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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.


A 90-day oral repeated dose study in rats (key, K1; Chase, 2022) performed according to OECD 408 guideline and to GLP requirements. A NOAEL value for systemic toxicity of 25 mg/kg/day was derived.


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

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017-02-15 to 2017-03-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was designed to provide information for further repeated dose toxicity studies:
The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for up to fourteen consecutive days, at dose levels of 75, 125 and 250 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Distilled water). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- chemical name: 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-N-methylethanamine
- Lot/batch number of test material: DR74271215
- Expiry Date : 30 December 2018
- Appearance : Clear colorless liquid
- Purity: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study 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 or test system and environmental conditions:
TEST ANIMALS
- Source: 12 male and 12 female Wistar Han™:RccHan™:WIST strain rats obtained from Envigo RMS (UK) Limited, Oxon, UK.
- Females (if applicable) nulliparous and non-pregnant: not indicated
- Age at study initiation: approximately thirteen weeks old.
- Weight at study initiation: males: 327 to 388g; females: 187 to 221g.
- Fasting period before study: not indicated
- Housing: The animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet: ad libitum, pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Water: ad libitum, mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: thirteen days

DETAILS OF FOOD AND WATER QUALITY: The diet, drinking water, bedding and environmental enrichment was 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 fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: 2017-03-07 To: 2017-03-21
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study will aid dose level selection for subsequent studies.
Vehicle:
water
Remarks:
Distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The test item was administered by gavage using a stainless steel cannula attached to a disposable plastic syringe.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted on days 4, 8 and 11.

VEHICLE
- Concentration in vehicle: 0, 15, 25, 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Duration of treatment / exposure:
for up to fourteen consecutive days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
12 males and 12 females,
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on preliminary toxicity work; In the absence of any toxicity data, preliminary toxicity work was undertaken, treating one male and one female rat each at 150, 500 or 1000 mg/kg bw/day for up to three consecutive days. At 1000 mg/kg bw/day one male and one female were each dosed for up to three consecutive days. By the second day of treatment the female showed hunched posture, which continued to Day 3. On Day 2, the male exhibited signs of ataxia, noisy respiration, dehydration, hunched posture, decreased respiratory rate and lethargy, due to the severity of these clinical signs this animal was killed in extremis. The female was killed as scheduled on Day 4. Both animals showed slight body weight losses at termination. At necropsy, the male had red patches on the stomach and purple coloured contents in the intestines. The female had gaseous distension in the stomach and intestines, red patches on the stomach and sloughing on the non-glandular region of the stomach. At 500 mg/kg bw/day both male and female completed 3 days of treatment, however, both animals showed actual body weight losses of 6g and 20g, respectively by Day 4. The female also showed increased salivation (Day 1) and noisy respiration (Days 1 and 2). At necropsy, both animals had compacted food in the stomach, red patches on the stomach and gaseous distension in the intestines. The female also had fluid filled intestines. days. No clinical signs of toxicity were evident in either animal, however, the female showed a slight body weight loss (2g) on Day 4. At necropsy, the female had gaseous distension in the caecum.

- Rationale for animal assignment: The animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.


Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS AND CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made four hours following dosing (not at weekends).

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15.

FOOD EFFICIENCY: Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On completion of the dosing period, all surviving animals were killed by carbon dioxide asphyxiation, followed by exsanguination and subjected to an internal and external macroscopic examination. Animals that died during the study were also necropsied. No tissues were retained.

HISTOPATHOLOGY: No

Statistics:
No statistics available.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of systemic toxicity in animals of either sex at 75, 125 or 250 mg/kg bw/day. An incidental observation of post-dose noisy respiration was noted in one male treated with 250 mg/kg bw/day on Day 4 only. This clinical sign was considered to reflect a difficulty in dosing this particular animal rather than any underlying toxicological effect of the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 250 mg/kg bw/day, animals of either sex were terminated on Day 8 (relative to the start of dosing) due to marked body weight losses and marked reduction in food intake between Days 4 and 8 of study. There were no further unscheduled deaths in this study at 75 or 125 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a clear adverse effect on body weight development by Day 8 of treatment for animals of either sex treated with 250 mg/kg bw/day as these animals showed marked body weight loss by Day 8, hence, these animals were prematurely terminated. In order to assess the body weight development further, from Day 9 all surviving animals were weighed daily. Animals of either sex from all dose groups showed instances of actual body weight loss, with some animals showing improvement. However, the overall body weight gains showed a dose related reduction in relation to controls. At 125 mg/kg bw/day, animals of either sex showed fluctuations throughout the treatment period, with lower overall body weight gains (44% and 94%, males and females, respectively).
At 75 mg/kg bw/day, animals of either sex also showed fluctuations in body weight development during the treatment period, and overall showed 20% and 59% lower body weight gains compared to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a reduction in food intake for animals of either sex treated with 250 or 125 mg/kg bw/day in a dose related manner, this was particularly apparent in males treated with 250 mg/kg bw/day as this group showed a marked reduction in food consumed, reductions of 33% to 70% were noted in these animals from Days 1-4 and 4-8 respectively when compared to control. Food consumption in animals treated with 75 mg/kg bw/day were considered unaffected by treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 250 mg/kg bw/day showed marked reductions in food conversion efficiencies from Days 1 to 8 as negative values were apparent. Males treated with 125 or 75 mg/kg bw/day showed fluctuations in food conversion efficiency throughout the treatment period. Females treated with 250 or 125 mg/kg bw/day also showed a reduction in food efficiency in relation to controls. Females treated with 75 mg/kg bw/day showed reduced food conversion efficiency during the first week of treatment, improvement was evident during the second week but remained lower overall (54%). These intergroup differences were considered to be associated with the body weight changes and/or diet intake.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Variation in water consumption was identified in prematurely terminated 250 mg/kg bw/day animals, males showed a decrease relative to controls from Day 3 whilst the females generally showed a slight increase. Animals of either sex treated with 125 mg/kg bw/day (in particular the females), showed an overall increase (58%) when compared to the control, females treated with 75 mg/kg bw/day also showed sporadic increases which resulted in an overall increase (16%).
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All animals treated with 250 mg/kg bw/day were humanely killed on Day 8. The macroscopic examination revealed gaseous distension of the large intestines in 2/3 males and 2/3 females. No further macroscopic abnormalities were detected in any other animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The oral administration of the test substance to rats for a period of up to fourteen consecutive days at dose levels of 75, 125 and 250 mg/kg bw/day resulted in the early termination of animals treated with 250 mg/kg bw/day on Day 8 of the treatment period and treatment related effects being noted across all other treatment groups which were generally in a dose related manner.
Animals of either sex treated with 250 mg/kg bw/day exhibited adverse effects on body weight gain throughout the eight day treatment period. This was particularly apparent in the male animals as body weight losses were seen throughout this treatment period. Food consumptions were also reduced in these animals when compared to control. Both body weight and food consumption for these animals became progressively worse during the treatment period such that it was considered necessary to terminate these animals early (on Day 8 of treatment) and as such this dose level is too high for investigation in the upcoming Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 - Envigo Study Number: HB11QL).
The effects noted on body weight development and food consumption also extended to animals of either sex treated with 125 mg/kg bw/day. However, the effects seen were not quite as marked as those effects noted at 250 mg/kg bw/day but still resulted in significant reductions in overall body weight gain at the end of the treatment period (45% for males and 94% for females). Reductions in body weight gain were particularly apparent in the female animals as continual mean body weight losses were noted throughout the first ten days of the treatment period. The effects noted (especially in body weight development in female animals) in animals treated with 125 mg/kg bw/day also precludes the use of this dose level from further investigation.
Whilst there were effects noted on body weight development noted in animals of either sex treated with 75 mg/kg bw/day (particularly in female animals) there are no such effects on food consumption when compared to control. It was, therefore, considered that these effects on body weight development were not of a sufficient severity to preclude the use of this dose level for further investigation in the upcoming OECD 422 study.
Conclusions:
Based on these findings, 20, 60 and 100 mg/kg bw/day are recommended as the low, intermediate and high dose level, respectively for the Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 Envigo Study Number: HB11QL).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-06-1 to 2021-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
June 25, 2018
Deviations:
no
Principles of method if other than guideline:
The purpose of this study was to assess the systemic toxic potential of the test substance when administered orally, by gavage, to Han Wistar rats for 13 weeks. Recovery from any effects were evaluated during a 4-week recovery period.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): 2-(2-(dimethylamino)ethoxy)-N-(2-(2-(dimethylamino)ethoxy)ethyl)-Nmethylethanamine, N,N,2,8-Tetramethyl-5,11-dioxa-2,8-diazatridecan-13-amine
- Lot/batch number: PFW200553
- Physical appearance: Clear colourless liquid
- Retest date: 2022-11-06
- Purity: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature (15 to 25°C) in the dark and protected from air (eg nitrogen blanket)
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: Formulations prepared at 2 and 20 mg/mL were found to be homogeneous and stable when refrigerated (2 to 8°C) for up to 15 days (Covance study number HB11QL). Formulations prepared at 3 and 7.5 mg/mL were found to be homogeneous and stable at ambient temperature (15 to 25°C) for up to 23 hours (Labcorp study number 8460938).
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified
- Storage of formulation: refrigerated (2 to 8 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing : not specified

FORM AS APPLIED IN THE TEST: liquid
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 65 males and 65 females, Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 41 to 47 days
- Weight at study initiation: males 130-175 g; females: 106-144g
- Fasting period before study: not specified
- Housing: Up to four of the same sex, (unless reduced by mortality) were housed in polycarbonate cages with a stainless-steel mesh lid, with wood based bedding which was changed at appropriate intervals each week. Males and females were blocked by sex and the cages constituing each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations. Aspen gnawing material and plastic shelter was provided to each cage throughout the study and replaced when necessary.
- Diet: ad libitum, Teklad 2014C, pelleted diet
- Water: ad libitum, Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 12 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24°C
- Humidity (%): Monitored and maintained within the range of 40-70%
- Air changes (per hr): A minimum of 15 air changes per hour; filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12 (Artificial lighting)

IN-LIFE DATES: From: 2021-06-02 To:2021-09-14 (main study)
From: 2021-06-02 To:2021-10-11 (recovery study)
Route of administration:
oral: gavage
Details on route of administration:
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. Formulations were administrated by oral gavage, using a suitably graduated syringe and a flexible cannula inserted via the mouth. A 5% sugar solution (glucose or sucrose) was used to lubricate the cannula immediately before administration.
- Rationale for route of administration: oral, to simulate the conditions of potential human exposure.
Vehicle:
water
Remarks:
purified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Starting with the lowest concentration, the required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogenous. The formulation was transferred to the final containers, via syringe whilst magnetically stirring.
- Dosing solutions were prepared weekly.
- A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE
- Concentration in vehicle: 0, 3.125, 3.125, 5 mg/ml for groups G1, G2, G3 and G4 respectively.
- Amount of vehicle (if gavage): 10, 4, 8, 10 mL/kg/day for groups G1, G2, G3 and G4 respectively.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analyzed for achieved concentration of the test item. On sampling days, two samples (2 x 10 mL) of each test formulation and vehicle control were taken from the middle of the bulk container, transferred to glass vials and stored refrigerated (2 to 8ºC). Samples were sent as soon as possible on cool packs (approximately 2 to 8ºC) by courier to the Principal Investigator for analysis.
Duration of treatment / exposure:
13 weeks followed by a 4-week recovery period
All surviving main study animals were treated throughout the necropsy period; cessation of treatment for the recovery study animals started on the first day of the necropsy of the Main study animals
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
G1, vehicle control
Dose / conc.:
12.5 mg/kg bw (total dose)
Remarks:
G2, low dose
Dose / conc.:
25 mg/kg bw (total dose)
Remarks:
G3, mid dose
Dose / conc.:
50 mg/kg bw (total dose)
Remarks:
G4, High dose
No. of animals per sex per dose:
10 males and 10 females (main study)
5 males and 5 females (recovery period)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 12.5 (G2), 25 (G3) and 50 (G4) mg/kg/day were selected in conjunction with sponsor.
The conclusion drawn from the OECD 422 study (Covance study number HB11QL) with rats dosed at 0, 20, 60 or 100 mg/kg bw/day, was that it was not possible to determine a NOAEL in the study. At dose levels of 60 or 100 mg/kg bw/day, the histopathological examination of the tissues revealed vacuolation and hypertrophy of the choroid plexus of the brain and macrophage vacuolation within the spleen, thymus and uterus. The significance of the
vacuolation for many of these tissues was unclear, however centrilobular vacuolar degeneration of the liver was apparent for both sexes at these dosages and, such degeneration was regarded as a clear adverse finding. At 20 mg/kg bw/day, vacuolation was still apparent for some tissues, but to a lesser extent, and there were no incidences of vacuolation and hypertrophy of the choroid plexus of the brain or incidences of macrophage vacuolation observed. However, centrilobular vacuolar degeneration of the liver persisted for males and the presence of this degeneration precluded the classification of this dosage as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity. There was also a considerable effect noted at 60 or 100 mg/kg bw/day on body weight, particularly for males, and food consumption was also reduced which may prove to be unsustainable in a longer-term study.
No clinical signs were seen but noisy respiration was noted occasionally. Given that on the OECD 422 the animals were dosed for approximately 6/7 weeks it was expected that the same histopathological changes (extensive vacuolation of tissues), possibly at a greater extent, would be seen in animals dosed for 90-days at 50 mg/kg bw/day and it was very likely that these changes would also be seen, albeit to a lesser extent, at 25 mg/kg bw/day. However, at the dose levels chosen for this 13 week toxicity study it was very likely that the animals would not exhibit any significant effects during the in-life phase and were expected to complete the 90 days of treatment. The OECD 408 test guideline indicated that consideration should be given to including an additional satellite group of at least ten animals (five per sex) in the control and in the top dose group for observation after the treatment period, for the potential reversibility or persistence of any toxic effects. For this study, permission was sought and was granted by the Home Office to also allocate recovery animals to the low and intermediate dose groups, thereby ensuring information relating to the reversibility of the pathological findings was available for all dose groups that survived to termination and would not be lost if it proved necessary to prematurely terminate the high dose group due to adverse toxicity. It also
allowed more robust assessment of the reversibility of some of the histopathological findings to be established, as it was possible to correlate recovery against the severity of findings observed at the end of treatment for some of the organs. Considering all the above, the dose levels selected for this study were 0, 12.5, 25 and 50 mg/kg bw/day.
- Route of administration and justification: Route of test item administration was through oral gavage to simulate the conditions of potential human exposure.
Observations and examinations performed and frequency:
MORTALITY: Yes
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.
- Signs associated with dosing: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose observation.
- 1 to 2 hours after completion of dosing all groups.
- As late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

FUNCTIONAL OBSERVATIONS:
- Sensory reactivity and grip strength:
Sensory reactivity and grip strength assessments were performed (before dosing) on all Main and Recovery study animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:
* Approach response
A blunt probe was brought towards the head until it was close to the nose (but not touching the whiskers). The reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction (e.g. approaches and/or sniffs probe)
3 Abnormally fearful or aggressive reaction
* Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response (e.g. ear twitches/flattens or head shake)
3 Active avoidance, abnormally fearful or aggressive response
* Auditory startle reflex
The response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response (e.g. ear twitch only)
3 Normal response (e.g. obvious flinch or startle)
4 Exaggerated response (e.g. all feet off floor)
* Tail pinch response
The tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response (e.g. turns around slowly or weak vocalization without moving away)
3 Normal response (e.g. jumps forward or turns around sharply, usually with vocalization)
4 Exaggerated response (e.g. excessive vocalization, body movement or aggression)
* Grip strength
Forelimb and hindlimb grip strength were measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.
As there were no treatment-related changes seen in Week 12, the observations and measurements were not performed at the end of the recovery period.

- Motor Activity
During Week 12 of treatment (before dosing), the motor activity of all Main and Recovery study animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Labcorp. In addition, all Recovery study animals were tested during Week 4 of recovery.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the treatment and recovery periods, and before necropsy.
- Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the treatment and recovery periods.

WATER CONSUMPTION : No
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
Pretreatment - All Main and Recovery study animals (including spares)
Week 12 - All Main study animals of Groups 1 and 4
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined. As there were no treatment-related changes seen in Week 12, the examination was not extended to include Main study animals of Groups 2 and 3 or Recovery study animals during Week 12, or to the Recovery study animals at the end of the recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on week 13 (main study), week R4 (recovery study), and at necropsy (after 4 weeks recovery)*, prior to dosing, blood samples, of nominally 0.5 ml, were collected from the sublingual vein and collected into tubes containing EDTA anticoagulant .

*Due to the number of clotted EDTA samples at the Week R4 sampling occasion, additional EDTA samples were taken from the Recovery study animals at necropsy (without overnight deprivation of food).

- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, fasted overnight
- How many animals: 80 (main study) 40 (recovery study)
- The following Parameters were examined: Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute, reticulocyte count (Retic), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)), Platelet count (Plt).
* Derived values calculated in ClinAxys.
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters were performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
- Prothrombin time (PT) - using IL PT Fibrinogen reagent.
- Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on week 13 (main study) and week R4 (recovery study), prior to dosing, blood samples, of nominally 0.7 ml, were collected from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant .
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, fasted overnight
- How many animals: 80 (main study), 40 (recovery study)
- The following parameters were examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile acids (Bi Ac), Urea (numerically equivalent to blood urea nitrogen), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Cholesterol - high density lipoprotein (HDL), Cholesterol - low density lipoprotein (LDL), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Total protein (Total Prot), Albumin (Alb).
- Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: on week 14 (main study) and weak R5 (recovery study) blood samples of 2.0 ml were taken from the Sublingual vein and collected into Grenier Minicollect tubes with clotting activator (no anticoagulant)
- Anaesthetic used for blood collection: Yes (isoflurane), the animals did not recover from the anesthesia
- Animals fasted: No overnight depravation of food
- How many animals: 80 (main study) 40 (recovery study)
- Treatment of samples: samples were kept at ambient temperature (15 to 25 °C) for a minimum of 30 minutes prior to centrifugation. Centrifugation was performed at 2000g for 10 minutes at 4 °C. Samples are deep frozen (-60 to -80°C) pending analysis
- Hormones estimated: rodent thyroid stimulating hormone (TSH), rodent thyroxine (T4) and rodent triiodothyronine (T3)
As the TSH concentrations were unaffected after 13 weeks of treatment, the serum samples taken from the recovery study animals after 4 weeks were not analyzed and subsequenty discarded.

NEUROBEHAVIOURAL EXAMINATION: Yes, functional battery tests, as described above.

IMMUNOLOGY: No

OTHER:
Oestrous Cycle evaluation:
- Wet smears were taken from the vagina of all females using pipette lavage for four consecutive days before each scheduled termination. The last smear was taken on the morning of necropsy. Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination and were used to assist in the histological evaluation of estrogen sensitive tissues.
- Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Method of kill: Carbon dioxide asphyxiation with subsequent exsanguination.
- Schedule: Main study animals were killed following 13 weeks of treatment. Recovery study animals were killed following 13 weeks of treatment and 4 weeks of recovery.
- All Main and Recovery study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
- Tissue preservation and examination: the required tissue samples preserved in appropriate fixative that is 10% Neutral Buffered Formalin except for testes (preserved in modified Davidson’s fluid), eyes (preserved in Davidson's fluid) and Bone marrow smears (preserved in methanol). The retained tissues were checked before disposal of the carcass.

ORGAN WEIGHTS: Yes
- The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows in table in "additional information" field. For bilateral organs, left and right organs were weighed together.
- Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

HISTOPATHOLOGY: Yes
- Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal 4 to 5 μm thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- The full list of organs (see table in "additional information" field) was examined for the animals killed prematurely and all Main study animals of Groups 1 and 4 killed after 13 weeks of treatment.
- the following list of organs was examined for animals of Groups 2 and 3 killed after 13 weeks of treatment and all recovery study animals of group 1,2, 3, and 4 killed after 4 weeks of recovery:
Adrenals, aorta, brain, eyes, esophagus, heart, kidneys, liver, pancreas, trachea, lungs/bronchi, thyroids and parathyroids, pituitary, spleen, stomach, cecum, colon, duodenum, ileum, jejunum, rectum, thymus, urinary bladder, uterus, prostate, seminal vesicles and abnormalities.
- Staining: Sections were stained with hematoxylin and eosin. Additional 4-5 μm sections were prepared from the liver of two male and two female Main study animals in each of Groups 1 and 4 selected by the study pathologist. These sections were stained with LAMP-2 antibody using an optimized method and subsequently evaluated by the pathologist. The LAMP-2 staining method was previously validated on additional liver sections taken from the controls. LAMP-2 is a heavily glycosylated membrane protein expressed by lysosomes. It is thought to have a function in protecting the lysosomal membrane from proteolytic enzymes. LAMP-2 is expressed throughout the cell.
Tissues preserved for examination were examined as follows:
-Premature death: All animals from all groups - all organs listed in table in "additional information" field
-Scheduled kill: All Main study animals of Groups 1 and 4, - all organs listed in table in "additional information" field
All Main study animals of Groups 2 and 3 and All Recovery study animals of Groups 1, 2, 3 and 4 -following list :
Adrenals, aorta, brain, eyes, esophagus, heart, kidneys, liver, pancreas, trachea, lungs/bronchi, thyroids and parathyroids, pituitary, spleen, stomach, cecum, colon, duodenum, ileum, jejunum, rectum, thymus, urinary bladder, uterus, prostate, seminal vesicles and abnormalities.
- Detailed qualitative examination was made of the right testis, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
- Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Other examinations:
BONE MARROW
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study. The smears were air dried and subsequently fixed in methanol. No examinations were performed, however, the smears were retained for possible future examination.

SPERM ANALYSIS
- A sperm sample was taken from the left vas deferens at necropsy of all Main study males (killed after 13 weeks of treatment) and all Recovery study males (killed after 4 weeks of recovery) and assessed for motility using a computer assisted sperm analyzer (CASA).
- The residual sperm samples taken from the left vas deferens were retained in neutral buffered formalin. The left testis and left cauda epididymis (see Section 4) were also weighed and retained deep frozen (-10 to -30°C).
- As no treatment-related changes were identified from organ weight or histopathology investigations, an assessment of sperm count and sperm morphology was not performed and all retained samples were discarded when the report was finalized. The results of the sperm motility assessment were retained in the raw data but not reported.

Statistics:
See section "Any other information on materials and methods"
Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behavior of the animals were unaffected by treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No. 132 (Group 1, Control) was killed for welfare reasons in Week 5, as the animal
accidentally bit and swallowed the end of the cannula. There were no other significant
macroscopic or microscopic findings. This death was considered accidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall bodyweight gain (Week 0 to 13) was slightly low, when compared to the
controls, for males receiving 50 mg/kg/day (91% of control) and females receiving 25 or 50 mg/kg/day (92 and 89% of control, respectively). These inter-group differences were still apparent at the end of the recovery period (Week R0 to R4), although there was some improvement compared to controls for males which previously received 50 mg/kg/day.
The overall bodyweight gain (Week 0 to 13) of males receiving 12.5 or 25 mg/kg/day and females receiving 12.5 mg/kg/day was unaffected by treatment.
See data tables for detailed information.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The overall food consumption (Week 1 to 13) was slightly low, when compared to the controls, for males and females receiving 50 mg/kg/day (93 and 91% of control, respectively). During the recovery period, the overall food consumption (Week R1 to R4) for males and females which previously received 50 mg/kg/day was generally similar to that of the controls.
The overall food consumption (Week 1 to 13) of males and females receiving 12.5 or
25 mg/kg/day was unaffected by treatment.
See data tables for detailed information.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmoscopic findings in Week 12 for males and females
receiving 50 mg/kg/day.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination during Week 13 revealed, when compared to the controls, slightly low hematocrit, hemoglobin concentration and erythrocyte counts in males and females receiving 50 mg/kg/day and females receiving 25 mg/kg/day. Mean cell volume was slightly high for males receiving 25 or 50 mg/kg/day and reticulocyte counts were slightly low for females receiving 50 mg/kg/day. Slightly low hematocrit and erythrocyte counts were seen in females receiving 12.5 mg/kg/day but there was no effect on hemoglobin concentrations. After 4 weeks of recovery, hematocrit, hemoglobin concentration and erythrocyte counts were still slightly low for females which previously received 50 mg/kg/day and reticulocyte counts were slightly high for males which previously received 50 mg/kg/day. All other differences were no longer apparent at the end of the recovery period.
Lymphocyte counts were slightly high, when compared to the controls, in males and females receiving 50 mg/kg/day. As a consequence, there was also an increase of the total leucocyte counts of males and females receiving 50 mg/kg/day. In addition, large unstained cell counts were slightly high in females receiving 25 or 50 mg/kg/day. After 4 weeks of recovery, neutrophil, basophil and large unstained cell counts were slightly high for males which previously received 50 mg/kg/day.
Prothrombin time was slightly reduced in males and females receiving 50 mg/kg/day, and in males receiving 12.5 or 25 mg/kg/day. This inter-group difference was not apparent at the end of the recovery period. All other inter-group differences from controls were minor, confined to one sex or were without dose-relationship and were therefore considered to represent normal biological variation.
Due to the small number of clotted EDTA samples at the recovery Week 4 sampling occasion, additional EDTA samples were taken at necropsy and these generally confirm the original results. They are presented for information only.
See data tables for detailed information.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma after 13 weeks of treatment revealed, when compared to the controls, high urea concentrations in males and females receiving 50 mg/kg/day and this associated with a small increase of creatinine concentration in males. Urea concentrations were still high after 4 weeks of recovery for males and females which previously received 50 mg/kg/day.
Potassium concentrations were high in males and females receiving 50 mg/kg/day. This intergroup difference was not apparent at the end of the recovery period.
Total plasma protein and albumin concentrations were low in males and females receiving 50 mg/kg/day although there was no effect on the albumin:globulin ratio. Albumin concentration was still low after 4 weeks of recovery for females which previously received 50 mg/kg/day.
All other inter-group differences from controls were minor, confined to one sex or were without dose-relationship and were therefore considered to represent normal biological variation. Such differences included the slightly reduced high density lipoprotein concentrations in Week 13 of treatment in all groups of treated females as there was no dose response and no similar change in males. These also included the parameters which attained statistical significance after 4 weeks of recovery (chloride for males and alkaline phosphatase, aspartate amino-transferase and bilirubin for females) as there were no similar changes after 13 weeks of treatment.
See data tables for detailed information.
Endocrine findings:
no effects observed
Description (incidence and severity):
The serum thyroid stimulating hormone (TSH) concentrations were unaffected by treatment.
See data tables for detailed information
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Sensory reactivity and grip strength: There were no treatment-related findings at the sensory reactivity and grip strength assessments in Week 12.
- Motor activity: The total low beam scores (cage floor activity) in Week 12 were statistically significantly low, when compared to the controls, for females receiving 50 mg/kg/day, although lower values only attained statistical significance at the end of the 6, 12 and 30 minute recording intervals. Lower high beam scores (rearing activity) were also observed for females receiving 50 mg/kg/day for the 6 minute and 54 minute recording intervals although slightly lower total high beam scores did not attain statistical significance. Both low and high total beam scores for males receiving 50 mg/kg/day were lower than control, but statistical significance was not attained. These inter-group differences were not apparent at the end of the recovery period. There was no effect of treatment on motor activity for males and females receiving 12.5 or 25 mg/kg/day.
See data tables for detailed information
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Higher body weight adjusted liver weights were seen in males and females receiving 50 mg/kg/day with statistical significance, which correlated with microscopic centrilobular vacuolated degeneration. Higher body weight adjusted kidney weights were seen in of males receiving 50 mg/kg/day with statistical significance, which correlated with microscopic vacuolated macrophages in the glomeruli.
- Following 4 weeks of recovery, body weight adjusted liver weights were still higher than controls in males which had previously received 50 mg/kg/day, and body weight adjusted kidney weights were statistically significantly higher than controls in males which had previously received 50 mg/kg/day.
- All other differences in organ weight parameters were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small. These included the bodyweight adjusted weights which attained statistical significance after 4 weeks of recovery (heart, kidney, pituitary and thyroid for males) as there were no similar changes after 13 weeks of treatment.

See data tables for detailed information
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment and after 4 weeks of recovery revealed no test item-related findings.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Han Wistar rats of this age. Therefore, they were considered not test item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Vacuolation was observed in numerous tissues at all doses and in both sexes. These tissues included the liver, pancreas, parotid salivary glands, gastrointestinal tract, heart, smooth muscle cells of the aorta and pulmonary artery base, media of the small arteries, kidneys, urinary bladder, adrenals glands, pituitary, thyroids, parathyroids, spleen, lungs, respiratory epithelium, respiratory epithelium of the trachea, brain, eyes , prostate and seminal vesicles (males), the smooth muscle cells of the myometrium (females).
Adverse changes were noted in the liver and kidneys at 50 mg/kg/day.
See data tables for detailed information.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on estrous cycle at the end of the treatment or recovery periods.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Formulation analysis


The analysis of formulation samples prepared for administration in week 1, 6 and 13 indicated that the mean achieved concentration values were 105-125% of the nominal concentrations.










































































  concentration found
weeknominal concentration (mg/mL)individual (mg/mL)mean (mg/mL)mean (as a % of nominal)
10NDND-
3.133.49, 3.463.47111
55.24, 5.295.27105
60NDND-
3.133.95, 3.873.91125
56.04, 5.825.93119
130NDND-
3.133.39, 3.493.44110
55.56, 5.595.58112

-: not applicable


ND: none detected


The concentrations in week 6 were slightly higher than expected. A review of the formulation data confirmed that the doses were prepared correctly. The test item was not detected in the control forumations, demonstrating that there had been no inadverted cross-contamination during the formulation procedures.


 

Conclusions:
It is concluded that the oral (by gavage) administration of the test substance to Han Wistar rats at doses of 12.5, 25 or 50 mg/kg/day for 13 weeks resulted in adverse changes to the liver and kidney of males and females at 50 mg/kg/day. The no-observed- adverse-effect level (NOAEL) was 25 mg/kg/day.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-15 - 2020-07-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: (EC) No 440/2008
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: 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 or test system 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.
See data tables for detailed information
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-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.

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. Body weight gains for males treated at 20 mg/kg bw/day remained similar to controls throughout the study. These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item. Body weights and body weight gains for females were unaffected by treatment at 20 mg/kg bw/day during two week pre-pairing, the gestation and lactation phases. 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.
See data tables for detailed information
Food consumption and compound intake (if feeding study):
effects observed, non-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. However, throughout gestation statistically significantly (p<0.01-p<0.001) lower food consumption was apparent. This trend continued during lactation in females treated with 100 mg/kg bw/day as lower food consumption was observed which achieved statistical significance (p<0.01) from Day 1 to 14. In contrast, females treated with 60 mg/kg bw/day showed signs of recovery durign lactation as food consumptions were only slightly lower during this phase of the study when compared to controls. No effect on food consumtion was detected for 20 mg/kg bw/day females during gestation or lactation.
These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item.
See data tables for detailed information
Food efficiency:
effects observed, non-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.
These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item.
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.
See data tables for detailed information
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.
See data tables for detailed information
Endocrine findings:
effects observed, non-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.
See data tables for detailed information
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral assessments: 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 Tests:
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.
See data tables for detailed information
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):
Vacuolation was observed in numerous tissues for both sexes treated at 60 and 100 mg/kg bw/day, however special staining with Oil-Red-O and PAS did not give any indication of the identity of the contents of the vacuoles. Vacuolation and hypertrophy of the choroid plexus within the brain was apparent for both sexes at these dosages. Macrophage vacuolation was apparent within the spleen (both sexes) and the uterus at these dosages and, for one female at 100 mg/kg bw/day, also within the thymus. Centrilobular vacuolar degeneration was apparent within the liver for both sexes at 60 and 100 mg/kg bw/day, such degeneration is regarded as a clear adverse finding.
At 20 mg/kg bw/day, vacuolation was still observed, but both the number of tissues affected and the incidence of findings was lower than seen at higher dosages, with findings often being restricted to a single sex. There were no incidences of vacuolation and hypertrophy of the choroid plexus within the brain or incidences of macrophage vacuolation in the spleen, uterus or thymus apparent at this dosage. However, centrilobular vacuolar degeneration was apparent within the liver for 2/5 males at 20 mg/kg bw/day and, as previously indicated, such degeneration is regarded as an adverse finding.
This interpretation is overruled 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).

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
Details on results:
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 six weeks (males) and up to eight 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. Extensive functional observations were performed 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 toxicologically significant changes in the behavioral parameters in animals of either sex treated with 20, 60 and 100 mg/kg bw/day. 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 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. 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. 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 resulting in lower overall body weight gains during the pre-pairing phase, compared to controls. Lower body weight gains, compared to control, were apparent during the first week of gestation and, to a lesser extent, the rest of the gestation period and this pattern of lower body weight gain persisted throughout the lactation period. 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, resulting in lower overall body weight gain during the pre-pairing phase, compared to controls. Lower body weight gains, compared to control, were apparent during the first week of gestation, but subsequent body weight gains during gestation were similar to controls. During lactation, body weight gains were lower than control from Day 4 of lactation, resulting in lower overall body weight gain during the lactation period. These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item. There was no effect of treatment on body weights gains 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 generally showed lower efficiency than controls. However, throughout gestation females treated with 60 or 100 mg/kg bw/day showed statistically significantly lower food intake. This trend continued during lactation in females treated with 100 mg/kg bw/day. In contrast, females treated with 60 mg/kg bw/day showed signs of recovery during lactation as food consumptions were only slightly lower than controls. No effect was detected for 20 mg/kg bw/day females during pre-pairing, gestation or lactation. These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item. Daily visual assessment of water consumption did not reveal any significant intergroup differences.
At 100 mg/kg bw/day males had a statistically significant increase (p<0.01) in neutrophils and platelet count in relation to controls, without a dose related response, and also exhibited a statistically significant reduction (p<0.05) in hematocrit. 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.
Test item 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).
Although some liver enzymes were statisticaly significantly altered, the differences and patterns observed were considered not to be biologically relevant due to the contradictions in the effects seen. Additionally, the pattern for these enzymes (AST elevated in males at 100 mg/kg bw/day but not females; ALT reduced in females at 60 and 100 mg/kg bw/day but not males; ALP reduced in males at 60 and 100 mg/kg bw/day but not females) was not consistent with adverse cellular damage.
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.
Vacuolation was observed in numerous tissues for both sexes treated at 60 or 100 mg/kg bw/day, with vacuolation and hypertrophy of the choroid plexus being apparent within the brain. Macrophage vacuolation was also apparent within the spleen, thymus at these dosages and uterus at 100 mg/kg bw/day. The exact cause of the vacuolation could not be determined, but where this vacuolation occurred alone, with no degenerate changes and at a minimal level, it may be considered to be non-adverse. As vacuolation is seen as part of a degeneration process and vacuolar degeneration of hepatocytes was observed in the liver of both sexes at 60 or 100 mg/kg bw/day, the study director considered this effect to be toxicologically significant and adverse. This interpretation is complemented by an expert statement prepared by the sponsor. 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.
To conclude, within this study, the oral administration of the test item to rats at dose levels of 60 or 100 mg/kg bw/day was associated with vacuolation in numerous tissues, vacuolation and hypertrophy of the choroid plexus of the brain and macrophage vacuolation within the spleen, thymus and uterus. The significance of the vacuolation for many of these tissues is unclear, and can be attributed to the test item characteristics. The NOAEL for systemic toxicity is considered to be 100 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Any observations made are considered to be non-adverse (justification included in the endpoint summary)
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 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:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1 scoring, according to OECD guideline, GLP-compliant
Organ:
kidney
liver

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:


DRF study


A dose range finding study (K1; Edwards, 2017) was performed in three groups, consisting each of three male and three female Wistar Han™:RccHan™:WIST strain rats. The test item was administared by gavage for up to fourteen consecutive days, at dose levels of 75, 125 and 250 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Distilled water). At 250 mg/kg bw/day, animals of either sex were terminated on Day 8 (relative to the start of dosing) due to marked reductions in body weight and food intake. There were no further unscheduled deaths in this study. There were no clinical signs of toxicity considered to be related to toxicity of the test item. There was an adverse effect on body weight development for treated animals of either sex, in a dose related manner, which ultimately resulted in lower overall body weight gains across all treatment groups when compared to controls. At 250 or 125 mg/kg bw/day animals of either sex showed lower dietary intake when compared to controls throughout their respective treatment periods. Animals treated with 75 mg/kg bw/day generally showed similar food intake to controls. There were variations in water consumption in the early terminated 250 mg/kg bw/day animals. Males showed a decreased water intake relative to controls from Day 3 whilst the females generally showed a slight increase. Animals of either sex treated with 125 mg/kg bw/day (in particular the females) showed overall increases when compared to control, with females treated with 75 mg/kg bw/day also showing sporadic increases. All animals treated with 250 mg/kg bw/day were humanely killed on Day 8. The macroscopic examination revealed gaseous distension of the large intestines in 2/3 males and 2/3 females. No further macroscopic abnormalities were detected in any other animal.
Based on these findings, 20, 60 and 100 mg/kg bw/day are recommended, respectively for the Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 Envigo Study Number: HB11QL).


 


Combined repeated dose toxicity with screening reproductive/developmental toxicity OECD 422


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 six weeks (males) and up to eight 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. Extensive functional observations were performed 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 toxicologically significant changes in the behavioral parameters in animals of either sex treated with 20, 60 and 100 mg/kg bw/day. 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 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. 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. 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 resulting in lower overall body weight gains during the pre-pairing phase, compared to controls. Lower body weight gains, compared to control, were apparent during the first week of gestation and, to a lesser extent, the rest of the gestation period and this pattern of lower body weight gain persisted throughout the lactation period. 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, resulting in lower overall body weight gain during the pre-pairing phase, compared to controls. Lower body weight gains, compared to control, were apparent during the first week of gestation, but subsequent body weight gains during gestation were similar to controls. During lactation, body weight gains were lower than control from Day 4 of lactation, resulting in lower overall body weight gain during the lactation period. These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item. There was no effect of treatment on body weights gains 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 generally showed lower efficiency than controls. However, throughout gestation females treated with 60 or 100 mg/kg bw/day showed statistically significantly lower food intake. This trend continued during lactation in females treated with 100 mg/kg bw/day. In contrast, females treated with 60 mg/kg bw/day showed signs of recovery during lactation as food consumptions were only slightly lower than controls. No effect was detected for 20 mg/kg bw/day females during pre-pairing, gestation or lactation. These observations are commonly seen when dosing an irritant test item/formulation and are considered not to be toxicologically significant as they do not specifically relate to systemic toxicity of the test item. Daily visual assessment of water consumption did not reveal any significant intergroup differences.


At 100 mg/kg bw/day males had a statistically significant increase (p<0.01) in neutrophils and platelet count in relation to controls, without a dose related response, and also exhibited a statistically significant reduction (p<0.05) in hematocrit. 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.


Test item 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).


Although some liver enzymes were statisticaly significantly altered, the differences and patterns observed were considered not to be biologically relevant due to the contradictions in the effects seen. Additionally, the pattern for these enzymes (AST elevated in males at 100 mg/kg bw/day but not females; ALT reduced in females at 60 and 100 mg/kg bw/day but not males; ALP reduced in males at 60 and 100 mg/kg bw/day but not females) was not consistent with adverse cellular damage.


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. 


Vacuolation was observed in numerous tissues for both sexes treated at 60 or 100 mg/kg bw/day, with vacuolation and hypertrophy of the choroid plexus being apparent within the brain. Macrophage vacuolation was also apparent within the spleen, thymus at these dosages and uterus at 100 mg/kg bw/day. The exact cause of the vacuolation could not be determined, but where this vacuolation occurred alone, with no degenerate changes and at a minimal level, it may be considered to be non-adverse. As vacuolation is seen as part of a degeneration process and vacuolar degeneration of hepatocytes was observed in the liver of both sexes at 60 or 100 mg/kg bw/day, the study director considered this effect to be toxicologically significant and adverse. This interpretation is complemented by an expert statement prepared by the sponsor. 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.


To conclude, within this study, the oral administration of the test item to rats at dose levels of 60 or 100 mg/kg bw/day was associated with vacuolation in numerous tissues, vacuolation and hypertrophy of the choroid plexus of the brain and macrophage vacuolation within the spleen, thymus and uterus. The significance of the vacuolation for many of these tissues is unclear, and can be attributed to the test item characteristics. The NOAEL for systemic toxicity is considered to be 100 mg/kg bw/day. 


 


Long-term repeated dose toxicity (OECD408)


A 90 day repeated dose toxicity study (K1, OECD 408; Chase, 2022) was performed in male and female Han Wistar rats. Three groups, each comprising ten male and ten female, received the test substance at doses of 12.5, 25 or 50 mg/kg bw/day. A similarly constituted control group received the vehicle, purified water, at the same volume dose as the high dose group. A further five male and five female Han Wistar rats were assigned to each group. These animals were treated for 13 weeks, followed by a four-week period without treatment to assess the potential for any treatment-related change to recover.
During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone, estrous cycle, organ weight, macropathology and histopathology investigations were undertaken.


The general appearance and behavior of the animals were unaffected by treatment, and there were no treatment-related deaths.
Sensory reactivity and grip strength were unaffected by treatment. Motor activity was slightly reduced (both high and low beam scores) for males and females receiving 50 mg/kg/day. This change was not apparent at the end of the recovery period. Bodyweight gain was slightly low for males receiving 50 mg/kg/day and females receiving 25 or 50 mg/kg/day. These differences were still apparent at the end of the recovery period. Food consumption was slightly low for males and females receiving 50 mg/kg/day. This change was not apparent at the end of the recovery period. There were no treatment-related ophthalmoscopic findings in Week 12.


The hematological examination during Week 13 revealed slightly low hematocrit, hemoglobin concentration and erythrocyte counts in males and females receiving 50 mg/kg/day and females receiving 25 mg/kg/day. Mean cell volume was slightly high for males receiving 25 or 50 mg/kg/day and reticulocyte counts were slightly low for females receiving 50 mg/kg/day. Slightly low hematocrit and erythrocyte counts were seen in females receiving 12.5 mg/kg/day but there was no effect on hemoglobin concentrations. After 4 weeks of recovery, hematocrit, hemoglobin concentration and erythrocyte counts were still slightly low for females which previously received 50 mg/kg/day and reticulocyte counts were slightly high for males which previously received 50 mg/kg/day. Lymphocyte counts were slightly high in males and females receiving 50 mg/kg/day and large unstained cell counts were slightly high in females receiving 25 or 50 mg/kg/day. After 4 weeks of recovery, neutrophil counts were slightly high for males which previously received 50 mg/kg/day, with slightly high basophil and large unstained cell counts. Prothrombin time was slightly reduced in males and females receiving 50 mg/kg/day, and in males receiving 12.5 or 25 mg/kg/day. This was not apparent at the end of the recovery period.


The biochemical examination of the blood plasma in Week 13 revealed high urea concentrations in males and females receiving 50 mg/kg/day and this associated with a small increase of creatinine concentration in males; urea concentrations were still high after 4 weeks of recovery. Potassium concentrations were high in males and females receiving 50 mg/kg/day but this was not apparent at the end of the recovery period. Total plasma protein and albumin concentrations were low in males and females receiving 50 mg/kg/day (although albumin/globulin ratio was unaffected) and these differences were still low after 4 weeks of recovery for females which previously received 50 mg/kg/day.


 


High liver weights were seen in males and females which received 50 mg/kg/day and high kidney weights in males which received 50 mg/kg/day. Following 4 weeks of recovery, the changes were still apparent in males which had previously received 50 mg/kg/day.
The macroscopic examination performed after 13 weeks of treatment and after 4 weeks of recovery revealed no effects of treatment.


Histopathology revealed centrilobular vacuolated degeneration in the liver of all males and females receiving 50 mg/kg/day and some males receiving 25 mg/kg/day, and focal hepatocyte necrosis was also seen in six females receiving 50 mg/kg/day. Following a 4-week recovery period, centrilobular vacuolated degeneration had fully recovered in males and partially recovered in females; focal hepatocyte necrosis had fully recovered in females. Vacuolated macrophages in the glomeruli capillaries of the kidneys were seen in males and females at 25 or 50 mg/kg/day and following 4 weeks of recovery, this finding persisted at a similar incidence and severity. These findings in the liver and kidneys were considered adverse in males and females receiving 50 mg/kg/day. Vacuolation was also seen in numerous tissues generally in males and females at 25 or 50 mg/kg/day but in all other tissues the vacuolation was considered to be non-adverse as they occurred alone with no associated degenerative changes or adverse effect on the organ.


It is concluded that the oral (by gavage) administration of the test substance to Han Wistar rats at doses of 12.5, 25 or 50 mg/kg/day for 13 weeks resulted in adverse changes to the liver and kidney of males and females at 50 mg/kg/day. The no-observed- adverse-effect level (NOAEL) was 25 mg/kg/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.


 


 

Justification for classification or non-classification

The test item cause vacuolization in several organs and tissues that was to mostly transient in nature as shown in the OECD 408 study. These effects were largely reversible in regenerative organs, hence they are considered of minimal toxicological importance. Therefore, such effects do not, by themselves, indicate 'significant' or 'severe' 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.