544.9 |
589.9 |
689.5 |
633.4 |
756.1 |
842.2* |
880.1* |
3 |
513.0 |
299.8 |
510.7 |
486.6 |
448.7 |
482.1 |
673.6 |
403.5 |
4 |
532.2 |
402.8 |
593.3 |
534.5 |
414.9 |
373.1 |
415.8 |
256.0 |
5 |
345.0 |
228.0 |
397.5 |
418.1 |
368.9 |
353.4 |
217.5 |
207.1 |
6 |
280.8 |
116.9 |
298.7 |
230.6 |
152.3 |
314.7 |
256.8 |
282.8 |
Distance (b) |
|
|
|
|
|
|
|
|
1 |
3405.8 |
3758.2 |
3762.6 |
4679.8 |
3886.6 |
5300.8* |
5154.1 |
5873.6** |
2 |
2400.0 |
2006.3 |
2157.0 |
2527.4 |
2352.1 |
2688.2 |
2977.1 |
3078.8* |
3 |
1948.7 |
1082.4 |
1858.3 |
1769.5 |
1642.4 |
1730.9 |
2395.6 |
1414.5 |
4 |
1976.3 |
1532.4 |
2215.3 |
1936.2 |
1579.7 |
1356.0 |
1534.6 |
931.8 |
5 |
1289.2 |
872.2 |
1483.0 |
1530.3 |
1318.9 |
1294.8 |
745.5 |
700.7 |
6 |
1032.6 |
466.4 |
1034.4 |
866.2 |
581.9 |
1130.5 |
954.7 |
1037.5 |
At rest (c) |
|
|
|
|
|
|
|
|
1 |
291.4 |
274.2 |
285.5 |
228.0 |
272.1 |
223.4 |
234.1 |
195.0** |
2 |
358.5 |
391.9 |
391.7 |
359.1 |
364.5 |
357.1 |
356.8 |
330.9 |
3 |
398.1 |
473.6 |
422.4 |
420.6 |
427.5 |
436.5 |
395.2 |
470.4 |
4 |
406.9 |
445.2 |
387.6 |
407.1 |
435.3 |
469.8 |
447.4 |
510.5 |
5 |
456.4 |
496.9 |
452.6 |
455.5 |
460.7 |
460.8 |
520.8 |
531.7 |
6 |
482.1 |
540.1 |
496.1 |
513.4 |
529.3 |
483.2 |
501.6 |
502.9 |
(a) movement counts over 10 min periods (b) distance travelled (cm) over 10 min periods (c) time at rest (sec) over 10 min periods
* Statistically significant difference from control group mean, p<0.05
** Statistically significant difference from control group mean, p<0.01
Table 6: Intergroup comparison of forelimb grip strength
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
511.87 |
470.73 |
461.13 |
402.30* |
419.53 |
360.70 |
394.40 |
390.07 |
* Statistically significant difference from control group mean, p<0.05
Table 7: Intergroup comparison of selected organ weights (g)
|
|
Dose level (mg/kg/day) |
|
|
|
|
|
|
|
|
|
Males |
|
|
|
Females |
|
|
|
Organ |
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
Liver |
actual |
14.001 |
13.949 |
15.586 |
19.445** |
8.265 |
8.654 |
8.885 |
12.008** |
|
adjusted |
13.793 |
14.329 |
15.375** |
19.484** |
8.485 |
8.644 |
8.992 |
11.656** |
Thyroid |
actual |
0.0192 |
0.0229 |
0.0238* |
0.0257** |
0.0191 |
0.0180 |
0.0175 |
0.0215 |
|
adjusted |
0.0191 |
0.0232* |
0.0237* |
0.0257** |
0.0197 |
0.0181 |
0.0178 |
0.0205 |
Adrenal |
actual |
0.0683 |
0.0643 |
0.0694 |
0.0691 |
0.0883 |
0.0814 |
0.0745* |
0.0941 |
|
adjusted |
0.0676 |
0.0655 |
0.0687 |
0.0692 |
0.0890 |
0.0815 |
0.0749* |
0.0929 |
* Statistically significant difference from control group mean, p<0.05
** Statistically significant difference from control group mean, p<0.01
Table 8: Intergroup comparison of treatment-related histopathological findings in liver
Finding |
|
Dose level |
|
|
|
|
|
|
|
|
|
M |
M |
M |
M |
F |
F |
F |
F |
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
No. tissues examined |
|
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Hyper- |
min. |
0 |
0 |
0 |
2 |
0 |
0 |
0 |
0 |
|
slight |
0 |
0 |
0 |
8 |
0 |
0 |
0 |
10 |
|
total |
0 |
0 |
0 |
10 |
0 |
0 |
0 |
10 |
Table 9: Intergroup comparison of treatment-related histopathological findings in thyroids
Finding |
|
Dose level |
|
|
|
|
|
|
|
|
|
M |
M |
M |
M |
F |
F |
F |
F |
|
|
0 |
15 |
150 |
1000 |
0 |
15 |
150 |
1000 |
No. tissues examined |
|
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
Hyper- |
min. |
0 |
0 |
0 |
9 |
0 |
0 |
0 |
7 |
|
total |
0 |
0 |
0 |
9 |
0 |
0 |
0 |
7 |
Table 10: Intergroup comparison of treatment-related histopathological findings in adrenals
Finding |
|
Dose level |
|
|
|
|
|
M |
M |
M |
M |
|
|
0 |
15 |
150 |
1000 |
No. tissues examined |
|
10 |
10 |
10 |
10 |
Vacuolation, fasciculate/glomerulosa |
min. |
0 |
0 |
0 |
4 |
|
slight |
0 |
0 |
0 |
4 |
|
total |
0 |
0 |
0 |
8 |
Table 11: Intergroup comparison of treatment-related histopathological findings in ovaries
Finding |
|
Dose level |
|
|
|
|
|
0 |
15 |
150 |
1000 |
No. tissues examined |
|
10 |
10 |
10 |
10 |
Hypertrophy/vacuolation, interstitial gland |
min. |
0 |
0 |
0 |
2 |
|
slight |
0 |
0 |
0 |
2 |
|
mod. |
0 |
0 |
0 |
3 |
|
total |
0 |
0 |
0 |
7 |
Applicant's summary and conclusion
- Conclusions:
- The test substance administered once daily by oral gavage to the rat at doses of 0, 15, 150 or 1000 mg/kg/day was tolerated for at least 90 days, but elicited test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level. The No Observed Adverse Effect Level (NOAEL) for both sexes was considered to be 150 mg/kg/day.
- Executive summary:
The oral repeated dose toxicity of the substance to the rat was investigated in a GLP study according to the OECD TG 408. Groups of 10 male and 10 female Crl:WI(Han) rats were dosed with 0 (vehicle), 15, 150 or 1000 mg/kg/day test substance, once daily by gavage, for at least 90 days, until the day before necropsy. All animals were observed daily from the start of treatment. Body weights and food intake were recorded daily for the first week of treatment and weekly thereafter. The eyes of all animals were examined before the start of treatment, with Control and high dose animals also examined in Week 13. Blood samples were taken during Week 13 for clinical pathology assessment. All animals were subjected to standard arena observations pre-dose and once weekly thereafter; in addition, grip strength, motor activity and sensorimotor responses to visual, acoustic, or proprioceptive stimuli were assessed once in Week 13. All animals were subjected to a gross necropsy, specified organs were weighed and a full list of tissues was examined microscopically from the Control and high dose animals. In addition, the liver and thyroids from both sexes, adrenals from males and ovaries from females were examined microscopically from intermediate and low dose animals.
There were no deaths during the study. Excessive salivation was seen after dosing on a number of occasions throughout the treatment period for both sexes given 15, 150 or 1000 mg/kg/day, number and frequency of these occasions increased in a dose related manner. Yellow stained bedding was observed in cages for males given 150 mg/kg/day and both sexes given 1000 mg/kg/day. Throughout the study females given 1000 mg/kg/day consistently gained more weight than Controls. Their cumulative mean body weight gains and mean body weights at the end of the dosing period were 30% and 10% higher, respectively, than the Control values. There were no effects on body weight in males at 1000 mg/kg/day or in either sex at 15 or 150 mg/kg/day. A slight increase (+9%) in mean food intake was observed for females given 1000 mg/kg/day during the study. There was no notable effect on food intake during the study for males given any dose level and females given 15 or 150 mg/kg/day. There were no test item-related effects on functional observations or on sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli at any dose level. All treated females were more active and travelled a greater distance than Controls for the first and second 10 minutes of motor activity monitoring, which was more notable for animals given 1000 mg/kg/day but with no differences from Controls for subsequent intervals. Forelimb grip strength was reduced for males given 1000 mg/kg/day, but their hind limb grip strength was similar to Controls. There were no effects on grip strength for females given 1000 mg/kg/day or either sex at any other dose level. There were no ocular changes that were considered to be related to test substance administration. At 1000 mg/kg/day females had a statistically significant increase in white blood cells count due to increase in neutrophils and monocytes. Males had statistically significant increase in platelets and reduced prothrombin time, compared with Controls. Bile acids were notably increased for females given 1000 mg/kg/day. Activity of aspartate aminotransferase was lower for both sexes given 1000 mg/kg/day and for females given 15 or 150 mg/kg/day. Males given 1000 mg/kg/day also had low alkaline phosphatase activity when compared with Controls. Group mean total plasma protein was increased (with high levels of albumin and globulin for males and only globulin for females) and associated albumin/globulin ratio was low for both sexes given 1000 mg/kg/day compared with Controls. Cholesterol was increased in both sexes given 1000 mg/kg/day, compared with Controls. Coupled with the microscopic findings in the liver of high dose animals, these changes in this group were considered to be test item-related. Females given 1000 mg/kg/day also showed increase in inorganic phosphorus and triglyceride concentration. Plasma chloride was slightly lower for both sexes given 1000 mg/kg/day. There were no test item-related macroscopic findings. Group mean absolute and adjusted liver weights were notably higher than Controls for both sexes given 1000 mg/kg/day (39% and 41% for males, and 45% and 37% for females, respectively) and slightly higher for males given 150 mg/kg/day (11%). Microscopic evaluation indicated hepatic centrilobular hepatocyte hypertrophy in animals given 1000 mg/kg/day. Group mean absolute and adjusted thyroid weights were higher than Controls for all treated males (21% to 35%). Group mean absolute and adjusted thyroid weights were also higher for females given 1000 mg/kg/day (13% and 4%). Microscopic evaluation indicated follicular cell hypertrophy in the thyroids of males and females given 1000 mg/kg/day. Group mean adjusted adrenal weights were higher than the Control mean for females given 1000 mg/kg/day and lower for females given 150 mg/kg/day. However, vacuolation in the zona fasciculata and the zona glomerulosa were observed in the adrenals of males only, given 1000 mg/kg/day. Hypertrophy/vacuolation of the interstitial gland was observed in the ovary in females given 1000 mg/kg/day; there were no changes to ovarian weights.
The test substance administered once daily by oral gavage to the rat at doses of 0, 15, 150 or 1000 mg/kg/day was tolerated for at least 90 days, but elicited test-item related ovarian changes in females and adrenal changes in males at 1000 mg/kg/day. In the absence of any functional investigations the adverse nature of these changes remains equivocal. In addition, significant increases in liver and thyroid weights in the presence of histopathological correlates were observed in males and females at this dose level. The No Observed Adverse Effect Level (NOAEL) for both sexes was considered to be 150 mg/kg/day.
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.