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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
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- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- Auto flammability
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- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- 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
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- 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.
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- 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.
- Principles of method if other than guideline:
- An integrated standard genotoxicity assessment into a general toxicology study (OECD, 2016) & ICH S2(R1) (2011) methodolgy was used during this test.
- 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-18 - 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
- Vehicle:
- corn oil
- Details on 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. - Details on mating procedure:
- Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Weekly on Day 7 and 15, or until mating is confirmed.
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear or a retained vaginal plug.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females which have not shown evidence of mating by Day 10 of the pairing period will be paired with proven males of the same treatment group. Likewise, males not showing any evidence of mating by Day 10 of the pairing period may be paired with untreated females. - 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 25 °C) 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:
- 5-8 weeks
- Frequency of treatment:
- Once daily
- Details on study schedule:
- 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. - 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 (Imtermediate)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Gropu 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
- Parental animals: Observations and examinations:
- 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 examined
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 - Oestrous cyclicity (parental animals):
- ESTROUS CYCLE: Yes
-Daily vaginal lavage (washings) was conducted for all females during acclimation (predose), from 1 week after arrival until the day prior to dosing. The stage of estrous was recorded, and only females with regular 4- to 5-day cycles were included on study.
- Daily vaginal lavage was performed on females from the start of dosing until the confirmation of mating, and on LD 14, prior to necropsy. - Sperm parameters (parental animals):
- Sliterections of testes and epididymides were also stained with periodic acid‑Schiff (PAS) for spermatogenic staging for all Group 1 and 4 males
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
PARAMETERS EXAMINED: Yes
The following parameters were examined in [F0 / F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other.
GROSS EXAMINATION OF DEAD PUPS: Yes
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No
ASSESSMENT OF DEVELOPMENTAL: Yes - Postmortem examinations (parental animals):
- SACRIFICE: Yes
- Male animals: Post-Pairing Day 22 (Day 43).
- Maternal animals: LD 14 (those that littered) or Day 26 post‑coitum (those that did not litter).
GROSS NECROPSY: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS: Yes - Postmortem examinations (offspring):
- SACRIFICE: Yes
- Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed
GROSS NECROPSY: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS: Yes - Statistics:
- Statistical analyses were performed, where appropriate - refer to the attached report.
- Reproductive indices:
- Number of indices were used, where appropriate, to evaluate reproductive function: mating index, fecundity index, fertility index, mean duration of gestation, mean number of implantation sites
- Offspring viability indices:
- Number of indices were used, where appropriate, to evaluate reproductive function: Mean number of pups born, Mean number of pups alive PND 1, % male pups PND 1 (litter), % male pups PND 1 (mean), % Post-implantation survival index (litter), % Post-implantation survival index (mean), % Live birth index (litter) and Live birth index.
- 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. - Dermal irritation (if dermal study):
- not examined
- 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. These three death were unrelated to treatment.
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. This was considered a low incidence and not treatment related. - 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, similar to controls.
Significantly 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 were also observed in females at these doses, (approximately -11% in the first 2 weeks), compared with controls. The mean overall food consumption during the study, for females administered 100 mg/kg/day was 12% lower than controls during lactation.
- 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):
- 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.
All remaining intergroup differences were transient in nature or only affected one or two animals and, as such, these observations were considered to have arisen incidentally.
No test article-related effects were evident for either sex administered 30 mg/kg/day. - Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- The number and length of estrous cycles was unaffected by test article administration
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Pup body weights were essentially similar to controls on PND 1 and 4; however, lower mean male body weights were evident on PND 7 (-5%) and PND 13 (-9%) from litters maternally administered 100 mg/kg/day, compared with controls. Female body weights were also lower on PND 7 (‑5%) and PND 13 (-9%), compared with controls.
For litters in the group administered 50 mg/kg/day, lower male body weights were also evident on PND 7 (-5%) and PND 13 (-6%), compared with controls. Following maternal administration of 50 mg/kg/day, female body weights were lower from PND 1 onwards (-4 to -7%) compared with controls.
No effect on pup body weights was evident following maternal administration of 30 mg/kg/day. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Description (incidence and severity):
Upon macroscopic examination, no findings considered related to the test article were recorded for adults or the subsequent offspring. Tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.- Other effects:
- not specified
- Behaviour (functional findings):
- no effects observed
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- ca. 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: There was no test item related effects observed.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive 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, reproductive performance and offspring development were unaffected as such, the no observed adverse effect level NOAEL for reproductive toxicity was considered as 100 mg/kg/day.
- Executive summary:
In an OECD 422 study, following formulation analysis (by analysing dibutylamine), 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.
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 mortalities were observed; one male in the control group, a female in the 50 mg/kg/day group and a male at 100 mg/kg/day group.
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.
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 test article-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 nipples/areolae were present for male offspring that survived to PND 13.
No effect on ano-genital distance was observed in litters from dams administered any test article dose level, compared with controls.
No test article-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%).
Table 2: 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 2. 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 |
16.9 |
20.8 |
19.2 |
20.3 |
19.9 |
21.0 |
24.6 |
25.4*H |
27.5*H |
*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 Mean Estrous Cycles
Group |
Pre‑dose |
Pre‑pairing |
||
|
No. of Estrous Cycles |
Mean Cycle Length (days) |
No. of Estrous Cycles |
Mean Cycle Length (days) |
control |
2.5 |
4.3 |
2.2 |
4.2 |
2 |
2.8 |
4.6 |
2.6 |
4.3 |
3 |
2.3 |
4.8 |
2.2 |
4.4 |
4 |
1.9 |
4.4 |
2.2 |
4.7 |
Estrous cycle = from 1st Oestrus to the next nonconsecutive stage of the same type
@ = Number examined reduced due to Female R0800 (Group 4) excluded
Table 9. 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 10. Summary of Functional Observational Battery in female
Group |
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 |
0.0 |
|
Table 11. 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
Code |
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 12. 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.
- Reason / purpose for cross-reference:
- reference to same study
Reference
- 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.
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.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for the Testing of Chemicals, No. 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
- Version / remarks:
- 29 July 2016
- Deviations:
- yes
- Remarks:
- The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- yes
Test material
- Reference substance name:
- N,N,N',N'-tetrabutylmethylenediamine
- EC Number:
- 243-678-5
- EC Name:
- N,N,N',N'-tetrabutylmethylenediamine
- Cas Number:
- 20280-10-8
- Molecular formula:
- C17H38N2
- IUPAC Name:
- dibutyl[(dibutylamino)methyl]amine
- Reference substance name:
- Dibutylamine
- EC Number:
- 203-921-8
- EC Name:
- Dibutylamine
- Cas Number:
- 111-92-2
- Molecular formula:
- C8H19N
- IUPAC Name:
- 1-Butanamine, N-butyl-
- Test material form:
- liquid
- Details on test material:
- Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19
Constituent 1
Constituent 2
- 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-18
Test animals
- Species:
- rat
- Strain:
- other: Crl:WI(Han)
- 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
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on 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 25°C) and homogenous (Covance Study 8359299).
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 achieved concentration of formulations was 85 to 115% of nominal. Results were within this range. Test article was not detected in Group 1 control samples.
The formulation prepared for use in Week 6 of dosing was also analyzed to determine stability when stored at 2-8 °C. For stability, the change in achieved concentration should be no greater than 15% from the initial time point. Results were within this range and therefore the dose formulations are considered stable for 5 days when stored at 2-8 °C.
The mean % target range for the preparation of the formulations was 85 to 115% of the nominal concentration. - Details on mating procedure:
- Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Weekly on Day 7 and 15, or until mating is confirmed.
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear or a retained vaginal plug.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females which have not shown evidence of mating by Day 10 of the pairing period will be paired with proven males of the same treatment group. Likewise, males not showing any evidence of mating by Day 10 of the pairing period may be paired with untreated females.
On Pairing Day 0, Animal R0306 (Group 4 male) was sent to necropsy. Animal R0706 (Group 4 female) was paired with a proven male (Animal R0308, Group 4), after a 5-day rest period - Duration of treatment / exposure:
- 5-8 weeks
- Frequency of treatment:
- Once daily
- Duration of test:
- 5-8 weeks
Doses / concentrationsopen allclose all
- 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 no treatment
- 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.
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: YES
POSTDOSE OBSERVATIONS:
- For the first 3 days of dosing, postdose observations were to be recorded upon return to the cage and 0.5, 1, 2, and 4 hours postdose. On Pre-Pairing Day 1, the return to home cage postdose observations were not recorded onto the data capture system for Animals R0001, R0002, R0003, and R0004 (Group 1 males), in error
DETAILED CLINICAL OBSERVATIONS: Yes
- pre-test: Twice
- treatment to lactation: weekly
BODY WEIGHT: Yes
- pre-test: once
Note: Female body weights were recorded once during acclimation (Day-7); before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 7, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- pre-test: weekly
- treatment to lactation: 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 (female) from GD 0 to 21.
SENSORY REACTIVITY & GRIP STRENGTH:
- Week 6 (before dosing)
MOTOR ACTIVITY:
- Week 6 (before dosing)
- Days 7 of lactation
HAEMATOLOGY: Yes
- Week 8/9 of dosing (LD 14 for females).
BLOOD CHEMISTRY: Yes
- 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 females 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.
PARTURITION CHECKS:
- Three times each working day, at the beginning, middle, and end of the day from GD 21. Animal R0800 (Group 4 female) had GD 21 parturition checks omitted, in error but proceeded on GD 22.
THYROID HORMONE ANALYSIS: Yes (T3/4 and TSH)
- Time schedule: 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 - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:n/a - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter] - Statistics:
- Statistical analyses were performed, where appropriate - refer to the attached report.
- Indices:
- Number of indices were used, where appropriate, to evaluate reproductive function: mating index, fecundity index, fertility index, mean duration of gestation, mean number of implantation sites.
Other inndices used were: Mean number of pups born, Mean number of pups alive PND 1, % male pups PND 1 (litter), % male pups PND 1 (mean), % Post-implantation survival index (litter), % Post-implantation survival index (mean), % Live birth index (litter) and Live birth index. - Historical control data:
- Laboratory historical control data was available to the Study Director.
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- 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 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.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- 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):
- 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):
- Effects was 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):
- 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.
All remaining intergroup differences were transient in nature or only affected one or two animals and, as such, these observations were considered to have arisen incidentally.
No test article-related effects were evident for either sex administered 30 mg/kg/day. - 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,
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- 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.
All remaining intergroup differences were transient in nature or only affected one or two animals and, as such, these observations were considered to have arisen incidentally.
No test article-related effects were evident for either sex administered 30 mg/kg/day. - Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Details on results:
- 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 test article-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%).
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Other effects:
- not specified
Effect levels (maternal animals)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Pup body weights were essentially similar to controls on PND 1 and 4; however, lower mean male body weights were evident on PND 7 (-5%) and PND 13 (-9%) from litters maternally administered 100 mg/kg/day, compared with controls. Female body weights were also lower on PND 7 (‑5%) and PND 13 (-9%), compared with controls.
For litters in the group administered 50 mg/kg/day, lower male body weights were also evident on PND 7 (-5%) and PND 13 (-6%), compared with controls. Following maternal administration of 50 mg/kg/day, female body weights were lower from PND 1 onwards (-4 to -7%) compared with controls.
No effect on pup body weights was evident following maternal administration of 30 mg/kg/day. - Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- effects observed, non-treatment-related
- Description (incidence and severity):
- For litters in the group administered 50 mg/kg/day, lower male body weights were also evident on PND 7 (-5%) and PND 13 (-6%), compared with controls. Following maternal administration of 50 mg/kg/day, female body weights were lower from PND 1 onwards (-4 to -7%) compared with controls.
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- not specified
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- ca. 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Fetal abnormalities
- Key result
- Abnormalities:
- no effects observed
Overall developmental toxicity
- Key result
- Developmental effects observed:
- no
Any other information on results incl. tables
Table 2: 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 2. 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 |
16.9 |
20.8 |
19.2 |
20.3 |
19.9 |
21.0 |
24.6 |
25.4*H |
27.5*H |
*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 Mean Estrous Cycles
Group |
Pre‑dose |
Pre‑pairing |
||
|
No. of Estrous Cycles |
Mean Cycle Length (days) |
No. of Estrous Cycles |
Mean Cycle Length (days) |
control |
2.5 |
4.3 |
2.2 |
4.2 |
2 |
2.8 |
4.6 |
2.6 |
4.3 |
3 |
2.3 |
4.8 |
2.2 |
4.4 |
4 |
1.9 |
4.4 |
2.2 |
4.7 |
Estrous cycle = from 1st Oestrus to the next nonconsecutive stage of the same type
@ = Number examined reduced due to Female R0800 (Group 4) excluded
Table 9. Summary of Functional Observattional Battery in male
roup |
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 10. Summary of Functional Observational Battery in female
Group |
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 |
0.0 |
Table 11. 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
Code |
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 12. 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.
Applicant's summary and conclusion
- 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, reproductive performance and offspring development were unaffected as such, the no observed adverse effect level NOAEL for systemic and developmental 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.
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.
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