Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral: A K1 GLP compliant 90-day repeated dose toxicity study according to OECD guideline 408 was performed in rats via oral gavage. Dose levels tested were 10, 30 and 100 mg/kg. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day (highest dose tested).

 

Repeated dose toxicity - inhalation/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 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:
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
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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.

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 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
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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (vehicle only - Arachis oil BP)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
30 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:
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
Positive control:
no
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)
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
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
Key result
Critical effects observed:
no

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

10.7

2.6

10.7

4.2

9.6

4.0

7.4

3.0

4.6

8.9

216.4

34.1

108.5

19.4

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

0.4

2.8

81.5

11.6

52.2

8.6

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

20.2

20.2

20.2

19.8

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

13.2

Day numbers relative to start date

Table 4: Food efficiency

Group

(sex)

From

To 

1

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

6.9

7.5

6.7

5.3

3.7

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

-1.9

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

16.02

1.70

10

8.462

0.747

10 

45.26

3.65

10 

18.83

1.18

53.62

1.70

35.12

1.96

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

15.93

1.22

10 

8.481

0.590

10 

45.5

3.21

10 

18.87

0.63

53.94

1.28

10 

34.83

1.15

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

 16.28

1.65

9

 8.617

0.789

9

47.58

4.65

18.87

0.82

55.20

1.74

34.19

0.75

8.92

3.24

3.618

1364

5.286

1.923

0.012n

0.037

0.010

0.023

0.000n

0.000

9.54

0.72

600.8

118.7

15.29

1.50

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

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

15.33

1.26

8.110

0.674

44.74

3.27

18.90

0.24

55.23

1.05

34.23

0.47

8.10

2.70

2.079

0.772

5.945

1.984

0.000n

0.000

0.078

0.128

0.000n

0.000

8.98

0.79

577.3

228.2

13.76

1.37

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

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.
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2006 - 28 September 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422 and EPA/OPPTS 870.3650, using the hydrochloride salt of the submission substance. Erroneously, several females of all test groups including the controls were gavaged with application volumes being 0.1 -0.3 mL above or below the target volumes on two days. These marginal and transient deviations from the study protocol are not considered to have any influence on the outcome of this study.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
dosing volumes (see field 'Any other information on materials and methods incl. tables)'
Qualifier:
according to
Guideline:
other: EPA/OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
yes
Remarks:
dosing volumes (see field 'Any other information on materials and methods incl. tables)'
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Hydrochloride salt of 3-Methoxypropylamine. For repeated dose tests, the salt of the substance under evaluation is used in order to counteract the corrosive properties of the substance. The salt is not considered to have any impact on the hazardous effects of the substance.
- Molecular formula (if other than submission substance): C4 H12 Cl N O
- Molecular weight (if other than submission substance): 125.6 g/mole
- Substance type: Aqueous solution / liquid, orange, clear
- Physical state: Aqueous solution/liquid
- Lot/batch No.: BA 1464
- Storage conditions: Room temperature
- Other:
Test substance No.: 05/0675-1
Concentration of stock solution: 67.2 g 3-Methoxypropylamine hydrochloride per 100 g aqueous solution
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Crl:WI(Han)
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: male animals (264.0 g) and female animals (200.2 g)
- Fasting period before study: food and water were available ad libitum except during the fasting period (16 to 20 hours prior to necropsy) and measurement of motor activity)
- Housing: The rats were housed individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2), with following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until sacrifice, the pregnant animals and their litters were housed in Makrolon type M III cages (floor area about 800 cm2). The M III cages were also supplied by Becker & Co. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy. The cages with the test animals were arranged on the racks in such way that uniform experimental conditions (ventilation and light) were ensured. Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, Germany (floor area about 800 cm2) and small amounts of absorbant material.
- Diet (e.g. ad libitum): The food used was ground Kliba maintenance diet mouse/rat "GLP", supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): from water bottles available ad libitum (except during the fasting period and measurement of motor activity).
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For preparation of the administration solutions, the test substance was weighed in a graduated measuring flask (corresponding to the dose group), topped up with tap water and dissolved by shaking.

The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals that took into account the stability of the test substance preparation.

The pH-value of each test substance preparation was measured before dosing. All dosing solutions were in the pH range of 6.0 to 7.5.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical studies were carried out in compliance with GLP.
Method: Potentiometric titration
Discussion of results: Assuming a content of the test item of 100% at 0h, at least approximately 94% of the test item were stable in the vehicle for at least 11 days at ambient temperature.
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period and 4 days of lactation in females.
Frequency of treatment:
Daily at the same time in the morning (exception: no administration to animals being in labor).
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected as it is considered to be the route whereby the majority of exposure would occur.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- At least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined:
1. abnormal behavior during "handling", fur, skin, salivation, nose discharge, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
- In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (day 0 post coitum) and on days 7, 14 and 20 post coitum.
- Females with litter were weighed on the day of parturition (day 0 post partum) and on day 4 post partum.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
Food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period
- Food consumption of the F0 females with evidence of sperm was determined on days 0, 7, 14 and 20 post coitum
- Food consumption of F0 females, which gave birth to a litter was determined on days 0 and 4 post partum.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Blood from 5 F0 males was sampled under Isoflurane anesthesia on study day 31
- Blood from 5 F0 females was sampled under Isoflurane anesthesia on study day 49 followed by necropsy of all female animals and their pups under CO2 anesthesia
- Following parameters were determined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes

CLINICAL CHEMISTRY: Yes
- Blood from 5 F0 males was sampled under Isoflurane anesthesia on study day 31
- Blood from 5 F0 females was sampled under Isoflurane anesthesia on study day 49 followed by necropsy of all female animals and their pups under CO2 anesthesia
- Following parameters were determined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
- With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the urine constituents were determined semiquantitatively using test strips and a reflection photometer. The specific gravity was determined using an urine refractometer. The sediment was evaluated microscopically. The following examinations were carried out: volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on study day 28, a functional observational battery and motor activity measurement were carried out in the first five male animals per group.
- Battery of functions tested: motor activity, sensorimotor tests/reflexes, home cage observations, open field observations
- Functional observation battery: performed in the first five animals per sex and group at the end of the administration period starting at about 10.00 a.m. The test started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations performed were performed at random.

Sacrifice and pathology:
Necropsy: The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Organ weights: the weights of the following organs were determined in all animals sacrificed at scheduled dates: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart
Organ/Tissue fixation: the following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumbar cord), sciatic nerve, pituitary gland, salivary glands, thyroid glands, parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (axillar and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), eyes with optic nerve, femur with knee joint, skin and skeletal muscle.
Histopathology: all gross lesions, trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, oviducts, uterus, vagina, prostate glands, seminal vesicles, coagulation glands, thymus, lymph nodes, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, bone marrow.
Statistics:
Food consumption, body weight and body weight change: Dunnet's test (two-sided) for the hypothesis of equal means)
Feces, rearing, grip strength of forelimbs and hindlimbs, landing footsplay test, motor activity: non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Clinical pathology parameters, except reticulocytes and differential blood count: non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using wilcoxon-test (two-sided) for the equal medians.
Urinalysis, except volume, color, turbidity and specific gravity: pairwise comparison of each dose group with the control group using Fisher's exact test for the hypothesis of equal proportions
Weight of anesthesized animals and absolute and relative organ weights: Kruskal-Wallis and Wilcoxon test
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations for signs of general toxicity revealed post-dose salivation in 5 high dose males and all high dose females. Moreover, urine discoloration was observed for all mid and high dose rats. Both findings are considered to be without toxicological relevance and are, if seen in isolation, not assessed as being adverse.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increased bilirubin and triglyceride and decreased protein and albumin values in the F0 females of the high dose group were observed. Clinical pathology examinations suggested a slightly impaired liver function of the high dose females. The production of albumin and some other proteins, which are synthesized in the liver cells, was probably decreased. Additionally, the ability of the hepatocytes to eliminate bilirubin from the body by conjugation was probably diminished. Moreover, higher triglyceride levels were found in the blood of these dams. The signs indicating a disturbed liver function are considered as adverse substance-induced effects. However, this original findings are challenged. Taking into account all the other information available on this study (no histophatological correlation as liver observations are similar to the controls), these changes seem the result of an adaptive process rather than an adverse effect. This assumption is also supported by the fact that no changes have been observed in ALT, AST, ALP and GCP levels.
See section 'Any other information on results' for more details.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced urine volumes with subsequently increased specific gravity were excreted by female rats of the top dose group (1000 mg/kg body weight/day). It is likely, that this was caused by a decreased water intake. It is concluded that the changes in urinalysis are not caused by a direct toxic effect of the test compound on the kidney and are not considered adverse in nature. This assessment is supported by the fact, that kidney weights as well as gross and histopathological evaluations of this organ did not give any indications for substance-induced alterations in the high dose females.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
no

Blood chemistry (mean+/-SD (number of animals)) - statistical method: Kruskal-Wallis + Wilcoxon-test, Two sides, *p<0.05, ** p<0.010

 

 Male         

Female          

   0 mg/kg bw  100 mg/kg bw  300 mg/kg bw  1000 mg/kg bw  0 mg/kg bw  100 mg/kg bw  300 mg/kg bw  1000 mg/kg bw
 ALT µkat/l  0.49 +/-0.10 (5)  0.52 +/-0.09 (5)  0.49 +/-0.06 (5) 0.51 +/-0.11 (5)   0.46 +/-0.24 (5)  0.31 +/-0.07 (5)  0.30 +/-0.05 (5)  0.38 +/-0.09 (5)
 AST µkat/l  1.31 +/-0.30 (5)  1.46 +/-0.22 (5)  1.42 +/-0.23 (5)  1.32 +/-0.11 (5)  1.52 +/-0.18 (5)  1.38 +/-0.31 (5)  1.19 +/-0.20 (5)  1.20 +/-0.19 (5)
 ALP µkat/l  1.52 +/-0.27 (5)  1.46 +/-0.17 (5)  1.32 +/-0.17 (5)  1.53 +/-0.10 (5)  0.57 +/-0.16 (5)  1.66 +/-2.14 (5)  0.58 +/-0.07 (5)  0.71 +/-0.20 (5)
 SGGT nkat/l  0 +/-0 (5)

 0 +/-0 (5)

 0 +/-0 (5)  0 +/-0 (5)  0 +/-0 (5)  19 +/-42 (5)  0 +/-0 (5)  0 +/-0 (5)
 INP mmol/l  1.83 +/-0.06 (5)  1.91 +/-0.18 (5)  1.88 +/-0.06 (5)  2.06 +/-0.08** (5)  1.55 +/-0.15 (5)  1.64 +/-0.12 (5)  1.48 +/-0.17 (5)  1.74 +/-0.15 (5)
 Bilirubine µmol/l  1.96 +/-0.45 (5)  2.04 +/-0.23 (5)  2.13 +/-0.47 (5)  2.70 +/-0.62 (5)  2.51 +/-0.29 (5)  2.88 +/-0.28 (5)  2.76 +/-0.39 (5)  3.18 +/-0.43* (5)
 Protein 64.80 +/-1.86 (5)   63.86 +/-1.61 (5)  62.99 +/-2.20 (5)  62.54 +/-0.78 (5)  69.39 +/-1.43 (5)  67.35 +/-4.27 (5)  68.07 +/-1.71 (5)  64.12 +/-1.53** (5)
 Albumin  37.65 +/-0.98 (5)  36.59 +/-0.58 (5)  36.82 +/-1.41 (5)  36.57 +/-0.61 (5)  40.96 +/-0.87 (5)  38.99 +/-3.73 (5)  40.10 +/-0.77 (5)  37.50 +/-0.85** (5)
 Globulin  27.15 +/-1.04 (5)  27.27 +/-1.07 (5)  26.17 +/-1.48 (5)  25.97 +/-0.76 (5) 28.43 +/-1.10 (5)   28.53 +/-1.06 (5)  27.97 +/-1.07 (5)  26.62 +/-1.30 (5)
 Triglycerides  0.66 +/-0.45 (5)  0.52 +/-0.16 (5)  0.58 +/-0.34 (5)  0.55 +/-0.18 (5)  0.32 +/-0.05 (5)  0.32 +/-0.04 (5)  0.43 +/-0.10 (5)  0.42 +/-0.06* (5)
 Cholesterol  1.40 +/-0.19 (5)  1.74 +/-0.36* (5)  1.72 +/-0.14* (5)  1.47 +/-0.18 (5)  1.49 +/-0.22 (5)  1.55 +/-0.18 (5)  1.61 +/-0.32 (5)  1.57 +/-0.33 (5)
Conclusions:
The NOAEL for general, systemic toxicity of the substance is 1000 mg/kg body weight/day for the F0 generation as no adverse effects were observed in F0 rats of both sexes.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
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:
Critical effects observed:
not specified
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:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

90-day repeated dose toxicity study

A 90-day repeated dose toxicity study was performed in rats. Male and female animals were dosed via oral gavage at dose levels of 0, 10, 30 or 100 mg/kg/day, according to OECD guideline 408 (Allt, 2019; K1, GLP).

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day (highest dose tested).

No treatment related effect was seen in mortality, clinical signs, behaviour, functional performance, sensory activity, body weight, food and water consumption, ophthalmology, hematology, blood chemistry organ weight.

Three unscheduled deaths occurred during the study.

- One female treated with 100 mg/kg bw/day were 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. This female had gaseous distension in the stomach, red contents in the ileum and jejunum and dark lungs, focal, minimal hyperplasia in the non-glandular stomach. Examination of the nasopharynx proved inconclusive.

- 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. Gaseous distention in the cecum, colon and ileum and red lungs were observed, as well as 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 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. Necropsy revealed gaseous distention in the cecum, duodenum, ileum, jejunum and stomach, red lungs and enlarged adrenals. Ulceration with necrosis/inflammation in the trachea (marked) and mild inflammatory change in the lungs were observed ; the tracheal change is considered to be the cause of the poor clinical condition.

Effects such as increased salivation and noisy respiration, hunched posture and lethargy were observed; observations of this nature are commonly observed following the oral administration of an unpalatable or irritant test item formulation and represent difficulties dosing particular animals rather than evidence of true systemic toxicity.

The terminal necropsy on other animals revealed that 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.

Ulceration of the trachea with necrosis/inflammation was present in one male treated with 100 mg/kg bw/day.

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 and used as key value for the chemical safety assessment.

Combined repeated dose toxicity with screening reproduction/developmental study

BASF (2007, K2) investigated the oral toxicity of the test substance after repeated exposure in male/female Wistar rats. The test has been performed according the guideline OECD 422 (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test) with the hydrochloride salt of 3-methoxypropylamine. For repeated dose tests, the salt of the substance under evaluation was used in order to counteract the corrosive properties of the substance. The salt was not considered to have any impact on the hazardous effects of the substance. The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period and 4 days of lactation in females. 12 males and 12 females (F0 animals) received daily (except animals being in labor) doses of 100, 300 and 1000 mg/kg bw/day. Clinical observations for signs of general toxicity revealed post-dose salivation in 5 high dose males and all high dose females. Moreover, urine discoloration was observed for all mid and high dose rats. Both findings are considered to be without toxicological relevance and are, if seen in isolation, not assessed as being adverse. The test compound did not affect food consumption and body weight gain at dose levels as high as 1000 mg/kg body weight/day. Detailed clinical examinations in an open field, detailed observations in a functional observational battery (FOB) and measurements of motor activity did not reveal any indications of test substance-induced effects in low, mid and high dose rats. Clinical pathology examinations suggested a slightly impaired liver function of the high dose females. The production of albumin and some other proteins, which were synthesized in the liver cells, was probably decreased. Additionally, the ability of the hepatocytes to eliminate bilirubin from the body by conjugation was probably diminished. Moreover, higher triglyceride levels were found in the blood of these dams. The signs indicating a disturbed liver function were considered as adverse substance-induced effects. Reduced urine volumes with subsequently increased specific gravity were excreted by female rats of the top dose group (1000 mg/kg body weight/day). It is likely, that this was caused by a decreased water intake. It is concluded that the changes in urinalysis have not been caused by a direct toxic effect of the test compound on the kidney and were not considered adverse in nature. This assessment was supported by the fact, that kidney weights as well as gross and histopathological evaluations of this organ did not give any indications for substance-induced alterations in the high dose females. No treatment-related effects were noted in the clinical pathology investigations of the substance-treated males (100, 300 and 1000 mg/kg body weight/day) and the low and mid dose females (100 and 300 mg/kg body weight/day). The test substance did not cause adverse effects regarding terminal body and organ weights, macroscopic evaluation at necropsy or histopathological evaluation. The NOAEL for general, systemic toxicity of the test substance was 300 mg/kg body weight/day for the F0 parental females based on some indications of disturbed liver function. The NOAEL for F0 parental males was found to be 1000 mg/kg body weight/day. The systemic NOAEL of 300 mg/kg for females as originally determined in this test could be challenged. This value was proposed for females based on minimal clinical chemistry changes observed (total proteins, triglycerides and albumin). Taking into account that there was no histopathological correlation (livers of the high dose females were similar to controls), these changes seemed to be the result of an adaptive process rather than being an adverse effect. This assumption was also supported by the fact that no changes have been observed in ALT, AST, ALP and GCP levels. Therefore, a NOAEL of 1000 mg/kg body weight/day was proposed.

Repeated dose toxicity - dermal/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.

 

Justification for classification or non-classification

According to the criteria of the CLP Regulation and based on the study results, the substance should not be classified for STOT repeated exposure.