Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity

- oral: A key K1 study in male and female Wistar rats in a combined repeated dose toxicity test with reproductive/developmental screening was conducted according to OECD guideline 422 (Edwards, 2018). Based upon the study data, the NOAEL is considered to be 100 mg/kg bw/day since no adverse effects have been identified at or below this dose level.

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-15 - final report date
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: (EC) No 440/2008
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Jeffcat LE 30; 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Physical State/appearance: clear colorless liquid
- CAS number: 65286-55-7
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 30 December 2018
- Purity test date: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature and humidity in darkness
- Stability under test conditions: used/formulated in light
- Solubility and stability of the test substance in the solvent/vehicle: stable in distilled water for at least 15 days when stored refrigerated
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected as it is a readily available rodent species, historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 11 weeks
Females: approx. 12 weeks
- Weight at start of treatment:
Males: 270g - 335g
Females: 196g - 240g
- Fasting period before study: No
- Housing:
Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of succesful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum, a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water (e.g. ad libitum): ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE dates: 2017-09-12 to 2017-11-15
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in Distilled Water.
- The formulations were shown to be stable for at least 15 days when stored refrigerated. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 4, 12, 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg of Distilled water. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
- Lot/batch no. (if required): not specified
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined at the test facility and results showed that the formulations to be stable for at least 15 days when stored refrigerated.
Samples of test item formulations were taken on three occasions and analyzed for concentration and the results indicate that the prepared formulations were within 98-104% of the nominal concentration.
Duration of treatment / exposure:
Males: approx. 6 weeks
Females: up to 8 weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
Intermediate dose group
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat (Study No.:QS12HY)
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
- Treatment volume: 5mL/kg body weight.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).
- Parameters: signs of toxicity, ill-health and behavioral change

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION : Yes
- During the pre-pairing period: weekly food consumption was recorded for each cage of adults.
- For males after the mating phase: weekly food consumption was recorded for each cage of adults.
- For females showing evidence of mating: food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.
- For females with live litters: food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY: Yes
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.


WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (Day 44 for males and Day 13 post partum for females)
- Anaesthetic used for blood collection: not specified. Blood samples were obtained from the lateral tail vein.
- Animals fasted: no
- How many animals: five males and five females selected from each test and control group.
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination (Day 44 for males and Day 13 post partum for females)
- Animals fasted: no
- How many animals: five males and five females selected from each test and control group.
- Parameters checked: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++).

OTHER:
FUNCTIONAL OBSERVATION
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIORAL ASSESSMENT:
- Detailed individual clinical observations were performed for each animal using a purpose built arena.
- The parametes observed were : Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE
- Motor activity: purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period.
- Forelimb/Hindlimb Grip Strength: An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

SENSOR REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed : Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.
Sacrifice and pathology:
SACRIFICE
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS NECROPSY
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHT
- For five males and five females selected from each test and control group.
- The following organs were examined: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), Spleen, Testes, Thymus, Thyroid (weighed partially fixed with parathyroid), Uterus (weighed with cervix and oviducts).
- For all remaining animals, the following organs were weighed: Epididymides, Prostate, Seminal Vesicles (with coagulation gland), Ovaries, Pituitary (weighed after partial fixation), Thyroid (weighed after partial fixation with parathyroid), Uterus (weighed with Cervix).

HISTOPATHOLOGY
- From five males and five females selected from each test and control group had the following organs preserved in buffered 10% formalin.
- The tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination.
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator ani-bulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.
The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the KruskalWallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs observed that indicated any systemic effect of treatment at dosage of 20, 60 and 100 mg/kg bw/day.
At 100 mg/kg bw/day, five males and seven females, respectively, exhibited sporadic instances of noisy respiration between Days 8 and 52 of treatment. Four females at 60 mg/kg bw/day exhibited noisy respiration between Days 8 and 45. At this incidence this observation is likely to be associated with difficulty during the dosing procedure for these particular animals, rather than any underlying effect of systemic toxicity.
One 100 mg/kg bw/day male exhibited pilo-erection and hunched posture on Day 24 of treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 60 or 100 mg/kg bw/day had statistically significantly lower body weight gains (p<0.01 - p<0.05) throughout the treatment period in a dose related manner. This resulted in overall body weight gains which were 40% and 50% lower than controls for males treated with 60 or 100 mg/kg bw/day, respectively. Body weight gains for males treated at 20 mg/kg bw/day remained comparable to controls throughout the study.

Females at 100 mg/kg bw/day showed a mean body weight loss during the first week of treatment, and a reduced body weight gain during the second week. This resulted in overall body weight gains during the pre-pairing phase being 97% lower when compared to controls. At 60 mg/kg bw/day, females showed a statistically significant (p<0.05) group mean body weight loss during the second week of treatment, this resulted in overall body weight gains to be 15% lower compared to controls. There was no effect of treatment in females treated with 20 mg/kg bw/day, a statistically significantly higher (p<0.05) body weight gain was noted during the first week of treatment, however, an increase in body weight gain is not considered to be an adverse effect of treatment, and is therefore of no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Male food consumption at 60 or 100 mg/kg bw/day was generally lower in relation to controls from Week 2 of treatment onwards. No such effects were detected for males treated with 20 mg/kg bw/day.

For females, there were no adverse effects noted on food consumption during the pre-pairing phase.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The food conversion efficiency showed a similar trend to the male body weight changes and food intake, as males treated with 60 or 100 mg/kg bw/day values were generally lower than controls. No such effects were detected for males treated with 20 mg/kg bw/day.

For females, the food conversion efficiency showed a similar trend to the female body weight changes, as females treated with 60 or 100 mg/kg bw/day were generally lower than controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual assessment of water consumption did not reveal any significant intergroup differences.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day males had a statistically significant increase (p<0.01) in neutrophils in relation to controls, without a dose related response. All individual values from these males exceeded the background control ranges. Females treated with 60 and 100 mg/kg bw/day showed a statistically significant increase in neutrophil count (p<0.05 and p<0.01, respectively) compared to controls in a dose related response. These females also showed a statistically significant increase in lymphocyte counts (p<0.05) in a dose related response. The increased levels of neutrophil and lymphocyte counts for these females, therefore, contributed to the statistically significant increase in the total leukocyte count (p<0.05 and p<0.01, respectively) in a dose related manner. The majority of individual values were within the background control ranges, with isolated individuals exceeding these ranges. The increased values in these parameters may be a response to the extensive vacuolation/vacuolated macrophages of numerous tissues observed at histopathological examination.

Males treated with 100 mg/kg bw/day showed a statistically significant decrease (p<0.05) in hematocrit, however all individual values for these males were within the background control ranges. These males also showed a statistically significant increase (p<0.05) in platelet counts. 3/5 individual male values at 100 mg/kg bw/day exceeded the background control ranges. Due to the effects noted at histopathological examination an association with treatment cannot be discounted.

Treated males and females showed a dose related increase in reticulocyte count, attaining statistical significance for males treated with 60 and 100 mg/kg bw/day (p<0.01) and females at 100 mg/kg bw/day (p<0.05). 4/5 and 5/5 individual male values at 60 and 100 mg/kg bw/day respectively, and 2/5 individual female values at 100 mg/kg bw/day exceeded the background control ranges. Although there is no evidence of red blood cell destruction or anemia which would trigger increased reticulocyte production, and no corresponding histopathological changes in the bone marrow, a relationship to treatment cannot be excluded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Both 60 and 100 mg/kg bw/day males showed a decrease (p<0.05) in alkaline phosphatase (AP) in relation to the controls. All individual values were within the background control range. At 100 mg/kg bw/day, males also showed an increase (p<0.01) in aspartate aminotransferase (ASAT), however 3/5 individual values exceeded the background control range; including one value which was extremely high, and was likely to be the reason for this statistical significance. Due to the histopathological correlates to the liver an associated response to treatment cannot be ruled out.
Females treated with 60 and 100 mg/kg bw/day also showed a statistically significant reduction in alanine aminotransferase (ALAT) in relation to controls. Due to histopathological correlates to the liver an associated response to treatment cannot be ruled out.
At 100 mg/kg bw/day the males showed statistically significant reductions in glucose (p<0.05), albumin (p<0.01), total protein (p<0.01) and bilirubin (p<0.05) compared to controls. These males, as well as all female treatment groups, showed a statistically significant increase (p<0.05) in sodium concentration compared to controls. However, none of these parameters showed a dose related response, and all individual values were within background control ranges. These findings are likely to be the result of normal biological variation and of no toxicological significance.
An increase (p<0.05) in bile acids was noted for females treated with 100 mg/kg bw/day, without a dose relationship when compared to controls. All individual values were within the background control ranges, with 1/5 of the values at the upper end of this range may have influenced the significance of this parameter. This finding is likely to be the result of normal biological variation, however, due to histopathological changes in the liver a relationship to treatment cannot be discounted.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral assessment: There were no toxicologically significant changes in the behavioral parameters at 20, 60 and 100 mg/kg bw/day. There were isolated incidences of noisy respiration from one male each from 60 and 100 mg/kg bw/day dose groups on Day 14 and three 100 mg/kg bw/day males on Day 42 of treatment. Also noisy respiration was noted for three females treated with 100 mg/kg bw/day on four separate occasions. Due to the sporadic nature of these observations this finding was considered to be of no toxicological significance.

Functional performance :
There were no changes in functional performance considered to be related to treatment at 20, 60 and 100 mg/kg bw/day.
Males treated with 20 mg/kg bw/day showed a statistically significant increase (p<0.05) in hind limb grip strength. The intergroup difference was confined to one out of the three tests, in the absence of any similar effects at higher dosages, this finding was considered to be incidental and of no toxicological importance.
The final 20% of activity was reduced in males treated with 60 or 100 mg/kg bw/day which attained statistical significance (p<0.05), a dose relationship was not apparent. Overall activity was statistically significantly reduced (p<0.05 - p<0.01) in these animals when compared to controls in a dose related manner. In the absence of any clinical signs of neurotoxicity the intergroup difference was considered to be of no toxicological significance.

Sensory Reactivity Assessments:
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 20, 60 and 100 mg/kg bw/day.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 100 mg/kg bw/day showed statistically significant increases (p<0.01) in liver weights both absolute and relative to terminal body weight. The majority of individual values exceeded the background control ranges. Males treated with 60 mg/kg bw/day showed statistically significantly lower (p<0.05) absolute liver weights and a statistically significant increase (p<0.05) in relative liver weights to terminal body weight. A dose related increase was noted for the relative liver weights. All of the individual values for the absolute liver weights were within the background control ranges and 3/5 of the relative liver weights were above these ranges. Therefore an associated effect of treatment cannot be ruled.
No such effects were evident in females treated with 100 or 60 mg/kg bw/day or in animals of either sex treated with 20 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic necropsy findings did not show any effect of treatment for either sex at dosages of 20, 60 or 100 mg/kg bw/day.
Increased pelvic space (hydronephrosis) was observed in one male each treated with 60 and 100 mg/kg bw/day (right kidney and both kidneys respectively). Findings of this nature are consistent with normally expected low incidence findings in laboratory maintained rats within this laboratory. One female treated with 100 mg/kg bw/day exhibited pale discoloration of the uterus and cervix.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were extensive and unusual changes observed within this study, which were considered to be a direct effect of treatment with the test item.

Vacuolation/vacuolated macrophages were observed in numerous tissues from males treated with 20 mg/kg bw/day and above. The degeneration of hepatocytes in the liver was seen in males of all dose levels and females treated with 60 or 100 mg/kg bw/day. As vacuolation is a part of the degeneration process, this finding was considered to be adverse.

Reduced hematopoiesis in the spleen was present in all males and females treated with the test substance. The significance of this finding is unclear but unlikely to be significant. A further male treated with the test substance at 100 mg/kg bw/day had cellular depletion.

Atrophy of the thymus was present in two males and one female treated with 100 mg/kg bw/day.

Adrenal Glands: Vacuolation of the cells in the cortex was present in two males and females treated with 20, 60 and 100 mg/kg bw/day.
Aorta: Vacuolation in the wall of the aorta was present in two females given 20 mg/kg bw/day and all males and females given 60 or 100 mg/kg bw/day.
Brain: Vacuolation/vacuolated macrophages of the choroid plexus was present in 4/5 males and all females treated with 100 mg/kg bw/day. It was present in 4/5 males and one female treated with 60 mg/kg bw/day. It occurred in all brain regions where choroid plexus was present but was recorded under cerebrum for clarity.
Eyes: Vacuolation in the ciliary body was present in all females and 2/5 males treated with 100 mg/kg bw/day. It was also present in one female treated with 20 mg/kg bw/day.
Esophagus: Vacuolation in the muscle layer was present in all males and 3/5 females treated with 100 mg/kg bw/day, along with one female treated with either 20 or 60 mg/kg bw/day.
Heart: Vacuolation/vacuolated macrophages occurred in the cardiac muscle cells and/or the wall of the vessels of all males and females treated with 60 or 100 mg/kg bw/day. It was present in one male treated with 20 mg/kg bw/day.
Kidneys: Vacuolation/vacuolated macrophages in the glomeruli was present in 4/5 males and all females treated with 100 mg/kg bw/day. It was present in 3/5 males and females treated with 60 mg/kg bw/day.
Liver: Centrilobular, vacuolation and degeneration was present in all males and females treated with 60 or 100 mg/kg bw/day. It was present in 2/5 males treated with 20 mg/kg bw/day. Vacuoltation alone, multifocal and minimal was present in the other three males treated with 20 mg/kg bw/day.
Pancreas:Vacuolation was present in all males and females treated with 100 mg/kg bw/day along with all males and 2/5 females treated with 60 mg/kg bw/day.
Respiratory Tract: Vacuolation was present in the tracheal epithelium and/or the epithelium lining the bronchi of all males and females treated with either 60 or 100 mg/kg bw/day. Vacuolation in the muscle surrounding the bronchi/bronchioles was present in all males and females treated with 100 mg/kg bw/day, most males and females treated with 60 mg/kg bw/day and 3/5 males and females treated with 20 mg/kg bw/day.
Parathyroid Glands: Vacuolation was present in all males and females treated with either 60 or 100 mg/kg bw/day, in which the parathyroid glands were present.
Pituitary Gland: Vacuolation of the posterior lobe was present in seven males and eight females treated with 100 mg/kg bw/day. It was also present in one male treated with 20 mg/kg bw/day and two males and four females treated with 60 mg/kg bw/day.
Prostate: Vacuolation in the muscle tissue within the prostate was present in 8/12 animals treated with 100 mg/kg bw/day and 6/7 treated with 60 mg/kg bw/day.
Seminal Vesicles: Vacuolation in the muscle tissue within the seminal vesicles was present in 11/12 animals treated with 100 mg/kg bw/day and all males treated with 60 mg/kg bw/day which were examined.
Spleen: Vacuolated macrophages were present in all males and 4/5 females treated with 100 mg/kg bw/day and all males and females treated with 60 mg/kg bw/day.
Reduced hematopoiesis was present in all males and females treated with the test substance. A further male treated with 100 mg/kg bw/day had cellular depletion.
Stomach: Vacuolation in the glandular region of the stomach mucosa was present in all males and females treated with 100 mg/kg bw/day and most treated with 60 m/kg bw/day. Vacuolation in the muscularis of the stomach was present in 4/5 males and all females treated with 100 mg/kg bw/day, all males and females treated with 60 mg/kg bw/day along with most males and females treated with 20 mg/kg bw/day. Vacuolar degeneration in the muscularis was present in the remaining male treated with 100 mg/kg bw/day.
Intestines: Vacuolation in the muscularis was present in one or more areas of the intestine in all males and females treated with either 60 or 100 mg/kg bw/day.
Thymus: Atrophy was present in two males and one female treated with 100 mg/kg bw/day. Macrophage vacuolation was present in one further female treated with 100 mg/kg bw/day.
Thyroid Glands: Vacuolation was present in 10/12 males and 8/12 females treated with 100 mg/kg bw/day.
Urinary Bladder: Vacuolation in the muscle of the bladder wall was present in all males and females treated with 100 mg/kg bw/day along with all males treated with 60 and one male treated with 20 mg/kg bw/day.
Uterus: Vacuolated macrophages were present in the walls of the uterus in most animals treated with either 60 or 100 mg/kg bw/day.
Vacuolation of the cells of the uterine wall was present in most animals treated with either 60 or 100 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
An evaluation of Thyroxine (T4) in adults show a dose related reduction attaining statistical significance (p<0.001) for adult males treated with 60 and 100 mg/kg bw/day. This could be an associated effect due to the histopathological changes to the thyroid gland.
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

Analytical verification

The results indicate that the prepared formulations were within 98 -104% of the nominal concentration.

Summary of the results from the 14day repeated dose oral (gavage) range-finding toxicity study

In this study, effects on body weight development and reduced dietary intake was observed for animals treated with 250 and 125 mg/kg bw/day. Due to the severity of the body weight loss for animals of either sex treated with 250 mg/kg bw/day, these animals were terminated early on Day 8 of the study. At 75 mg/kg bw/day, effects on body weight development were considered not to represent an adverse effect of treatment.

Conclusions:
The oral administration of the test substance to rats by gavage at dose levels of 20, 60 and 100 mg/kg bw/day resulted in treatment related, non-adverse effects. The primary observation in the study was vacuolisation/vacuolated macrophages in numerous tissues from males treated with 20 mg/kg bw/day and above and degeneration of hepatocytes in the liver for males of all dose levels and females treated with 60 or 100 mg/kg bw/day. With regards to vacuolisation/vacuolated macrophages in numerous tissues, the vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines (which can be primary, secondary or tertiary amines) and supporting evidence from similar Huntsman compounds within the same chemical family of aliphatic amines. With regards to degeneration of hepatocytes in the liver for males and females, there are no other parameters within the study that supports this observation as being adverse or progressive (i.e., clinical pathology or hematology/clinical blood chemistry). Thus, with no obvious adverse effects at 100 mg/kg bw/day, a NOAEL of 100 mg/kg bw/day is considered acceptable (Lewis et al. 2012).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity - oral:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™: RccHan™: WIST strain rats, for approximately eleven weeks (males) and twelve weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 20, 60 and 100 mg/kg bw/day (OECD 422; Edwards, 2018). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) over the same period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Functional/sensory responses to various stimuli were evaluated on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post-partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post-partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

There was no mortality observed during the study. There were no clinical signs apparent that were considered to be related to systemic toxicity of the test item. Isolated occurrences of noisy respiration were noted in five males and seven females treated at 100 mg/kg bw/day as well as in four females at 60 mg/kg bw/day. No clinical signs were apparent in any animal of either sex treated with 20 mg/kg bw/day. 

There were no treatment-related changes in the behavioral parameters in animals of either sex treated with 20, 60 and 100 mg/kg bw/day. There were considered to be no treatment related changes in functional performance considered to be related to treatment at 20, 60 and 100 mg/kg bw/day. Males treated with 20 mg/kg bw/day showed a statistically significant increase (p<0.05) in hind limb grip strength but in the absence of any similar effects at higher dosages, this finding was considered to be incidental and of no toxicological importance. The final 20% of activity was reduced in males treated with 60 or 100 mg/kg bw/day which attained statistical significance (p<0.05) but a dose relationship was not apparent. Although the overall activity was statistically significantly reduced (p<0.05 - p<0.01) in these animals when compared to controls in a dose related manner, the absence of any clinical signs of neurotoxicity the intergroup difference lead to conclude no toxicological significance. In terms of body weight, the males treated with 60 or 100 mg/kg bw/day had statistically significantly lower body weight gains (p<0.01 - p<0.05) throughout the treatment period in a dose related manner. No differences, compared to control, were observed in males treated at 20 mg/kg bw/day throughout the study. Females at 100 mg/kg bw/day showed a mean body weight loss during the first week of treatment, and a reduced body weight gain during the second week. At 60 mg/kg bw/day, females showed a statistically significant (p<0.05) group mean body weight loss during the second week of treatment. There was no effect of treatment in females treated with 20 mg/kg bw/day.  

Male food consumption at 60 or 100 mg/kg bw/day was generally lower in relation to controls from Week 2 of treatment onwards. The food conversion efficiency showed a similar trend to the male body weight changes and food intake, as males treated with 60 or 100 mg/kg bw/day values were generally lower than controls. No such effects were detected for males treated with 20 mg/kg bw/day.

There were no adverse effects noted on food consumption during the pre-pairing phase, however, food conversion efficiency showed a similar trend to the female body weight changes, as females treated with 60 or 100 mg/kg bw/day were generally lower than controls.

Hematology:

Females treated with 60 and 100 mg/kg bw/day showed a statistically significant increase in neutrophil count (p<0.05 and p<0.01, respectively) compared to controls in a dose related response.These females also showed a statistically significant increase in lymphocyte counts (p<0.05) in a dose related response. Both factors contributed to the statistically significant increase in the total leukocyte count (p<0.05 and p<0.01, respectively) in a dose related manner.

Males treated at 100 mg/kg bw/day had a statistically significant increase (p<0.01) in neutrophils in relation to controls, but without a dose related response. These males also showed a statistically significant decrease (p<0.05) in hematocrit, however all individual values were within the background control ranges. These males also showed a statistically significant increase (p<0.05) in platelet counts with 3/5 individual exceeding the background control ranges.  Treated males and females showed a dose related increase in reticulocyte count, attaining statistical significance for males treated with 60 and 100 mg/kg bw/day (p<0.01) and females at 100 mg/kg bw/day (p<0.05). 

Blood chemistry:

Both 60 and 100 mg/kg bw/day males showed a decrease (p<0.05) in alkaline phosphatase (AP) in relation to the controls. All individual values were within the background control range. At 100 mg/kg bw/day, males also showed an increase (p<0.01) in aspartate aminotransferase (ASAT), however 3/5 individual values exceeded the background control range; including one value which was extremely high and was likely to be the reason for this statistical significance. 

Females treated with 60 and 100 mg/kg bw/day also showed a statistically significant reduction in alanine aminotransferase (ALAT) in relation to controls.

The evaluation of Thyroxine (T4) in adults showed a dose related reduction attaining statistical significance (p<0.001) for adult males treated with 60 and 100 mg/kg bw/day. Organ weight observations showed that males treated with 100 mg/kg bw/day showed statistically significant increases (p<0.01) in liver weights both absolute and relative to terminal body weight. The majority of individual values exceeded the background control ranges. Males treated with 60 mg/kg bw/day showed statistically significantly lower (p<0.05) absolute liver weights and a statistically significant increase (p<0.05) in relative liver weights to terminal body weight. A dose related increase was noted for the relative liver weights. 

Histopathology results revealed extensive and unusual changes which were considered to be a direct effect of treatment with the test item.

Vacuolation/vacuolated macrophages were observed in numerous tissues from males treated with 20 mg/kg bw/day and above. The degeneration of hepatocytes in the liver was seen in males of all dose levels and females treated with 60 or 100 mg/kg bw/day.The vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines (which can be primary, secondary or tertiary amines) and supporting evidence from similar compounds within the same chemical family of aliphatic amines (Smith et al., 2019, internal document). With regards to degeneration of hepatocytes in the liver for males and females, there are no other parameters within the study that supports this observation as being adverse or progressive (i.e., clinical pathology or hematology/clinical blood chemistry). Cytoplasmic vacuolization is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death. Hence, the vacuolization observed in the numerous tissues from the study was considered to be excessive and of potential toxicological concerns due to the number of tissues where the pathological findings was observed.

It can be demonstrated that aliphatic amines, such as the test item induce clear cytoplasmic vacuoles. That the induced vacuolization is most likely the result of osmotic effects associated with disturbed ionic balance in the organelles rather than an impact on proteins controlling cellular functions. These osmotic effects are considered to be transient in nature and thus non-adverse.

 

Reduced hematopoiesis in the spleen was present in all males and females treated with the test substance. The significance of this finding is unclear but unlikely to be significant. A further male treated with the test substance at 100 mg/kg bw/day had cellular depletion.

Atrophy of the thymus was present in two males and one female treated with 100 mg/kg bw/day.

Repeated dose toxicity - inhalation:

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

 

Repeated dose toxicity - dermal:

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

Annex IX testing:

A test proposal for a repeated dose 90 -day oral toxicity study in rats is included for this substance.

Justification for classification or non-classification

Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, this observation of vacuolization in the test item is considered also to be transient and non-adverse, and as such the effects are of minimal toxicological importance. Therefore, such effects do not, by themselves, indicate 'significant' toxicity in relation to Specific Target Organ Toxicity Repeated Exposure (STOT RE) classification in GHS. The observed effects of vacuolization are considered not to support STOT RE classification.