Registration Dossier
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EC number: 948-040-6 | 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
Endpoint summary
Administrative data
Description of key information
NOAEL (Systemic toxicity) = 100 mg/kg /day; OECD 422; N. Barraclough. (2018)
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 08 March 2017 - 29 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day oral gavage study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
- GLP compliance:
- no
- Limit test:
- no
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Homogenous suspension formed in corn oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspended in corn oil prior to administration
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
OTHER SPECIFICS: N/A - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:WI(Han)
- Details on species / strain selection:
- The rat was selected because it is a rodent species known to be accepted by various regulatory authorities.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 9-10 weeks
- Weight at study initiation: Males: 299.6 - 327.7 g; Females: 191 - 239.3 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of four by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to 5LF2 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: ≥13 days
DETAILS OF FOOD AND WATER QUALITY: As described above.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 10 April 2017 To: 20 June 2017 - Route of administration:
- oral: gavage
- Details on route of administration:
- Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing.
Formulations were dosed within 3 hours of preparation.
The test article was administered orally by gavage. Animals were dosed once daily for up to 14 consecutive days, excluding the day of necropsy. A dose volume of 5 mL/kg was used. Dose volumes were based on the most recent individual body weight for each animal. - Vehicle:
- corn oil
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing. Formulations were dosed within 3 hours of preparation.
Method of preparation not reported in range-finder.
- DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item formed homogenous formulation in corn oil. Guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): Batch numbers MKBP7039V and MKBZ9899V
- Purity: Not reported, assumed 100 % - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- N/A
- Duration of treatment / exposure:
- 14 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Dosing stopped after 1 day
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- Dosing stopped after 2 days due to 5/5 male deaths. Surviving females dosed at 175 mg/kg bw/day for 13 days (total 14 days)
- Dose / conc.:
- 175 mg/kg bw/day (nominal)
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 300 mg/kg bw/day: 5 males and 5 females
250 mg/kg bw/day: 5 males and 5 females
175 mg/kg bw/day: 3 males and 5 females (5 surviving females from 250 mg/kg bw/day group and 3 spare males used as all died at 250 mg/kg bw/d)
125 mg/kg bw/day: 5 males and 5 females
50 mg/ kg bw/day: 5 males and 5 females - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
A dose level of 300 mg/kg/day was considered an acceptable initial dose level, based on the preliminary findings of the acute toxicity study (Covance Study 8359313).
In the current study, due to adverse clinical observations after the first dose in two females administered 300 mg/kg/day (prostrate, respiratory pattern changes, semi closed eyes, and twitching), which resulted in their early termination, dosing was discontinued for Groups 1 and 2 after the first dose, until a further dose level was explored in the acute toxicity study.
A dose level of 50 mg/kg was well tolerated in the acute toxicity study (Covance Study 8359313), and was therefore used as the dose level to be explored.
In the absence of any effects of test article noted following administration of 50 mg/kg/day in this study, and in the absence of any findings noted in the acute toxicity study (Covance Study 8359313) following administration of 300 mg/kg, the next dose level to be investigated was 250 mg/kg/day.
Adverse effects were noted following the second dose of 250 mg/kg/day (subdued/sluggish behavior, tremors, convulsive episode, and twitching), which resulted in the death of all treated males; as such, dosing was ceased for Group 3 females, and animals were retained for possible future testing at a lower dose level.
An additional intermediate-dose level of 175 mg/kg/day was included to ensure enough information was obtained from this study for successful dose level selection for the subsequent OECD 422 study. This dose was administered for 14 days to observe any adverse effects.
In the absence of effects following administration of 50 mg/kg/day, a dose level of 125 mg/kg/day was chosen as it was anticipated to be tolerated for 14 consecutive days.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Positive control:
- N/A
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Twice Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Weely (Where necessary, unscheduled clinical examinations were performed; these data were reported).
BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded once weekly during the predose phase; on Days 1 and 4 (all animals) and 8, 11, and 14 (Control, 500 and 750 mg/kg/day groups only) of the dosing phase; and before each necropsy
FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The amount of food consumed by each cage of animals was measured at twice weekly intervals where possible. Consumption was calculated as g/animal/day.
WATER CONSUMPTION: Yes
- Time schedule for examinations:
Daily
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No - Other examinations:
- N/A
- Statistics:
- Statistical analyses were not performed.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Following administration of 175 mg/kg/day, clinical observations included red staining of the neck for two females and vocalization for two females on the final (14th) dosing occasion (Study Day 35). Postdose observations included mouth rubbing for both sexes; paddling for one female; minimal staggering, subdued/sluggish behaviour, twitching, decreased respiration, and reduced activity for one female following the 3rd and 6th dose (Study Days 24 and 27 respectively); and reduced activity and vocalization for one female after the 1st dose (Study Day 21). Postdose observations were mostly recorded immediately upon return to the cage, with the exception of some 1 animal, for which observations were recorded up to 4 hours postdose.
Clinical observations for animals administered 125 mg/kg/day were limited to mouth rubbing and (occasionally) salivation. These observations were recorded immediately after dosing upon return to the cage.
Clinical observations for animals administered 50 mg/kg/day included vocalization for two females on Day 8 or 15 of the dosing phase and postdose observations of mouth rubbing and (occasionally) paddling and salivation. These observations were recorded immediately postdose (upon return to the cage).
Mouth rubbing was also recorded for controls; however, the incidence was much lower than that observed for test article-treated animals.
Other clinical observations, including thinning fur, were considered incidental as they were observed infrequently or are commonly observed in this species. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Test article-related mortality was observed after a single dose for animals administered 300 mg/kg. One male and two females administered 300 mg/kg/day were found dead or were sacrificed in a moribund condition on Day 1 of the dosing phase. Clinical observations for animals administered 300 mg/kg/day included prostrate, twitching, unresponsive, semi-closed to closed eyes; and irregular, rapid, or labored respiration.
Following administration of 250 mg/kg/day, all five males were found dead or were sacrificed in a moribund condition on Day 2 of dosing. Dosing was ceased after Day 2 of the dosing phase for all remaining animals due to these deaths. Clinical observations for animals administered 250 mg/kg/day included convulsions, subdued/sluggish behaviour, prostrate, twitching (body and head), arched gait, and tremors.
Dosing of females previously administered 250 mg/kg/day recommenced on Study Day 21 at a lower dose level of 175 mg/kg/day. An additional set of three males was also added to the study as Group 5 and was administered 175 mg/kg/day. All animals administered 175 mg/kg/day survived following 14 days of dosing.
All animals administered 50 or 125 mg/kg/day survived following 14 days of dosing. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Following initial body weight losses following administration of 300 or 250 mg/kg/day, after which dosing was discontinued for these animals, to ensure they were suitable for use following a wash-out period, the mean body weight of animals administered 300 or 250 mg/kg/day was generally comparable with that of controls for the remainder of the observation period. Initial body weight decreases were attributed to general debilitation.
Test article-related body weight losses were observed throughout the dosing phase for males administered 175 mg/kg/day and during the first week of dosing for females administered 175 mg/kg/day, with the body weight gain comparable with that of controls during the second week.
Test article-related body weight losses were observed during the first week of dosing for animals administered 125 mg/kg/day. Although no further body weight loss was recorded during the second week of dosing, the mean body weight gain of animals administered 125 mg/kg/day was still lower than controls.
These reductions in body weight were correlated with reduced food consumption for animals administered 125 mg/kg/day.
No effect on body weight was observed for animals administered 50 mg/kg/day. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Following initial reductions in food consumption following administration of 300 or 250 mg/kg/day, after which dosing was discontinued; the mean food consumption for surviving animals was comparable with that of controls for the remainder of the observation period. The initial decrease in food consumption was attributed to general debilitation.
Lower mean food consumption was noted throughout the dosing phase, for males and females administered 175 or 125 mg/kg/day, compared with controls. Reduced mean food consumption was correlated with body weight loss or reduced body weight gain.
Mean food consumption of animals administered 50 mg/kg/day was generally comparable with controls. - Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- No data was present for animals administered 300 or 250 mg/kg/day due to the cessation of dosing.
Dose-related reductions in water consumption were evident with increasing dose levels for both sexes administered ≥125 mg/kg/day, compared with controls.
Mean water consumption of animals administered 50 mg/kg/day was generally comparable with that of controls. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No test article-related effects were recorded for organ weights.
Compared with concurrent controls, decreased group mean unadjusted and relative (to body weight weight) epididymis and testes weights were recorded for males administered 50 mg/kg/day. These decreased mean values were attributed to small epididymis and testes for one male. All other males of this group had epididymis and testes weights that were similar to those of controls. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following macroscopic abnormalities were noted:
Thick, cream coloured stomach contents (abnormal contents) were recorded for two females administered 300 mg/kg/day.
Distended jejunum was recorded for one male administered 250 mg/kg/day.
Large seminal vesicle was recorded for one male administered 175 mg/kg/day.
Red mandibular lymph node was recorded for one female administered 125 mg/kg/day.
Following administration of 50 mg/kg/day, pale and/or mottled liver findings were recorded for one male and one female; red thymus was recorded for one female, and small epididymis and testes were recorded for one male.
Findings in these tissues were observed in few animals or lacked a dose-response; therefore, the relationship to the test article was uncertain. - Neuropathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: non-neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Conclusions:
- Once daily oral gavage administration of 50, 125, 175, 250, or 300 mg/kg/day Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male and female rats resulted in mortality following 250 or 300 mg/kg/day administration.
Dose levels of 125 or 175 mg/kg/day resulted in test article-related effects including body weight losses and reduced food and water consumption, which would not be tolerated over longer dosing duration.
Based on the findings of this 14 day repeated dose study, dose level of 30, 50 and 100 mg/kg/day are suggested for the following OECD 422 study. - Executive summary:
Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was administration to the rat once daily via oral garvage for up to 14 days and to provide the basis for the selection of dose levels for a subsequent OECD 422 study. In dosing phase one, groups of rats (5/sex) were administered 300 mg/k/day test item due to two deaths observed, dosing was stopped allowing the test item formulations to wash out from the remaining animals, phase two dosing was then launched at doses of 50 and 250 mg/kg/day. Dosing stopped after Day 2 at 250 mg/kg/day, following the deaths of all Group 3 males. Females were retained on the study, and dosing recommenced at a lower dose level of 175 mg/kg/day from Day 21 until Day 34 of the dosing phase. Group 5 animals were dosed at the same dose level, using the 3 remaining spare males.
Clinical observations for animals administered 250 or 300 mg/kg/day included one or more of the following: prostrate, twitching, convulsions, tremors, subdued/ sluggish (slow/deliberate movements), unresponsive, semi-closed to closed eyes; and/or irregular, rapid, or labored respiration. In addition, these animals showed body weight loss and low food or water consumption.
All animals administered 50, 125, or 175 mg/kg/day survived to their scheduled sacrifice. Postdose observations for animals administered 50, 125, or 175 mg/kg/day included mouth rubbing, (occasionally) salivation, and paddling. For one female administered 175 mg/kg/day, minimal staggering, subdued/sluggish behavior, twitching, and decreased respiration were observed on one occasion. Reduced activity was observed on one occasion for two females administered 175 mg/kg/day. Other clinical observations included vocalization or red staining of the neck for animals administered 50 or 175 mg/kg/day; these observations were transient and considered not adverse. Test article-related body weight loss was observed throughout the dosing phase for males administered 175 mg/kg/day and during the first week of dosing for females administered 175 mg/kg/day. During the second week of dosing, body weight gain was comparable with controls for females administered 175 mg/kg/day.
For animals administered 125 mg/kg/day, test article-related body weight loss was observed during the first week of dosing, followed by reduced body weight gain during the second week of dosing.
Macroscopic observations were recorded in one or two animals from each test article-treated group (excluding controls). These consisted of changes in the stomach (abnormal contents), jejunum (distended), liver (pale and mottled), mandibular lymph nodes (red), seminal vesicles (large), thymus (red), epididymis (small), and testes (small). In the absence of a dose-response, the relationship to test article was uncertain.
In conclusion, once daily oral gavage administration of 50, 125, 175, 250, or 300 mg/kg/day Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male and female rats resulted in mortality following 250 or 300 mg/kg/day administration. Dose levels of 125 or 175 mg/kg/day resulted in test article-related effects including body weight losses and reduced food and water consumption, which would not be tolerated over longer dosing duration. Based on the findings of this 14 day repeated dose study, dose level of 30, 50 and 100 mg/kg/day are suggested for the following OECD 422 study.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23 May 2017 - 11 Oct 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- 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:
- 2016
- Deviations:
- yes
- Remarks:
- The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. See "Overall remarks / attachements" for more details.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19 - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl:WI(Han)
- Details on species / strain selection:
- The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 11 to 12weeks
- Weight at study initiation: Males: 307.4 and 385.4 g; Females: 174.1 and 218.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of five by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 16 days prior to initiation of dosing (males) or 9 days prior to initiation of smearing (females).
DETAILS OF FOOD AND WATER QUALITY: As described above.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): Minimum of 15 hrs
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 15th Feb 2018 To: 13th Apr 2018 - Route of administration:
- oral: gavage
- Details on route of administration:
- Oral, by gastric gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- Formulations (excluding the control group) were stirred continuously from at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed for up to 57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter. Females were sent to necropsy on LD 14 or 26 days post‑coitum).
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition.
A dose volume of 5 mL/kg was used. Dose volumes were calculated using the most recent recorded body weight for each animal. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Covance Laboratories Ltd. (2018, in reporting) Stand-Alone Method Validation and Stability Analysis for One Formulation Used in GLP Studies. Covance Study 8359299. Harrogate (UK): Covance Laboratories Ltd.
Suspensions of 500 mg/mL in corn oil, were found stable for 16 days at room temperature (15 to 25C) and homogenous (Covance Study 8359299).
The test article formulation prepared during Week 6 of dosing was analyzed to determine achieved concentration as soon as possible after preparation, and after 5 days stored at 2 to 8°C.
Formulations prepared for use in Week 1, Week 3 and Week 6 of dosing were analyzed to determine the achieved concentration. Triplicate samples of 0.5 mL were removed from the test article formulations and control formulations and were analyzed.
The mean % target range for the preparation of the formulations was 85 to 115% of the nominal concentration. - Duration of treatment / exposure:
- Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43.
Females were dosed for up to 57 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13). - Frequency of treatment:
- Once daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 1 (control)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- Group 2 (Low)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Remarks:
- Group 3 (Intermediate)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Group 4 (High)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Method: Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females. It was stated in the raw data.
Frequency: Once daily
-F0 males:42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]).
-F0 females:57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter.
-F1: Potential indirect exposure in utero and through the milk during lactation
Dose levels: The amount of test item to be administered was calculated according to the most recent body weight recorded.
Group 1: 0 mg/kg/day
Group 2: 30 mg/kg/day
Group 3: 50 mg/kg/day
Group 4: 100 mg/kg/day
Administration volume 5 mL/kg
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition. - Positive control:
- No
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- pre-test: Twice
- treatment to lactation: weekly
BODY WEIGHT: Yes
- pre-test: once
- treatment to lactation: on day one then weekly, gestation observation Day 0, 7, 14 & 20 and on lactation day 1, 4,7,13 &14 prior to necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- pre-test: weekly
- treatment to lactation: twice weekly (male) and daily (female) from GD 0 to 20 and LD 1 to 13.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- treatment to lactation: Daily (male) and daily (female) from GD 0 to 20.
SENSORY REACTIVITY & GRIP STRENGTH:
- Week 6 (before dosing)
MOTOR ACTIVITY:
- Week 6 (before dosing)
- Days 7 of lactation
HAEMATOLOGY: Yes
- Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).
BLOOD CHEMISTRY: Yes
- Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).
URINALYSIS: Yes
- Week 6 of dosing
IMMUNOLOGY: No
- Not examine
OTHER: Yes (see below);
ESTROUS CYCLE: Yes
- Dry smears - taken for 15 days before pairing (reproductive feamles only).
- Wet smears - taken for 14 days before treatment (all females); females that failed to exhibit 4-5 day cycles were not allocated to the reproductive group.
- daily after pairing until mating (reproductive females only) and on LD 14, prior to necropsy.
THYROID HORMONE ANALYSIS: Yes (T3/4 and TSH)
- Time schedule: Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).
- All culled pups on PND 4
- Pups (2/sex) sacrificed on PND 13
TERMINAL INVESTIGATIONS: yes - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Other examinations:
- Quality Assessment: five selected animals/sex/group (five males with the lowest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post‑Pairing Day 19) for males and on LD 7 for females.
Quantitative assessment parameters are as follows:
Hind limb foot splay
Fore and/or hind limb grip strength - Statistics:
- Statistical analyses (ANOVA) were performed, where appropriate.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Transient instances of raised fur were noted for up to four males administered 100 mg/kg/day and one male administered 50 mg/kg/day.
An isolated instance of hunched posture was recorded from GD 5 to GD 7 for one female administered 100 mg/kg/day (Animal R0710). One male administered 100 mg/kg/day was observed with a tilted head and was sent to necropsy on Day 15. One female administered 100 mg/kg/day was observed with protruding eyes on LD 13 and 14. These findings were isolated and, as such, were considered unrelated to test article toxicity. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One male administered 100 mg/kg/day (Animal R0306) died on day 15 due to congenital abnormality in the brain.
At 50 mg/kg/day two female R0602 and R0603 found dead on GD 8 and 12, respectively. At necropsy, no macroscopic abnormalities were recorded for Animal R0602 but Animal R0603 had dark lungs, a distended caecum, and an enlarged and dark thymus.
One control female (Animal R04010), one female administered 30 mg/kg/day (Animal R0502) and one female administered 50 mg/kg/day (Animal R0610) did not produce a litter; as such, they were sent to necropsy on GD 26. - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Actual body weight loss and lower body weight gains were observed during the first week of dosing for males administered 50 or 100 mg/kg/day (P < 0.01 and P < 0.001 respectively), compared with controls. Overall body weight gain for test article-treated males over the study period was, however, essentially similar to controls.
Lower body weight gains were noted from the 1st week of dosing for females administered 100 mg/kg/day (-17.8 compared with controls) and were still evident for the remainder of the study. Overall body weight gain from the start to end of dosing (LD 13) was significantly lower for females administered 100 mg/kg/day compared with controls (P < 0.001).
No test article-related body weight changes were evident for females administered 30 or 50 mg/kg/day or males administered 30 mg/kg/day. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower mean food consumption was noted during the first 2 weeks of dosing for males administered 50 or 100 mg/kg/day i.e ‑11 or ‑18% respectively. The mean food consumption over the study duration was approximately -10% compared with controls. Similar effects was also observed in female at these doses, (approximately -11%), compared with controls. The mean overall food consumption during the study, for females administered 100 mg/kg/day was 12% lower than controls.
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Alkaline phosphatase activity was significantly elevated in females administered 100 mg/kg/day, compared with controls (P < 0.001); total protein and globulin levels were significantly reduced in this group (P < 0.05), with a non-significant increase in A:G ratio, compared with controls.
No effects observed in males. - Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- Higher incidence of decreased activity for males administered 100 or 50 mg/kg/day, compared with controls.
Hind limb foot splay was shorter for males administered 100 mg/kg/day, compared with controls, with statistically significant differences noted for 1 of 2 tests (P < 0.001).
The mean number of rears was reduced during lactation for females administered 100 mg/kg/day, compared with control (LD 1: P < 0.05; LD 7: P < 0.01, LD 13: P < 0.001). Similar effects were also evident following administration of 50 mg/kg/day (LD 1: P < 0.05; LD 7 and 13: P < 0.01), compared with control.
Locomotor activity was unaffected by test article administration. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related decreases in thymus weights were recorded for females in all groups administered the test article; increases in liver weights were recorded for females administered 50 or 100 mg/kg/day, and increases in kidney weights were recorded for males administered 100 mg/kg/day, compared with concurrent controls.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Kidneys of males administered 100 mg/kg/day, increased severity of hyaline droplets characterized by small, generally round eosinophilic droplets in the proximal tubular epithelium were observed.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: Test article-related effects observed were considered not to have represented adverse health effects
- Key result
- Critical effects observed:
- no
- Conclusions:
- Once daily oral gavage administration of 30, 50, or 100 mg/kg/day Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test article related findings at all dose levels which were not considered adverse in nature, therefore, the no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day.
- Executive summary:
In an OECD 422 study, following successful formulation analysis, Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was administered via oral garage once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item. The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg.
Assessment of toxicity in the adults was based on clinical observations, body weights, food consumption, functional and behavioral assessments, estrous cycles, mating, fertility and pregnancy indices, and offspring parameters. For pups, clinical observations, litter size, sex, and body weights were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. One pup/sex/litter/dose group was selected to have thyroid weights recorded and were retained in fixative.
Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow were not induced following test article administration at any dose level.
Complete necropsy was performed on all animals (except PND 4 pups and selected PND 13 pups), and any macroscopic abnormalities were noted. Blood samples were also collected for clinical pathology and thyroid hormone assessment. Femur from five males and five females from each dose group were also processed for micronucleus testing.
No postdose observations were evident during the first 3 days of dosing, however, transient instances of raised fur were noted for up to four males administered 100 mg/kg/day and one male administered 50 mg/kg/day.
No test item related deaths occurred but three mortality was observed; one male in control group, a female in the 50 mg/kg/day and a male at 100 mg/kg/day .
Food consumption was also lower during the first 2 weeks of dosing in both sex at 50 or 100 mg/kg/day treatment groups as such actual body weight loss and lower body weight gains were observed during the first week of dosing. Overall body weight gain for treated males over the study period was essentially similar to that of controls but the overall female body weight gain was lower from the start of dosing to LD 13. No adverse effect on food consumption was evident following administration of 30 mg/kg/day in both sexes. No overt differences in water consumption were noted for treated animals, compared with controls.
Following 100 mg/kg/day in males, initial body weight loss, lower body weight gains and reduced food consumption were observed. Furthermore, raised hair and shorter hind limb foot splay, with reduced activity and reduced rearing observed during the weekly behavioral assessments. However, locomotor activities were unaffected and in the absence of any overt pathology observation, these findings were not considered as an indication of neurotoxicity. The group mean kidney weights (absolute and body weight-relative) were higher correlating with presence of hyaline droplets; this is a common response in the male rat to xenobiotics and represents accumulations of α2u globulin, a naturally occurring male rat protein. Chemicals that bind to α2u globulin form a complex that is more resistant to catabolism and will result in accumulations of hyaline droplets. This male rat-specific finding is of little relevance to risk assessment in humans.
For females administered 100 mg/kg/day; body weight gain was reduced over the duration of the study, mean absolute liver weights and body weight relative weight ratios were higher with elevated alkaline phosphatase activity, total protein and albumin:globulin (A:G) ratios. Furthermore, lower total protein and globulin levels were observed. In the absence of any overt clinical or pathological findings, these observations were considered to represent adaptive responses to administration of a xenobiotics. In addition to the above, rearing was reduced but locomotive activities were not affected.
Following 50 mg/kg/day administration Initial body weight losses, reduced body weight gains, and lower food consumptions were observed in both sexes. For females, initial reduction in lower food consumption was evident with reduced rearing during lactation. Finally, mean thymus weights (absolute and body weight relative) were lower for females administered at all dose levels without any correlating haematological or microscopic abnormalities. In the absence of any associated decline in physical health, clinical pathology or microscopic changes to indicate an adverse effect, these findings were not considered treatment related.
Reproductive performance was unaffected; the test article did not affect estrous cycles, mating, fertility, pregnancy or parturition. The number and length of estrous cycles was unaffected by test article administration. Mating performance was unaffected by test article administration; all animals mated within 7 days of pairing. Ten, nine, nine, or ten females administered control article (vehicle) or 30, 50, or 100 mg/kg/day, respectively, achieved pregnancy. Nine, nine, seven or ten dams administered control article (vehicle) or 30, 50, or 100 mg/kg/day successfully reared a live litter to LD 13. One control female (Animal R0410) had no viable fetuses (100% in utero litter losses).
No treatment related effects were noted on mean gestation lengths or the mean number of implantation sites. A slightly smaller litter size was noted following administration of 100 mg/kg/day, compared with controls (-13%). No treatment related effect on ano-genital distance, no nipples/areolae were present for male offspring, no effects on thyroid weight or thyroid hormone levels were observed in dose groups.
Test item related offspring effects following maternal exposure of 100 mg/kg/day were confined to slightly smaller litter sizes, compared with controls, and lower mean offspring weights in litters of the groups administered 50 or 100 mg/kg/day, although mean values were ± 10% of controls. In the absence of any test article-related offspring mortality noted at this dose level, these findings were considered as non-adverse. A medulloblastoma was present in one male administered 100 mg/kg/day (decedent Animal R0306). This tumor has been observed as a background finding in short term studies, although it is rare. As only one animal was affected, a relationship to the test article was considered unlikely. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
It was concluded that once daily oral gavage administration of 30, 50, or 100 mg/kg/day Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test item related findings at all dose levels which were considered none adverse. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day. A no observed effect level (NOEL) was considered as 30 mg/kg/day for the males, although a NOEL for females could not be established, due to the lower thymus weights observed in all dose groups. Reproductive performance and offspring development were unaffected as such, the no observed adverse effect level (NOEL) and NOAEL for reproductive toxicity was considered as 100 mg/kg/day.
Referenceopen allclose all
Table 3 Mean body weights and body weight gains/ losses
Sex / Day | Bodyweight (g) | |||||||||
Control | 300 / 50 mg/kg bw/day | 250 / 175 mg/kg bw/day | 125 mg/kg bw/day | 175 mg/kg bw/day | ||||||
MALES | ||||||||||
Pre-dose Day 1 | 280.7 | 284.9 | 325.7 | - | 300.1 | |||||
Pre-dose Day 2 | - | - | - | 351.7 | - | |||||
Pre-dose Day 8 | - | - | 347.5 | - | 334.9 | |||||
Pre-dose Day 15 | - | - | 369.3 | 396.5 | 354.4 | |||||
Pre-dose Day 22 | - | - | - | - | 365.7 | |||||
Pre-dose Day 26 | - | - | - | - | 372.0 | |||||
Pre-dose Day 29 | - | - | - | - | 377.7 | |||||
Pre-dose Day 30 | - | - | - | 436.7 | - | |||||
Pre-dose Day 35 | - | - | 422.8 | 448.0 | 404.0 | |||||
Dosing Phase | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 |
Dosing Day 1 | 310.3 | 365.0 | 314.9 | 367.7 | - | 418.8 | - | 451.4 | - | 430.7 |
Dosing Day 2 | 310.2 | - | 310.1 | - | - | - | - | - | - | - |
Dosing Day 3 | 316.3 | - | 311.4 | - | - | - | - | - | - | - |
Dosing Day 4 (Change from Day 1 (P2)) | 318.4 | 375.6 (10.6) | 314.2 | 375.0 (7.3) | - | - | - | 444.2 (-7.2) | - | 409.7 (-21.0) |
Dosing Day 5 | 320.5 | - | 319.2 | - | - | - | - | - | - | - |
Dosing Day 6 | 322.1 | - | 321.2 | - | - | - | - | - | - | - |
Dosing Day 7 | 326.5 | - | 325.3 | - | - | - | - | - | - | 405.3 |
Dosing Day 8 (Change from Day 1 (P1) or Day 4 (P2)) | 332.0 (21.8) | 390.1 (14.5) | 331.0 (15.2) | 387.7 (12.8) | - | - | - | 437.6 (-6.6) | - | 404.6 (-5.2) |
Dosing Day 9 | 333.0 | - | 332.8 | - | - | - | - | - | - | - |
Dosing Day 10 | 337.4 | - | 336.2 | - | - | - | - | - | - | - |
Dosing Day 11 (Change from Day 8 (P2)) | 341.6 | 392.1 (2.1) | 343.0 | 387.5 (-0.2) | - | - | - | 432.1 (-5.5) | - | 404.0 (-0.5) |
Dosing Day 12 | 344.7 | - | 349.2 | - | - | - | - | - | - | - |
Dosing Day 13 | 343.2 | - | 345.5 | - | - | - | - | - | - | - |
Dosing Day 14 (Change from Day 11 (P2)) | 345.0 | 399.1 (7.0) | 346.7 | 396.7 (9.2) | - | - | - | 437.6 (5.5) | - | 401.3 (-2.8) |
Dosing Day 15 (Change from Day 8 (P1)) | 351.7 (19.7) | - | 353.6 (22.6) | - | - | - | - | - | - | - |
Dosing Day 16 | 348.5 | - | 357.8 | - | - | - | - | - | - | - |
Dosing Day 17 | 354.3 | - | 358.0 | - | - | - | - | - | - | - |
Dosing Day 18 | 356.6 | - | 357.8 | - | - | - | - | - | - | - |
Dosing Day 19 | 363.1 | - | 362.5 | - | - | - | - | - | - | - |
Dosing Day 20 | 359.2 | - | 364.4 | - | - | - | - | - | - | - |
Dosing Day 21 (Change from Day 15 (P1)) | 364.9 (13.2) | - | 366.9 (13.3) | - | - | - | - | - | - | - |
Dosing Day 22 (Change from Day 21 (P1)) | 370.1 (5.2) | - | 372.1 (5.3) | - | - | - | - | - | - | - |
Change over course of dosing | 59.8 | 34.1 | 56.4 | 29.0 | - | - | - | -2.8 | - | -29.5 |
FEMALES | ||||||||||
Pre-dose Day 1 | 191.0 | - | 189.4 | - | 182.3 | - | - | - | - | - |
Pre-dose Day 2 | - | - | - | - | - | - | 217.3 | - | - | - |
Pre-dose Day 8 | - | - | - | - | 194.3 | - | - | - | - | - |
Pre-dose Day 15 | - | - | - | - | 202.2 | - | 234.4 | - | - | - |
Pre-dose Day 22 | - | - | 219.8 | - | - | - | - | - | - | - |
Pre-dose Day 26 | - | - | 224.3 | - | - | - | - | - | - | - |
Pre-dose Day 29 | - | - | 222.5 | - | - | - | - | - | - | - |
Pre-dose Day 30 | - | - | - | - | - | - | 238.2 | - | - | - |
Pre-dose Day 35 | - | - | - | - | 225.1 | - | 242.0 | - | - | - |
Dosing Phase | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 | P1 | P2 |
Dosing Day 1 | 201.2 | 220.5 | 208.3 | 221.6 | - | 222.9 | - | 243.5 | - | - |
Dosing Day 2 | 201.7 | - | 199.0 | - | - | - | - | - | - | - |
Dosing Day 3 | 200.2 | - | 198.5 | - | - | - | - | - | - | - |
Dosing Day 4 (Change from Day 1 (P2)) | 203.6 | 226.1 (5.7) | 203.3 | 223.1 (1.6) | - | 218.0 (-4.9) | - | 241.1 (-4.9) | - | - |
Dosing Day 5 | 203.2 | - | 201.2 | - | - | - | - | - | - | - |
Dosing Day 6 | 204.5 | - | 201.4 | - | - | - | - | - | - | - |
Dosing Day 7 | 204.5 | - | 205.7 | - | - | - | - | - | - | - |
Dosing Day 8 (Change from Day 1 (P1) or Day 4 (P2)) | 207.1 (5.9) | 231.3 (5.1) | 210.7 (8.8) | 231.9 (8.8) | - | 226.3 (8.3) | - | 239.7 (-1.4) | - | - |
Dosing Day 9 | 207.6 | - | 210.1 | - | - | - | - | - | - | - |
Dosing Day 10 | 209.3 | - | 211.0 | - | - | - | - | - | - | - |
Dosing Day 11 (Change from Day 8 (P2)) | 211.7 | 228.2 (-3.1) | 214.5 | 234.1 (2.1) | - | 221.3 (-5.0) | - | 238.2 (-1.6) | - | - |
Dosing Day 12 | 213.6 | - | 214.3 | - | - | - | - | - | - | - |
Dosing Day 13 | 212.6 | - | 213.0 | - | - | - | - | - | - | - |
Dosing Day 14 (Change from Day 11 (P2)) | 212.9 | 235.0 (6.9) | 212.4 | 234.1 (0.1) | - | 228.5 (7.3) (5.6)* | - | 239.7 (1.5) | - | - |
Dosing Day 15 (Change from Day 8 (P1)) | 213.0 (5.9) | - | 218.5 (7.8) | - | - | - | - | - | - | - |
Dosing Day 16 | 212.6 | - | 221.1 | - | - | - | - | - | - | - |
Dosing Day 17 | 214.2 | - | 219.6 | - | - | - | - | - | - | - |
Dosing Day 18 | 218.8 | - | 218.4 | - | - | - | - | - | - | - |
Dosing Day 19 | 216.3 | - | 223.1 | - | - | - | - | - | - | - |
Dosing Day 20 | 218.0 | - | 223.2 | - | - | - | - | - | - | - |
Dosing Day 21 (Change from Day 15 (P1)) | 225.0 (12.0) | - | 224.6 (6.1) | - | - | 233.6 | - | - | - | - |
Dosing Day 22 (Change from Day 21 (P1)) | 228.2 (3.2) | - | 221.9 (-2.7) | - | - | - | - | - | - | - |
Dosing Day 24 (Change from Day 21 (P2)) | - | - | - | 227.4 (-6.2) | - | - | ||||
Dosing Day 27 | - | - | - | 226.5 | - | - | ||||
Dosing Day 28 (Change from Day 24 (P2)) | - | - | - | 225.4 (-2.0) | - | - | ||||
Dosing Day 31 (Change from Day 28 (P2)) | - | - | - | 224.2 (-1.2) | - | - | ||||
Dosing Day 34 (Change from Day 31 (P2)) | - | - | - | 228.7 (4.4) (-5.0)^ | - | - | ||||
Change over course of dosing | 27.0 | 14.6 | 20.7 | 12.6 | - | - | - | -3.8 | - | - |
* change from Day 1 (i.e. 14 days)
^ change from Day 21 (i.e. 14 days)
Table 4 Unadjusted organ weights (relative to body weight %)
Doses (ppm) | Control | 50 mg/kg bw/day | 125 mg/kg bw/day | 175 mg/kg bw/day | ||||
Males | ||||||||
Number of animals/group | 5 | 5 | 5 | 3 | ||||
BODY WEIGHT (termination weight for toxicity males) | ||||||||
Weight (g) | 379.0 | 377.3 | 433.1 | 393.9 | ||||
EPIDIDMYSIS (termination weight for toxicity males) | ||||||||
Weight (g) (difference vs control %) | 1.285
| 1.258 (-2) | 1.468 (14) | 1.445 (12) | ||||
% body weight (difference vs control %) | 0.3397
| 0.3365 (-1) | 0.3399 (0) | 0.3661 (8) | ||||
TESTIS (termination weight for toxicity males) | ||||||||
Weight (g) (difference vs control %) | 3.482
| 3.124 (-10) | 3.820 (10) | 3.697 (6) | ||||
% body weight (difference vs control %) | 0.9220
| 0.8370 (-9) | 0.8851 (-4) | 0.9380 (2) |
Table 4: Summary of Absolute Organ Weights - Terminal Sacrifice
Organ |
Dose level mg/kg/day |
Male |
Female |
||||
2M |
3M |
4M |
2F |
3F |
4F |
||
30 |
50 |
100 |
30 |
50 |
100 |
||
Thymus |
group mean absolute weights |
0.309 |
0.350 |
0.320 |
0.208 |
0.171 |
0.159 |
% total body weight |
0.079 |
0.087 |
0.086 |
0.082 |
0.070 |
0.063 |
|
Liver |
group mean absolute weights |
9.584 |
9.633 |
9.772 |
9.603 |
9.781 |
10.255 |
% total body weight |
2.467 |
2.399 |
2.634 |
3.781 |
4.018 |
4.100 |
|
Kidney |
group mean absolute weights |
2.410 |
2.357 |
2.492 |
1.714 |
1.647 |
1.693 |
% total body weight |
0.620 |
0.590 |
0.671 |
0.676 |
0.676 |
0.677 |
F = Female; M = Male.
Note: Organ weights were not taken from decedent animals.
Table 5. Incidence of Selected Findings; Kidney - Terminal Sacrifice
Tissue and finding |
Dose level mg/kg/day |
Male |
Female |
||||||
1M |
2M |
3M |
4M |
1F |
2F |
3F |
4F |
||
30 |
|
50 |
100 |
30 |
|
50 |
100 |
||
Kidney |
No. examined: |
5 |
5 |
5 |
5 |
5 |
- |
- |
5 |
hyaline droplets |
Grade - |
- |
- |
- |
- |
5 |
- |
- |
5 |
1 |
5 |
5 |
5 |
1 |
- |
- |
- |
- |
|
2 |
- |
|
- |
4 |
- |
- |
- |
- |
- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male.
Table 3. summary of mean body weight by day (g)
Group |
Male |
Female |
||||||||||||||||||
Predose Day: 1 Session 1 |
Pre‑pairing Day: 1 Session 1 |
Day: 8 Session 1 |
Day: 15 Session 1 |
Pairing Day: 7 Session 1 |
Post Pairing Day: 8 Session 1 |
Day: 15 Session 1 |
Day: 21 Session 1 |
Predose Day: 8 Session 1 |
Pre‑pairing Day: 1 Session 1 |
Day: 8 Session 1 |
Day: 15 Session 1 |
Gestation Day: 0 Session 1 |
Day: 7 Session 1 |
Day: 14 Session 1 |
Day: 20 Session 1 |
Lactation Day: 1 Session 1 |
Day: 4 Session 1 |
Day: 7 Session 1 |
Day: 13 Session 1 |
|
Control |
333.7 |
356.3 |
371.1 |
382.9 |
389.1 |
403.3 |
414.1 |
413.7 |
180.4 |
189.4 |
195.1 |
204.3 |
204.6 |
225.3 |
252.3 |
309.3 |
238.8 |
247.2 |
254.4 |
271.0 |
2 |
323.5 |
343.6 |
355.8 |
370.4 |
377.0 |
389.2 |
401.4 |
404.1 |
185.9 |
194.1 |
201.2 |
210.3 |
211.1 |
227.9 |
251.3 |
310.9 |
245.6 |
252.6 |
261.0 |
277.0
|
3 |
331.4 |
353.0 |
360.2 |
377.3 |
382.3 |
400.0 |
413.0 |
413.6 |
185.2 |
191.1 |
197.1 |
205.8 |
205.0 |
224.6 |
253.2 |
312.4 |
238.3 |
246.4 |
256.1 |
272.0 |
4 |
323.9 |
345.3 |
344.2 |
355.6 |
362.9 |
381.4 |
394.4 |
398.6 |
191.3 |
200.2 |
204.9 |
213.6 |
209.5 |
228.4 |
253.0 |
306.0 |
240.7 |
244.2 |
252.1 |
261.7 |
Table 5. Summary of mean body weight gain per interval (g)
Group |
Male |
Female |
|||||||||||||||
PA:1‑PA:8 |
PA:8‑PA:15 |
PA:15‑PR:7 |
PR:7‑PP:8 |
PP:8‑PP:15 |
PP:15‑PP:21
|
PA:1‑PP:21 |
PA:1‑PA:8 |
PA:8‑PA:15
|
GD:0‑GD:7 |
GD:7‑GD:14 |
GD:14‑GD:20 |
GD:20‑LA:1 |
LA:1‑LA:4 |
LA:4‑LA:7 |
LA:7‑LA:13
|
PA:1‑LA:13 |
|
Control |
14.8 |
11.8 |
6.2 |
14.2 |
10.8 |
-4 |
57.4
|
5.7 |
9.2 |
20.8 |
26.9 |
57.0 |
‑70.5 |
8.4 |
7.3 |
16.6 |
81.5 |
2 |
12.2 |
14.5 |
6.6 |
12.2 |
12.2 |
2.8 |
60.5 |
7.1 |
9.0 |
16.8 |
23.4 |
59.6 |
‑65.3 |
7.0 |
8.4 |
16.0 |
84.1 |
3 |
7.2+H |
17.1 |
5.0 |
17.7 |
13.1 |
0.5 |
60.5 |
5.9 |
8.7 |
19.6 |
26.9 |
59.2 |
‑74.1 |
8.1 |
9.8 |
15.9 |
79.1 |
4 |
‑1.1#H |
11.3 |
5.9 |
18.5 |
13.0 |
4.2 |
56.7 |
4.7 |
8.7 |
18.9 |
24.6 |
53.0 |
‑65.3 |
3.5 |
7.9 |
9.6 |
61.5#r
|
PA ‑ Pre‑pairing, PR – Pairing, PP ‑ Post Pairing, LA – Lactation, GD ‑ Gestation
#r = Wilcoxon rank Sum Test Significant at 0.001 level, +H = Dunnett Exact Homogeneous Test Significant: 0.01 level, #H = Dunnett Exact Homogeneous Test Significant: 0.001 level
Table 6. Summary of mean food consumption
Group |
Male |
Female |
||||||||||||||||||||||||||||||||||||
Pre‑pairing |
Post Pairing |
Pre‑pairing |
Gestation |
Lactation |
||||||||||||||||||||||||||||||||||
1 ‑ 4 |
4 ‑ 8 |
8 ‑ 11 |
11 ‑ 15 |
1 ‑ 6 |
6 ‑ 8 |
8 ‑ 11 |
11 ‑ 15 |
15 ‑ 18 |
18 ‑ 21 |
1 ‑ 4 |
4 ‑ 8 |
8 ‑ 11 |
11 ‑ 15 |
GD:8‑GD:9 |
GD:9‑GD:10 |
GD:10‑GD:11 |
GD:11‑GD:12 |
GD:12‑GD:13 |
GD:13‑GD:14 |
GD:14‑GD:15 |
GD:15‑GD:16
|
GD:16‑GD:17 |
GD:17‑GD:18 |
GD:18‑GD:19 |
GD:19‑GD:20 |
LA:1‑LA:2 |
LA:2‑LA:3 |
LA:3‑LA:4 |
LA:4‑LA:5
|
LA:5‑LA:6 |
LA:6‑LA:7 |
LA:7‑LA:8 |
LA:8‑LA:9 |
LA:9‑LA:10 |
LA:10‑LA:11 |
LA:11‑LA:12 |
LA:12‑LA:13 |
|
1 |
19.2 |
21.2 |
18.7 |
18.0 |
18.1 |
17.6 |
18.7 |
8.6 |
17.8 |
17.8
|
13.5 |
14.9 |
13.5 |
13.9
|
12.7 |
15.7 |
16.0 |
14.6 |
16.2 |
16.1 |
16.1 |
15.7 |
16.8 |
16.1 |
17.5 |
22.1 |
17.8 |
23.3 |
25.1 |
19.3 |
19.1 |
19.9 |
20.2 |
21.6 |
28.1 |
28.1 |
32.3 |
34.6 |
2 |
18.7 |
18.7 |
17.3 |
16.9 |
17.0 |
16.3 |
17.2 |
6.2 |
16.6 |
17.5 |
15.6 |
14.2 |
12.5 |
15.4
|
14.1 |
15.0 |
16.0 |
14.0 |
16.2 |
14.8 |
15.3 |
16.6 |
16.0 |
16.4 |
16.8 |
17.8*H |
16.2 |
19.3 |
25.1 |
21.5 |
19.8 |
18.2 |
20.5 |
21.4 |
23.6 |
29.1 |
32.3 |
34.3 |
3 |
17.8 |
17.7 |
17.3 |
15.7 |
17.1 |
16.7 |
17.6 |
5.9 |
15.8 |
17.3 |
11.2 |
13.2 |
11.4 |
13.7 |
14.0 |
14.6 |
14.3 |
14.9 |
16.3 |
16.4 |
15.2 |
16.1 |
15.2 |
17.1 |
17.3 |
18.8 |
15.4 |
19.4 |
18.1 |
19.4 |
22.6 |
20.2 |
18.9 |
21.1 |
22.7 |
27.2 |
32.4 |
27.8 |
4 |
16.6 |
15.4 |
15.1 |
16.2 |
17.8 |
17.3
|
18.9 |
5.7 |
18.4 |
18.9 |
12.1 |
12.6 |
11.6 |
12.7 |
11.4 |
13.8 |
13.8 |
14.3 |
15.5 |
14.7 |
14.9 |
15.8 |
14.7 |
14.5 |
16.0 |
17.4*H |
16.4 |
20.8 |
17.3 |
*H = Dunnett Exact Homogeneous Test Significant: 0.05 level
@ = Number examined reduced due to excluded data
Table 7. Summary of Fertility and Reproductive indices
Treatment Group |
Control |
30 mg/kg |
50 mg/kg |
100 mg/kg |
Total males |
10 |
10 |
10 |
10 |
Unscheduled Deaths Prior to Cohabitation |
0 |
0 |
0 |
0 |
Males Cohabitated |
10 |
10 |
10 |
10 |
Unscheduled Deaths Prior to Cohabitation |
0 |
0 |
0 |
1 |
Males mating with at least 1 female |
10 |
10 |
10 |
9 |
Males impregnating at least 1 female |
10 |
9 |
9 |
9 |
Mating Index (%) |
100 |
100 |
100 |
90 |
Fecundity Index (%) |
100 |
90 |
90 |
100 |
Fertility Index (%) |
100 |
90 |
90 |
90 |
Total Females |
10 |
10 |
10 |
10 |
Unscheduled Deaths Prior to Cohabitation |
0 |
0 |
0 |
0 |
Females Cohabited |
10 |
10 |
10 |
10 |
Unscheduled Deaths During Cohabitation |
0 |
0 |
0 |
0 |
Females Mated |
10 |
10 |
10 |
10 |
Pregnant Females |
10 |
9 |
9 |
10 |
Found Dead |
0 |
0 |
2 |
0 |
Non Pregnant Females |
0 |
1 |
1 |
0 |
Matings Per Day Periods Of Cohabitation – Day 1
|
2 |
2 |
2 |
1 |
Matings Per Day Periods Of Cohabitation – Day 2 |
3 |
5 |
6 |
4 |
Matings Per Day Periods Of Cohabitation – Day 3 |
1 |
2 |
2 |
1 |
Matings Per Day Periods Of Cohabitation – Day 4 |
4 |
1 |
0 |
2 |
Matings Per Day Periods Of Cohabitation – Day 6 |
0 |
0 |
0 |
1 |
Matings Per Day Periods Of Cohabitation – Day 7 |
0 |
0 |
0 |
1 |
Mating Index % |
100 |
100 |
100 |
100 |
Fecundity Index % |
100 |
90 |
90 |
100 |
Fertility Index % |
100 |
90 |
90 |
100 |
Summary of Mean Parturition and Litter Data
|
||||
Duration of gestation (days) |
23.3 |
23.1 |
23.1 |
23.1 |
Number of implantation sites |
11.4 |
12.1 |
11.6 |
10.9 |
Number of pups born |
10.3 |
10.6 |
11.1 |
9.4 |
Number of pups alive PND 1 |
10.3 |
10.6 |
10.4 |
9.2 |
% male pups PND 1 |
49.1 |
56.0 |
52.9 |
48.4 |
Number of pups alive PND 4 before culling |
10.3 |
10.6 |
10.4 |
9.1 |
Number of pups culled PND 4 |
1.4 |
1.0 |
1.0 |
0.2 |
Number of pups alive PND 4 after culling |
8.9 |
9.6 |
9.4 |
8.9 |
Number of pups alive PND 7 |
8.9 |
9.6 |
8.9 |
8.9 |
Number of pups alive PND 13 |
8.9 |
9.6 |
8.9 |
8.9 |
Summary of Pup Survival (mean value) |
||||
Post-implantation survival index % |
93.9 |
87.4 |
96.3 |
86.7 |
Live birth index % |
94.4 |
100.0 |
94.9 |
98.2 |
Survival index PND 1-4 % |
100.0 |
100.0 |
100.0 |
98.6 |
Survival index PND 4-7 % |
100.0 |
100.0 |
94.3 |
100.0 |
Survival index PND 7-13 % |
100.0 |
100.0 |
100.0 |
100.0 |
Summary of Pup Clinical Observations (Number of litters with sign) |
||||
Abdomen; mass: |
|
|
1 |
|
Culled |
5 |
6 |
4 |
2 |
Discoloration: body |
|
|
|
1 |
Front legs; deformed, limited mobility |
|
|
1 |
|
Found dead. |
1 |
|
1 |
1 |
Front paws: swollen |
1 |
|
|
|
Front leg; bent: right |
1 |
|
|
|
Hemorrhagic area: neck |
|
|
1 |
|
Hind leg; bent: inwards, left. |
1 |
|
|
|
Hind leg; facing inwards, left |
1 |
|
|
|
Mis-sexed |
|
2 |
1 |
1 |
Missing, presumed cannibalized |
|
|
3 |
1 |
Nose; sore |
|
1 |
|
|
pale |
|
|
|
1 |
Respiration: noisy |
|
|
|
1 |
Small |
|
2 |
2 |
|
Sores/lesion: mouth and head. |
|
|
|
1 |
Sent to necropsy |
1 |
|
1 |
2 |
Thin |
1 |
|
|
1 |
Tail; bent |
1 |
|
|
|
Tail; damaged |
|
|
1 |
|
Unfed |
|
|
1 |
|
Summary of Functional Observational Battery in male
|
||||
Mild vocalization
|
1 |
2 |
2 |
1 |
Moderate vocalization |
1 |
1 |
|
|
Mild decreased activity |
2 |
1 |
2 |
1 |
Moderate decreased activity |
|
1 |
|
4 |
Severe decreased activity |
|
|
1 |
|
Posture (body/head) tilting, head, severe, right |
|
|
|
1 |
Behavior ‑ other head shaking, occasional |
|
|
|
1 |
Behavior ‑ other jaw chomping |
|
|
1 |
1 |
Table 8. Summary of Functional Observattional Battery in male
Group |
Latency sec |
Rears |
F boli |
Ur pools |
||||||||||||||||||||||||
Pre-Dose |
Pre‑pairing |
Pairing |
Post Pairing |
Pre-Dose |
Pre‑pairing |
Pairing |
Post Pairing |
Pre-Dose |
Pre‑pairing |
Pairing |
Post Pairing |
Pre-Dose |
Pre‑pairing |
Pairing |
Post Pairing |
|||||||||||||
Day 5 |
Day 5 |
Day 12 |
Day 4 |
Day 5 |
Day 12 |
Day 19 |
Day 5 |
Day 5 |
Day 12 |
Day 4 |
Day 5 |
Day 12 |
Day 19 |
Day 5 |
Day 5 |
Day 12 |
Day 4 |
Day 5 |
Day 12 |
Day 19 |
Day 5 |
Day 5 |
Day 12 |
Day 4 |
Day 5 |
Day 12 |
Day 19 |
|
1 |
0.0 |
3.0 |
1.0 |
0.0 |
1.0 |
1.0 |
0.0 |
5.0 |
4.0 |
2.0 |
3.0 |
4.0 |
5.0 |
1.0 |
2.0 |
1.0 |
1.0 |
0.0 |
0.0 |
1.0 |
1.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
2 |
0.0 |
3.0 |
2.0 |
1.0 |
1.0 |
3.0 |
1.0 |
5.0 |
4.0 |
3.0 |
3.0 |
4.0 |
5.0 |
7.0 |
1.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
3 |
0.0 |
5.0 |
14 |
2.0 |
1.0 |
1.0 |
1.0 |
6.0 |
2.0 |
1.0 |
1.0 |
4.0 |
3.0 |
3.0 |
3.0 |
2.0 |
|
0.0 |
1.0 |
1.0 |
1.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
4 |
0.0 |
7.0 |
5** |
3.0 |
1.0 |
2.0 |
1.0 |
6.0 |
1.0 |
1.0 |
1.0 |
3.0 |
2.0 |
3.0 |
1.0 |
1.0 |
0.0 |
0.0 |
0.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Table 9. Summary of Functional Observational Battery in female
oup |
Latency sec |
Rears |
F Boli |
Ur pools |
|||||||||||||||||||||||||||||||||||||||||||||
Pre-Dose |
Pre‑pairing |
Pairing |
Gestation |
Lactation |
Pre-Dose |
Pre‑pairing |
Pairing |
Gestation |
Lactation |
Pre-Dose |
Pre‑pairing |
Pairing |
Gestation |
Lactation |
Pre-Dose |
Pre‑pairing |
Pairing |
Gestation |
|
||||||||||||||||||||||||||||||
D13 |
D6 |
D13 |
D5 |
D0 |
D7 |
D14 |
D20 |
D1 |
D7 |
D13 |
D13 |
D6 |
D13 |
D5 |
D0 |
D7 |
D14 |
D20 |
D1 |
D7 |
D13 |
D13 |
D6 |
D13 |
D5 |
D0 |
D7 |
D14 |
D20 |
D1 |
D7 |
D13 |
D13 |
D6 |
D13 |
D5 |
D0 |
D7 |
D14 |
D20 |
|
||||||||
1 |
0.0 |
0.0 |
2.0 |
- |
1.0 |
1.0 |
1.0 |
1.0 |
0.0 |
1.0 |
0.0 |
8.0 |
9.0 |
6.0 |
- |
7.0 |
8.0 |
5.0 |
|
9.0 |
8.0 |
9.0 |
6.0 |
1.0 |
0.0 |
- |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
1.0 |
0.0 |
- |
0.0 |
0.0 |
0.0 |
0.0 |
|
|||||||
2 |
0.0 |
0.0 |
3.0 |
- |
1.0 |
3.0 |
2.0 |
2.0 |
0.0 |
0.0 |
1.0 |
7.0 |
5* |
5.0 |
- |
3.0 |
4.0 |
2* |
1.0 |
7.0 |
5.0 |
7.0 |
4.0 |
1.0 |
1.0 |
- |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
1.0 |
0.0 |
- |
1.0 |
0.0 |
0.0 |
0.0 |
|
|||||||
3 |
0.0 |
0.0 |
2.0 |
- |
0.0 |
1.0 |
2.0 |
2.0 |
0.0 |
0.0 |
1.0 |
7.0 |
8.0 |
5.0 |
- |
3.0 |
3* |
2* |
2.0 |
1.0 |
4* |
3** |
2** |
0.0 |
0.0 |
- |
0.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
1.0 |
1.0 |
- |
1.0 |
1.0 |
0.0 |
0.0 |
|
|||||||
4 |
0.0 |
1.0 |
1.0 |
3.0 |
0.0 |
2.0 |
4* |
4.0 |
0.0 |
0.0 |
0.0 |
7.0 |
5* |
5.0 |
4.0 |
3.0 |
4.0 |
2.0 |
2.0 |
1.0 |
4* |
3** |
2*** |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
1.0 |
0.0 |
1.0 |
0.0 |
0.0 |
0.0 |
0.0 |
|
|||||||
|
Lactation |
|
|||||||||||||||||||||||||||||||||||||||||||||||
D1 |
D7 |
D13 |
* P<=0.05 ** P<=0.01 *** P<=0.001
|
|
|||||||||||||||||||||||||||||||||||||||||||||
1 |
1.0 |
0.0 |
0.0 |
|
|||||||||||||||||||||||||||||||||||||||||||||
2 |
0.0 |
0.0 |
0.0 |
|
|||||||||||||||||||||||||||||||||||||||||||||
3 |
1.0 |
1.0 |
0.0 |
|
|||||||||||||||||||||||||||||||||||||||||||||
4 |
0.0 |
0.0 |
Table 10. Summary of Hematology
Male |
||||||||||||||||||||||||||||||
Group |
HB g/dL |
RBC 10E12/L |
PCV % |
MCV fL |
MCH pg |
MCHC g/dL
|
RETA % |
RABS 10E9/L |
RDW % |
HDW g/dL |
WBC 10E9/L |
N 10E9/L
|
L 10E9/L |
M 10E9/L |
E 10E9/L |
B 10E9/L |
LUC. 10E9/L |
N% % |
L% % |
M% % |
E% % |
B% % |
LUC% % |
PLT 10E9/L |
PCT % |
MPV fL |
PDW % |
PT sec |
APTS sec |
FIB g/L |
1 |
14.1 |
8.27 |
43.9 |
53.1 |
17.0 |
32.0 |
2.0 |
163.9 |
13.3 |
2.48 |
4.9 |
1.15
|
3.46 |
0.10 |
0.14 |
0.0 |
0.03 |
24 |
71 |
2 |
3 |
0 |
1 |
753 |
0.56 |
7.4 |
53.2 |
21.9 |
17.8 |
1.45
|
2 |
14.1 |
8.38 |
44.1 |
52.6 |
16.9 |
32.0 |
2.2 |
179.6 |
12.6 |
2.53 |
6.4 |
1.22
|
4.92 |
0.12 |
0.08 |
0.01 |
0.04 |
19
|
77 |
2 |
1 |
0 |
1 |
738
|
0.56 |
7.6 |
53.5 |
PT sec |
17.4 |
1.46 |
3 |
14.1 |
8.43 |
44.0 |
52.2 |
16.7 |
32.0 |
2.0 |
168.4 |
12.9 |
2.55 |
5.6 |
1.07
|
4.29 |
0.13 |
0.09 |
0.01 |
0.03 |
20 |
75 |
2 |
2 |
0 |
0 |
725 |
0.55 |
7.6 |
55.2 |
26.2 |
18.3 |
1.45 |
4 |
13.8 |
8.34 |
43.5 |
52.2 |
16.5 |
31.7 |
2.2 |
189.4 |
13.0 |
2.52 |
6.7 |
1.58
|
4.86 |
0.17 |
0.06 |
0.01 |
0.04 |
23 |
73 |
2 |
1* |
0 |
1 |
722
|
0.54 |
7.5 |
54.3 |
22.0 |
17.4 |
1.44 |
Female |
||||||||||||||||||||||||||||||
group |
HB g/dL |
RBC 10E12/L |
PCV % |
MCV fL |
MCH pg |
MCHC g/dL
|
RETA % |
RABS 10E9/L |
RDW % |
HDW g/dL |
WBC 10E9/L |
N 10E9/L
|
L 10E9/L |
M 10E9/L |
E 10E9/L |
B 10E9/L |
LUC. 10E9/L |
N% % |
L% % |
M% % |
E% % |
B% % |
LUC% % |
PLT 10E9/L |
PCT % |
MPV fL |
PDW % |
PT sec |
APTS sec |
FIB g/L |
1 |
14.3 |
7.51 |
43.8 |
58.4 |
19.1 |
32.8
|
3.7 |
276.6 |
13.6 |
1.88 |
4.8 |
2.04
|
2.51 |
0.21 |
0.04 |
0.0 |
0.03 |
42 |
52 |
5.0 |
1.0 |
0.0 |
1.0 |
965 |
0.68 |
7.0 |
47.5 |
23.8 |
16.0 |
1.64 |
2 |
14.4 |
7.67 |
44.1 |
57.5 |
18.7 |
32.5
|
3.7 |
280.4 |
12.5 |
1.82 |
5.1 |
1.95 |
2.84 |
0.18 |
0.06 |
0.0 |
0.04 |
43 |
56 |
4.0 |
1.0 |
0.0 |
1.0 |
977 |
0.69 |
7.1 |
49.7 |
23.8 |
16.3 |
1.65 |
3 |
14.4 |
7.73 |
43.2 |
55.9 |
18.6 |
33.3 |
4.0 |
312.3 |
14.0 |
1.96 |
5.1 |
2.22
|
2.62 |
0.18 |
0.06 |
0.0 |
0.03 |
34 |
52 |
4.0 |
1.0 |
0.0 |
1.0 |
915 |
0.64 |
7.0 |
53.4 |
25.0 |
17.1 |
1.69 |
4 |
14.4 |
7.48 |
43.6 |
58.3 |
19.3 |
33.1 |
3.6 |
266.0 |
14.0 |
1.97 |
6.1 |
2.45 |
3.25 |
0.27 |
0.08 |
0.0 |
0.04 |
40 |
54 |
5.0 |
2.0 |
0.0 |
1.0 |
981 |
0.68 |
6.9 |
49.3 |
22.9 |
16.1 |
1.62 |
Key to abbreviations
ode |
Parameter |
Method of determination |
HB |
Haemoglobin |
Flow Cytometry |
RBC |
Red Blood Cells |
Flow Cytometry |
PCV |
Packed Cell Volume |
Flow Cytometry Calculation |
MCV |
Mean Cell Volume |
Flow Cytometry |
MCH |
Mean Cell Haemoglobin |
Flow Cytometry Calculation |
MCHC |
Mean Cell Haemoglobin Concentration |
Flow Cytometry Calculation |
RETA |
Reticulocytes % |
Flow Cytometry Calculation |
RABS |
Absolute reticulocytes |
Flow Cytometry |
RDW |
Red Cell Distribution Width |
Flow Cytometry |
HDW |
Haemoglobin Distribution Width |
Colorimetry |
WBC |
White Blood Cells |
Flow Cytometry |
N |
Neutrophils |
Calculation |
L |
Lymphocytes |
Calculation |
M |
Monocytes |
Calculation |
E |
Eosinophils |
Calculation |
B |
Basophils |
Calculation |
LUC |
Large Unstained Cells |
Calculation |
N% |
Neutrophils % |
Flow Cytometry |
L% |
Lymphocytes % |
Flow Cytometry |
M% |
Monocytes % |
Flow Cytometry |
E% |
Eosinophils % |
Flow Cytometry |
B% |
Basophils % |
Flow Cytometry |
LUC% |
Large Unstained Cells % |
Flow Cytometry |
PLT |
Platelets |
Flow Cytometry |
PCT |
Platelet Crit |
Flow Cytometry Calculation |
MPV |
Mean Platelet Volume |
Flow Cytometry |
PDW |
Platelet Distribution Width |
Flow Cytometry |
PT |
Tox Prothrombin time |
Turbidometry |
APTS |
Toxicology activated partial thromboplastin time - Synthasil |
Turbidometry |
FIB |
Fibrinogen |
Turbidometry |
Table 11. Summary of Clinical Chemistry
Male |
||||||||||||||||||
group |
AST IU/L |
ALT IU/L |
HALP IU/L |
CHOL mmol/L |
T.BI umol/L |
TP g/L |
ALB g/L |
GLOB g/L |
A\G RATIO |
NA mmol/L |
K mmol/L |
CL mmol/L
|
CAL mmol/L |
P mmol/L |
HCRE umol/L |
UREA mmol/L |
GLUC mmol/L |
ISBA umol/L
|
1 |
14.3 |
74 |
60 |
83 |
1.5 |
<1.7 |
59 |
40 |
19 |
2.2 |
143 |
3.9 |
102 |
2.56 |
1.7 |
32 |
7.9 |
9.5 |
2 |
14.4 |
67 |
59 |
83 |
1.4 |
<1.7 |
58 |
40 |
18 |
2.3 |
142 |
3.9 |
101 |
2.55 |
1.7 |
30 |
8.5 |
9.4 |
3 |
14.4 |
65 |
44 |
83 |
1.4 |
<1.7 |
58 |
40 |
18 |
2.3 |
141 |
4.0 |
100* |
2.54 |
1.8 |
31 |
8.0 |
9.3 |
4 |
14.4 |
78 |
50 |
79 |
1.6 |
<1.7 |
58 |
39 |
17 |
2.4 |
142 |
4.28 |
100* |
2.57 |
1.8 |
32 |
9.1 |
10.4 |
Female |
||||||||||||||||||
group |
AST IU/L |
ALT IU/L |
HALP IU/L |
CHOL mmol/L |
T.BI umol/L |
TP g/L |
ALB g/L |
GLOB g/L |
A\G RATIO |
NA mmol/L |
K mmol/L |
CL mmol/L
|
CAL mmol/L |
P mmol/L |
HCRE umol/L |
UREA mmol/L |
GLUC mmol/L |
ISBA umol/L
|
1 |
130 |
64 |
65 |
2.1 |
<1.7 |
59 |
35 |
23 |
1.5 |
138 |
4.0 |
98 |
2.67 |
2.6 |
31 |
9.5 |
8.2 |
36.86 |
2 |
113 |
68 |
71 |
1.9 |
<1.7 |
57 |
36 |
21 |
1.7 |
138 |
3.8 |
97 |
2.75 |
2.6 |
30 |
10.6 |
8.8 |
57.92
|
3 |
103 |
69 |
70 |
1.9 |
<1.8 |
57 |
36 |
21 |
1.7 |
137 |
4.2 |
97 |
2.68 |
2.6 |
27 |
10.6 |
9.5 |
85.90 |
4 |
122 |
<82 |
111*** |
2.0 |
<1.7 |
55* |
34 |
20* |
1.8 |
138 |
4.4 |
97 |
2.73 |
2.4 |
30 |
11.6 |
9.5 |
84.74 |
Key to abbreviations
Code |
Parameter |
Method of determination |
AST |
Aspartate Aminotransferase |
Optimised UV method using alpha ketoglutarate without pyridoxal phosphate activation |
ALT |
Alanine Aminotransferase |
Optimised UV method using L-Alanine and alpha‑oxoglutarate as primary substrates |
HALP |
Alkaline Phosphatase |
Colorimetric method using p‑nitrophenyl phosphate as substrate standardised to IFCC |
CHOL |
Total Cholesterol |
Enzymatic method using cholesterol oxidase/esterase |
T.BI |
Total Bilirubin |
A colorimetric method. Indirect bilirubin is liberated by detergent: Total bilirubin is coupled with a diazonium compound to give corresponding azobilirubin. |
TP |
Total Protein |
Colorimetric method using Biuret reagent |
ALB |
Albumin |
Bromocresol Green method |
GLOB |
Globulin |
globulin = total protein - albumin |
A\G |
Albumin/Globulin Ratio |
Calculation |
NA |
Sodium |
Ion‑selective electrode |
K |
Potassium |
Ion‑selective electrode |
CL |
Chloride |
Ion‑selective electrode |
CAL |
Calcium Gen 2 |
Photometric using 5-nitro-5'-methyl-BAPTA (NM-BAPTA) |
P |
Inorganic Phosphate |
UV Assay utilising ammonium molybdate |
HCRE |
Enzymatic Creatinine |
Enzymatic Colorimetric method utilising 4-aminophenazone |
UREA |
Urea |
UV method using a coupled urease procedure |
GLUC |
Glucose |
UV method using a coupled hexokinase procedure |
ISBA |
Serum Bile Acids - ILab 650 |
Spectrophotometric/Enzymic with Bile Acids being converted to 3-keto steroids with the production of Thio-NADH |
NB: More tables containing raw data are attached in background material.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- other:
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
- other:
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
- other:
- Justification for type of information:
- JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no study available
Mode of Action Analysis / Human Relevance Framework
It is not possible to conclude on the mode of action of the test item since no adverse effect was observed following subacute exposure in rats. Although non-adverse effects were observed at all dose tested, these were not dose related and without any pathological effects. It was concluded that these effects were considered adaptive response to the exposure to the test item.
Additional information
In an OECD 422 study, following successful formulation analysis, Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was administered via oral garage once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item. The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg.
Observations from the study resulted in test item related findings at all dose levels which were considered non-adverse. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day. A no observed effect level (NOEL) was considered as 30 mg/kg/day for the males, although a NOEL for females could not be established, due to the lower thymus weights observed in all dose groups. Reproductive performance and offspring development were unaffected as such, the no observed effect level (NOEL) and NOAEL for reproductive toxicity was considered as 100 mg/kg/day.
Justification for classification or non-classification
Harmonised classification:
The substance has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).
Self classification:
The substance does not meet the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE) in accordance with Regulation (EC) No 1272/2008 (CLP).
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.