3-methoxypropylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-17 to 2018-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05/01/2018- 05/09/2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: 14-day dose range finding study

Principles of method if other than guideline:

The study was run at a GLP compliant facility, however the study doesn't claim GLP compliance

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: batch TE767F05G
- Purity, including information on contaminants, isomers, etc.: 99.7%

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 (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item formulations were determined by Covance CRS Research Limited, Shardlow, UK Analytical Services within Covance Study Number: WD30CK. Results showed formulations to be stable for four hours.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: six weeks
- Weight at study initiation: males weighed 177 to 213g, the females weighed 145 to 164g
- Fasting period before study: no
- Housing: housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 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): 15/h
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 05/01/2018 To: 21/02/2018

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0; 7.5; 18.75; 37.5mg/mL
- Amount of vehicle (if gavage): 4mL/kg
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

no

Remarks:

Results of previous study showed formulations to be stable for four hours. Formulations for this study were prepared daily and were dosed within 2 hours of preparation. No analysis was conducted to determine the concentration of the test item formulation.

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary toxicity work was undertaken, treating one male and one female rat at 300 mg/kg bw/day for four days. Treatment-related effects consisted of increased salivation, hunched posture, pilo-erection and noisy respiration and body weight losses in both animals. At necropsy both animals had dark lungs and thickening/sloughing on the non-glandular region of the stomach. The male also had a dark area on the non-glandular region of the stomach.
- Rationale for animal assignment (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.
Additionally observations were also made four hours following dosing (not at weekends).
All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: No


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 (if feeding study):
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to
15. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): 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

HISTOPATHOLOGY: No

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values
where appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male and two females treated with 150 mg/kg bw/day showed an isolated incidence of
increased salivation post dosing on Day 14.
No such effects were evident in animals of either sex treated with 75 or 30 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 150 mg/kg bw/day showed a slight reduction in body weight gain between
Days 4 and 8, however, recovery was evident thereafter.
No such effects were evident in females treated with 150 mg/kg bw/day or in animals of
either sex treated with 75 or 30 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males treated with 150 mg/kg bw/day showed a slight reduction in food consumption
between Days 4 and 8, however, recovery was evident thereafter.
No such effects were evident in females treated with 150 mg/kg bw/day or in animals of
either sex treated with 75 or 30 mg/kg bw/day.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Females from all treatment groups showed an increase in overall water consumption; however, a true dose related response was not evident. No such effects were evident in treated males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

All males and two females treated with 150 mg/kg bw/day showed ulcerated stomachs at necropsy.
No such effects were evident in animals of either sex treated with 75 or 30 mg/kg bw/day.

Conclusions:

Dose levels of 0 (Control), 10, 30 and 100 mg/kg bw/day are recommended for use in the planned Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat

Executive summary:

In this 14-day dose range finding study treatment at 150 mg/kg bw/day showed an incidence of reduced body weight gain and food consumption in males only (Days 4-8) and although recovery was evident thereafter, adverse macroscopic abnormalities (ulcerated stomachs) were evident in all males and two females from this treatment group. In view of these findings, 150 mg/kg bw/day is considered unsuitable for a longer administration period on future studies. Dose levels of 0 (Control), 10, 30 and 100 mg/kg bw/day are therefore recommended for use in the planned Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 21 September 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methoxypropylamine

EC Number:

226-241-3

EC Name:

3-methoxypropylamine

Cas Number:

5332-73-0

Molecular formula:

C4H11NO

IUPAC Name:

3-methoxypropan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TE767F05G
- Analytical purity: 99.7%. N ocorrectoin fo rpurity was made
- Expiration date of the lot/batch: 05 June 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Solubility and stability of the test substance in the solvent/vehicle: The stability and homogeneity of the test item formulations determinations showed the formulations to be stable for four hours. Formulations were therefore prepared daily and dosed within four hours of preparation.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RCCHan(TM):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: Envigo RMS (UK) Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Males 186-214 g, Females 134-165 g
- Fasting period before study: no data
- Housing: housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): ad libitum, Rodent 2014C Teklad Global Certified Diet
- Water (e.g. ad libitum): ad libitum, no data
- Acclimation period: six days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Once daily, by gavage, using a stainless steel dosing cannula attached to a disposable plastic syringe for ninety consecutive days. 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:

arachis oil

Remarks:

BP

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for four hours.
- Formulations were therefore prepared daily and dosed within four hours of preparation.
- 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.

VEHICLE
- Concentration in vehicle: 0, 2.5, 7.5 and 25 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulations were taken on four occasions and analyzed for concentration of test item at Covance CRS Research Limited. The results indicate that the prepared formulations were within 90% and 105% of the nominal concentration.
The homogeneity and stability, determined with respect to the concentration of Test Item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 100 mg/mL. The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a achromatographic profile consisting of a single peak. The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accurracy (recovery) and precision, limit of quantification (LOQ). The homogeneity and stability of Test Item in Arachis Oil BP formulations was assessed at nominal concentrations of 2.5 mg/mL and 100 mg/mL during ambient storage.The mean concentrations of Test Item in test formulations analyzed for the study were within applied limits ± 10%, confirming accurate formulation.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (vehicle only - Arachis oil BP)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Intermediate dose group

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on available toxicity data including a 14 day range finder toxicity study in the rat (Allt J. 2018)
In a preliminary study one male and one female were treated with 300 mg/kg bw/day for four consecutive days. Both animals were terminated on Day 5 due to clinical signs of toxicity and body weight losses. At necropsy, macroscopic stomach and lung findings were evident in both animals. In the 14-day dose range finding study, treatment at 150 mg/kg bw/day showed an incidence of reduced body weight gain and food consumption in males only (Days 4-8) and although recovery was evident thereafter, adverse macroscopic abnormalities (ulcerated stomachs) were evident in all males and two females from this treatment group. The local stomach and lung effects seen in the dose range finding study were likely caused by the corrosive nature of the test item. In view of these findings, 150 mg/kg bw/day is considered unsuitable for a longer administration period on future studies. Dose levels of 0 (Control), 10, 30 and 100 mg/kg bw/day are therefore recommended for use in the planned Ninety Day Repeated Dose Oral (Gavage) Toxicity Study in the Rat

Positive control:

no

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing.
- Parameters: overt signs of toxicity, ill-health or behavioral change

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Individual body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all Groups 1 to 4 animals were examined pre-treatment. During Week 12, the eyes of all control and high dose animals (Groups 1 and 4, respectively) were examined.
Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.


HAEMATOLOGY: Yes
- Hematological investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
- Parameters checked: hemoglobin, erythrocyte count, hematocrit, erythrocyte indices (mean corpuscular hemoglobin, volume and hemoglobin concentration), total leukocyte count, differential leukocyte count (neutrophyls, lymphocytes, monocytes, eosinophils, basophils), platelet count, reticulocyte count (methylene blue stained slides were prepared but reticulocytes were not assessed), prothrombin time was assessed by 'Innovin' and activated partial thromboplastin time 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:
Blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on day 91.
- Animals fasted: No
- How many animals: all animals
- Parameters checked: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids

FUNCTIONAL OBSERVATIONS
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Behavioral assessment: Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin color, respiration, palpebral closure, urination, defecation, transfer arousal, tail elevation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory reactivity Tests.
- Functional performance tests:
- motor activity : 20 purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least 2 hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, reiter and Macphail, 1979).
- forelimb/hindlimb grip strength: An automated grip strength 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. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
- Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed: gasp response, vocalization, toe pinch, tail pinch, fingers approach, touch escape, pupil reflex, blink reflex, startle reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint) - retained only and not processed, bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides - preserved in Modified Davidson's fluid, esophagus, eyes - fixed in Davidson's fluid, gross lesions, heart, ileum (including Peyer's patches), jejunum, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes - preserved in Modified Davidson's fluid, thymus, kidneys, liver, lungs (with bronchi) - lungs were inflated to approximately normal inspiratory volume with buffered 10% formaline before immersion in fixative, lymph nodes (mandibular and mesenteric), mammary glands, muscle (skeletal), thyroid/parathyroid, tongue - retained only and not processed, trachea, urinary bladder, uterus (with cervix), vagina
All tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Lewis). All tissues from control and 100 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Other examinations:

No data

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. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
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).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The observed clinical effects were considered not to be treatment-related.
Increased salivation was evident in all males treated with 100 mg/kg bw/day and in nine females treated with 100 mg/kg bw/day between Days 1 and 88 (males) and Days 12 and 89 (females). Noisy respiration was evident in all males treated with 100 mg/kg bw/day from Day 1 onwards and in all females from this treatment group between Days 3 and 87. These observations were mainly sporadic in frequency. Decreased respiratory rate, hunched posture, lethargy and labored respiration were also noted in one or two animals treated with 100 mg/kg bw/day on isolated occasions. On one occasion only, one male treated with 30 mg/kg bw/day and two females treated with 10 mg/kg bw/day showed noisy respiration. Observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and represent difficulties in dosing particular animals rather than evidence of true systemic toxicity.

The following observations were considered to be incidental and unrelated to treatment. Red/brown staining around the right eye in one male treated with 100 mg/kg bw/day and generalised fur loss in two females treated with 10 mg/kg bw/day and in one control female.

For more details, please refer to Table 1a and 1b in section 'Additional information on results'.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three unscheduled deaths occured during the study.
One female treated with 100 mg/kg bw/day was found dead on Day 80. One male and one female treated with 100 mg/kg bw/day were euthanized in extremis on Days 56 and 78 (respectively).

The female that was found dead on Day 80 showed noisy/gasping respiration, a decreased respiratory rate, pilo-erection and hunched posture on Day 80 and had previously shown increased salivation.

The male treated with 100 mg/kg bw/day that were euthanized in extremis on Day 56 showed noisy/gasping/labored respiration, a decreased respiratory rate, hunched posture, pilo-erection and lethargy and had previously shown increased salivation. The female treated with 100 mg/kg bw/day that was euthanized in extremis on Day 78 showed noisy/gasping/labored respiration, a decreased respiratory rate, hunched posture, pilo-erection, pallor of the extremities and increased salivation.

There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in body weight or body weight gain.
Males treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during Week 2. An increase in body weight gain is considered not to represent an adverse effect of treatment.
For more details, please refer to Table 2 in section 'Additional information on results'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food consumption.
There was no adverse effect of treatment on food consumption for either sex at 10, 30 or 100 mg/kg bw/day.
For more details, please refer to Table 3 in section 'Additional information on results'.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food conversion efficiencies.
There was no adverse effect of treatment on food conversion efficiency for either sex at 10, 30 or 100 mg/kg bw/day.
For more details, please refer to Table 4 in section 'Additional information on results'.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in water consumption.
Dialy visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes observed during ophthalmoscopic examination of animals of both sexes from the control group and surviving 100 mg/kg bw/day during Week 12 of the treatment period.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in hematology.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The observed clinical effects were considered not to be treatment-related.
Males from all treatment groups showed a statistically significant reduction (p<0.05) in alanine aminotransferase. A true dose related response was not evident, all of the individual values were within historical control range and no associated histopathological correlates were evident, therefore, the intergroup differences were considered not to be of toxicological importance. Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in glucose. The majority of individual values were within historical control range and there were no associated histopathological correlates, therefore the intergroup difference was considered not to be of toxicological importance.
For more details, please refer to Table 1a and 1b in section 'Additional information on results'.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral assessments: There were no treatment-related changes in behavioral assessments.
Instances of noisy respiration were evident in a total of nine males and seven females treated with 100 mg/kg bw/day between Weeks 1 and 13 (males) and during Weeks 2, 5-7, 9-13 (females). Hunched posture was evident in one of these males during Week 1 only and labored respiration and a decreased respiratory rate was evident in another of these males during Week 2 only. Noisy respiration was also evident in two males treated with 30 mg/kg bw/day during either Week 1 or Week 11 and in one control male during Week 7. These correlated with the daily clinical observations seen in these treatment groups. Pilo-erection and hunched posture was evident in one male treated with 10 mg/kg bw/day during Weeks 8 and 9 only.
No such effects were evident in females treated with 30 or 10 mg/kg bw/day.
Functional performance tests:There were no treatment-related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.
Sensor reactivity assessments: There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects detected in organ weights measured.
Males from all treatment groups showed a statistically significant reduction (p<0.05) in kidney weight both absolute and relative to terminal body weight. With the exception of one individual relative value at 30 and 100 mg/kg bw/day, all remaining individual values were within historical control ranges. In the absence of a true dose related response or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight and a statistically significant reduction (p<0.05) in spleen weight both absolute and relative to terminal body weight. Although the majority of individual brain and spleen weights for treated animals were outside of the historical control ranges, the majority of control values were also outside of the historical control ranges. In the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic abnormalities observed at terminal necropsy. The female treated with 100 mg/kg bw/day that was found dead on Day 80 had gaseous distension in the stomach, red contents in the ileum and jejunum and dark lungs. The male treated with 100 mg/kg bw/day that was euthanized in extremis on Day 56 had gaseous distension in the cecum, colon and ileum and red lungs. The female treated with 100 mg/kg bw/day that was euthanized in extremis on Day 78 had gaseous distension in the cecum, duodenum, ileum, jejunum and stomach, red lungs and enlarged adrenals. Reddened lungs may have been the result of possible aspiration of the test item, with both of these animals showing significant respiratory observations prior to being terminated.
One control male, one control female, one male and two females treated with 10 mg/kg bw/day, one male and one female treated with 30 mg/kg bw/day and two males and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examinations revealed agonal congestion in the lungs for a number of these animals and in view of the finding being present in a control animal, these observations were considered unrelated to treatment with the test item. One female treated with 100 mg/kg bw/day had a fluid filled left horn of the uterus and a mass on the cervix. In the absence of any associated
treatment-related histopathological correlates these findings were considered not to be of toxicological significance. One male treated with 10 mg/kg bw/day had pale kidneys. In the absence of a similar effect at the higher dosages the intergroup difference was considered not
to be of toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following microscopic abnormalities were detected:
- The male euthanized in extremis on Day 56 had ulceration with necrosis/inflammation in the trachea (moderate) and agonal congestion in the lungs. Examination of the nasopharynx showed inflammatory/necrotic exudate as well as atrophy.
The female that was found dead on Day 80 had focal, minimal hyperplasia in the nonglandular stomach. Examination of the nasopharynx proved inconclusive.
- The female euthanized in extremis on Day 78 had ulceration with necrosis/inflammation in the trachea (marked) and mild inflammatory change in the lungs. The tracheal change is considered to be the cause of the poor clinical condition.

Terminal Necropsy
- Stomach: Hyperplasia of the non-glandular epithelium, minimal or mild was present in five males and three females treated with 100 mg/kg bw/day. Minimal, focal hyperplasia of the non-glandular region was present in one male treated with 30 mg/kg bw/day but in the
absence of any other changes in this group at this low incidence and severity this is considered incidental.
- Trachea: Ulceration with necrosis/inflammation was present in one male treated with 100 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse treatment related effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1a: Summary incidence of daily clinical observations (male)

	Group 1 0 (control)	Group 2 10 mg/kg bw/day	Group 3 30 mg/kg bw/day	Group 4 100 mg/kg bw/day
Killed 'in extremis'	-	-	-	1 animal Day 56
Scheduled kill	10 animals Day 91	10 animals Day 91	10 animals Day 91	9 animals Day 91
Staining around the eyes	-	-	-	1 animal Day 9
Increased salivation	-	1 animal Day 88	-	10 animals From day 1 to day 88
Noisy respiration	-	-	1 animal Day 75	10 animals From day 1 to day 90
Laboured respiration	-	-	-	1 animal Day 56
Gasping respiration	-	-	-	1 animal Day 56
Decreased respiratory rate	-	-	-	2 animals From day 6 to day 68
Pilo-erection	-	-	-	1 animal Day 56
Lethargy	-	-	-	1 animal Day 56
Hunched posture	-	-	-	3 animals From day 6 to day 88

Day numbers relative to start date

Tabel 1b: Summary incidence of daily clinical observations (female)

	Group 1 0 (control)	Group 2 10 mg/kg bw/day	Group 3 30 mg/kg bw/day	Group 4 100 mg/kg bw/day
Killed 'in extremis'	-	-	-	1 animal Day 78
Scheduled kill	10 animals Day 91	10 animals Day 91	10 animals Day 91	8 animals Day 91
Found dead	-	-	-	1 animal Day 80
Increased salivation	-	-	-	9 animals From day 12 to day 89
Noisy respiration	-	2 animals From day 63 to day 84	-	10 animals From day 3 to day 87
Laboured respiration	-	-	-	2 animals From day 61 to day 78
Gasping respiration	-	-	-	2 animals From day 78 to day 80
Decreased respiratory rate	-	-	-	4 animals From day 6 to day 80
Pilo-erection	-	-	-	2 animals From day 78 to day 80
Lethargy	-	-	-	1 animal From day 6 to day 7
Hunched posture	-	-	-	4 animals From day 6 to day 80
Generalised fur loss	1 animal From day 57 to day 63	2 animals From day 37 to day 90	-	-
Pallor of the extremities	-	-	-	1 animal Day 78

Day numbers relative to start date

Table 2: Group Mean Body Weight Gains

Increase in Bodyweight (g) - Day numbers relative to start date

Groups (sex)	From Day To Day	1 8	8 15	15 22	22 29	29 36	36 43	43 50	50 57	57 64	64 71	71 78	78 85	85 91	Abs Gain 1 91	% Gain 1 91
control (M)	Mean SD N	31.5 6.5 10	24.8 4.9 10	24.8 3.8 10	22.7 3.9 10	16.8 4.1 10	12.7 5.2 10	11.6 2.2 10	15.0 7.1 10	11.3 7.2 10	10.0 3.4 10	9.7 3.2 10	8.5 4.3 10	5.4 2.2 10	204.8 25.2 10	103.3 11.4 10
10 mg/kg bw (M)	Mean SD N	34.2 4.5 10	26.9 5.7 10	25.4 5.4 10	21.9 4.8 10	15.3 4.5 10	13.4 4.4 10	14.2 5.6 10	15.0 6.4 10	13.3 3.1 10	14.1 4.6 10	9.2 2.9 10	10.7 4.4 10	6.0 3.8 10	219.6 22.5 10	111.0 10.9 10
30 mg/kg bw (M)	Mean SD N	35.9 6.0 10	25.8 4.1 10	23.3 5.5 10	22.4 5.0 10	14.7 5.6 10	13.2 7.3 10	12.4 5.9 10	14.7 4.4 10	10.8 4.2 10	12.4 6.3 10	7.1 5.1 10	9.2 4.0 10	6.3 4.4 10	208.2 34.9 10	105.0 18.9 10
100 mg/kg bw (M)	Mean SD N	29.1 10.1 10	30.1* 3.2 10	26.7 2.8 10	21.7 4.3 10	18.3 7.6 10	16.5 5.1 10	10.3 5.4 10	16.8 4.0 9	10.7 2.6 9	10.7 4.2 9	9.6 4.0 9	7.4 3.0 9	4.6 8.9 9	216.4 34.1 9	108.5 19.4 9
control (F)	Mean SD N	13.1 3.3 10	11.2 7.1 10	10.3 5.8 10	9.3 6.1 10	4.0 4.6 10	7.1 6.0 10	3.2 6.1 10	4.6 4.4 10	0.0 3.4 10	4.9 4.9 10	1.7 4.9 10	3.7 5.0 10	0.1 4.4 10	73.2 18.6 10	46.9 10.9 10
10 mg/kg bw (F)	Mean SD N	16.4 3.8 10	11.4 4.6 10	14.4 5.5 10	8.3 2.9 10	5.3 5.2 10	5.0 5.4 10	5.3 4.2 10	4.6 2.3 10	2.1 2.4 10	2.8 4.7 10	5.0 3.7 10	3.6 4.6 10	-1.8 5.4 10	82.4 14.1 10	53.2 9.4 10
30 mg/kg bw (F)	Mean SD N	15.0 5.3 10	13.2 6.6 10	11.9 4.4 10	8.1 3.5 10	2.5 4.2 10	7.9 6.2 10	6.5 3.4 10	1.7 2.6 10	3.1 4.0 10	4.8 4.7 10	3.8 3.3 10	1.7 4.1 10	-0.8 3.7 10	79.4 17.2 10	52.0 9.6 10
100 mg/kg bw (F)	Mean SD N	15.9 9.3 10	13.0 5.0 10	7.9 4.0 10	13.3 4.4 10	7.6 6.7 10	5.3 4.6 10	4.4 5.0 10	3.6 5.8 10	3.1 6.7 10	1.5 4.2 10	4.6 5.2 10	0.3 3.6 8	0.4 2.8 8	81.5 11.6 8	52.2 8.6 8

Probability values (p) are presented as follows: p<0.01 ** - very significant; p<0.05 * - significant; p>0.05 - not significant

Increase in Bodyweight (g)

Day numbers relative to start date

Table 3: Group Mean Food consumptions       

Group (Sex)	From To	1 8	8 15	15 22	22 29	29 36	36 43	43 50	50 57	57 64	64 71	71 78	78 85	85 91
control (M)	Mean N	19.3 10	20.4 10	20.6 10	21.1 10	20.3 10	20.3 10	20.0 10	20.0 10	20.1 10	19.8 10	19.7 10	19.3 10	19.4 10
10 mg/kg bw (M)	Mean SD	19.8 10	21.1 10	21.0 10	21.1 10	20.4 10	20.7 10	19.9 10	20.2 10	20.7 10	20.7 10	20.5 10	20.5 10	20.0 10
30 mg/kg bw (M)	Mean SD	21.5 10	22.0 10	21.8 10	22.1 10	21.0 10	21.4 10	21.1 10	21.3 10	21.5 10	21.7 10	20.5 10	20.5 10	19.8 10
100 mg/kg bw (M)	Mean SD	18.3 10	21.9 10	21.3 10	20.8 10	19.8 10	21.1 10	20.1 10	20.2 10	22.2 9	20.2 9	20.2 9	20.2 9	19.8 9
control (F)	Mean SD	14.8 10	15.8 10	15.8 10	16.0 10	15.4 10	15.6 10	15.4 10	14.7 10	14.7 10	15.1 10	15.1 10	15.0 10	13.7 10
10 mg/kg bw (F)	Mean SD	15.0 10	16.3 10	16.7 10	16.5 10	16.0 10	16.1 10	15.4 10	15.1 10	15.4 10	15.2 10	15.7 10	15.3 10	14.3 10
30 mg/kg bw (F)	Mean SD	14.4 10	14.6 10	14.9 10	14.6 10	14.7 10	14.6 10	14.4 10	13.6 10	14.2 10	14.5 10	14.1 10	14.1 10	12.5 10
100 mg/kg bw (F)	Mean SD	13.9 10	15.5 10	15.5 10	15.5 10	15.7 10	15.0 10	14.7 10	14.4 10	14.0 10	14.1 10	14.5 10	14.3 9	13.2 8

Day numbers relative to start date

Table 4: Food efficiency

Group (sex)	From To	1 8	8 15	15 22	22 29	29 36	36 43	43 50	50 57	57 64	64 71	71 78	78 85	85 91
control (M)	Mean N	23.2 10	17.4 10	17.2 10	15.4 10	11.9 10	8.9 10	8.2 10	10.8 10	8.0 10	7.2 10	7.0 10	6.3 10	4.7 10
10 mg/kg bw (M)	Mean SD	24.6 10	18.1 10	17.4 10	14.9 10	10.7 10	9.2 10	10.0 10	10.6 10	9.2 10	9.7 10	6.4 10	7.6 10	5.0 10
30 mg/kg bw (M)	Mean SD	23.8 10	16.8 10	15.2 10	14.5 10	9.9 10	8.8 10	8.5 10	9.9 10	7.2 10	8.0 10	5.0 10	6.5 10	5.1 10
100 mg/kg bw (M)	Mean SD	22.4 10	19.6 10	17.9 10	14.8 10	13.1 10	11.1 10	7.4 10	11.8 9	6.9 9	7.5 9	6.7 9	5.3 9	3.7 9
control (F)	Mean SD	12.6 10	10.2 10	9.2 10	8.4 10	3.8 10	6.7 10	2.8 10	4.5 10	-0.1 10	4.8 10	1.5 10	3.5 10	0.3 10
10 mg/kg bw (F)	Mean SD	15.6 10	10.0 10	12.3 10	7.2 10	4.8 10	4.4 10	4.8 10	4.4 10	2.0 10	2.6 10	4.5 10	3.4 10	-2.0 10
30 mg/kg bw (F)	Mean SD	15.1 10	12.9 10	11.3 10	8.0 10	2.5 10	7.6 10	6.3 10	1.9 10	3.1 10	4.8 10	3.8 10	1.8 10	-1.0 10
100 mg/kg bw (F)	Mean SD	16.3 10	11.9 10	7.2 10	12.3 10	7.0 10	5.0 10	4.3 10	3.5 10	3.1 10	1.5 10	4.5 10	2.3 8	-1.9 8

Days numbers relative to start date

Table 5: Group Mean Hematological Values

Group (sex)		Hb g/dl	RBC 10^12/l	Hct %	MCH pg	MCV fl	MCHC g/dl	WBC 10^9/l	Neut 10^9/l	Lymph 10^9/l	Mono 10^9/l	Eos 10^9/l	Bas 10^9/l	C Seconds	PLT 10^9/l	APTT Seconds
control (M)	Mean SD N	16.47 1.56 10	8.807 0.644 10	47.26 3.8 10	18.68 0.88 10	53.64 1.91 10	34.79 0.59 10	7.85 1.56 10	2.842 0.688 10	4.959 1.278 10	0.000n 0.000 10	0.051 0.062 10	0.000n 0.000 10	9.39 0.56 10	542.5 101.5 10	14.70 1.71 10
10 mg/kg bw (M)	Mean SD N	16.02 1.70 10	8.462 0.747 10	45.26 3.65 10	18.83 1.18 9	53.62 1.70 9	35.12 1.96 9	7.90 2.05 10	3.122 0.984 10	4.745 1.107 10	0.000n 0.000 10	0.035 0.049 10	0.000n 0.000 10	9.55 0.48 10	593.3 129.7 10	14.48 1.96 10
30 mg/kg bw (M)	Mean SD N	15.93 1.22 10	8.481 0.590 10	45.5 3.21 10	18.87 0.63 9	53.94 1.28 10	34.83 1.15 9	7.67 2.62 10	3.090 1.251 10	4.538 1.583 10	0.010n 0.032 10	0.033 0.061 10	0.000n 0.000 10	9.26 0.84 10	510.8 139.2 10	14.73 1.96 10
100 mg/kg bw (F)	Mean SD N	16.28 1.65 9	8.617 0.789 9	47.58 4.65 9	18.87 0.82 9	55.20 1.74 9	34.19 0.75 9	8.92 3.24 9	3.618 1364 9	5.286 1.923 9	0.012n 0.037 9	0.010 0.023 9	0.000n 0.000 9	9.54 0.72 9	600.8 118.7 9	15.29 1.50 9
control (F)	Mean SD N	15.39 0.55 10	8.151 0.347 10	44.49 1.65 10	18.91 0.58 10	54.59 1.59 10	34.62 0.49 10	6.45 2.46 10	1.608 0.435 10	4.776 2.021 10	0.000n 0.000 10	0.068 0.073 10	0.000n 0.000 10	8.63 1.06 10	631.5 108.3 10	14.52 1.41 10
10 mg/kg bw (F)	Mean SD N	15.24 0.34 10	8.091 0.300 10	44.27 0.93 10	18.83 0.54 10	54.73 1.39 10	34.39 0.35 10	8.10 2.29 10	2.008 0.346 10	6.033 2.072 10	0.000n 0.000 10	0.060 0.070 10	0.000n 0.000 10	9.18 0.85 10	650.1 85.7 10	15.98 1.92 10
30 mg/kg bw (F)	Mean SD N	14.85 1.44 10	7.647 0.820 10	43.06 4.31 10	19.41 0.88 10	56.41 2.94 10	34.48 0.33 10	8.41 2.67 10	2.054 0.668 10	6.293 2.093 10	0.008n 0.025 10	0.057 0.061 10	0.000n 0.000 10	8.97 1.03 10	613.3 49.6 10	16.33 2.02 10
100 mg/kg bw (F)	Mean SD N	15.33 1.26 8	8.110 0.674 8	44.74 3.27 8	18.90 0.24 8	55.23 1.05 8	34.23 0.47 8	8.10 2.70 8	2.079 0.772 8	5.945 1.984 8	0.000n 0.000 8	0.078 0.128 8	0.000n 0.000 8	8.98 0.79 8	577.3 228.2 8	13.76 1.37 8

Abbreviations: Hb - hemoglobin, GBC - Erytrhocyte count, Hct - hematocrit, MCH - mean copuscular hemoglobin, MCV - mean corpuscular volume, MCHC - mean corpuscular hemoglobin concentration, WBC - total leukocyte count, Neut - neutrophils, Lymph - lymphocytes, Mono - monocytes, Eos - eosinophils, Bas - basophils, C - prothrombin time, PLT - platelet count, APTT - activated partial thromboplastin time

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of the test substance for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 30 or 100 mg/kg bw/day resulted in the unscheduled death of one male and two females treated with 100 mg/kg bw/day.
These sporadic deaths were not associated with target organ histopathological changes which could have suggested that mortality or morbidity was associated with, or a consequence of, systemic toxicity. The evidence of degenerative and inflammatory changes in the upper respiratory tract of these animals supports the conclusion that these deaths were associated with the process of gavage dose administration of an irritant material and the pathology was due to gastric reflux. The microscopic stomach changes identified in surviving animals of either sex at 100 mg/kg bw/day were also considered to be the result of gastric irritancy rather than attributable to true systemic toxicity. Therefore, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day.