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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Version 2018
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tributylamine
EC Number:
203-058-7
EC Name:
Tributylamine
Cas Number:
102-82-9
Molecular formula:
C12H27N
IUPAC Name:
N,N-dibutylbutan-1-amine
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 151-191 g, females: 108-143 g
- Fasting period before study:
- Housing: Polycarbonate cages ( Makrolon type IV, height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group together. During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. The rooms in which the animals were kept was documented in the study records. Cages were arranged on the racks according to a Latin-square model. Colour-coded cage card indicating at least Test Facility Study No., group, animal identification number(s).
- Diet: Ad libitum, except during designated procedures
- Water: Freely available to each animal via water bottles.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days before the commencement of dosing.

DETAILS OF FOOD AND WATER QUALITY:
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. Phytoestrogen content was below 350 mg total genestein equivalents (TGE)/kg diet (mean: 281.4 mg TGE/kg diet).

Water: Municipal tap water. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that could interfere with the outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Targeted: 18 to 24°C. Actual mean: 20 to 21°C
- Humidity (%): Targeted: 40 to 70%. Actual mean: 49 to 74%
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES
From: 22 JUN 2020 To: 19 OCT 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected at request of ECHA (see Decision No.: CCH-D-2114440251-64-01/F), due to insufficient stability of the test item in diet, as determined in a pre-study, oral application via gavage was selected.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The highest dose formulation was used as stock for the other groups. Vehicle was weighed, hereafter test item was pipetted in the vehicle and formulations (w/w) were homogenized for at least 15 minutes to visually acceptable levels. Headspace was minimized and a cap with septum or a screw cap with parafilm was used to close of the vials. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in a refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5, 15 and 45 mg/mL for low, mid and high dose. 30 mg/mL for females from day 50 onwards.
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected at week 1, 6 and 13 for analysis of concentration and homogeneity using an Acquity UPLC system.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%), except for Group 2 prepared in week 6 which was slightly above the target concentration (i.e. 112% of target concentration)
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%).

A small response at the retention time of the test item was observed in one of the chromatograms of the Group 1 formulation prepared for use in Week 1 and 13. It was considered not to derive from the formulation since it was an estimated value calculated using the mean response factor of the lowest calibration solution and the maximum contribution calculated was 0.13% and 0.57%, respectively. The maximum contribution of the test item in the Group 1 formulations was low and had therefore no effect on study validity. In addition, the small response in Week 13 was considered to derive from a “carry over” effect, since a similar response was observed in the blank solvent (acetonitrile). No test item was detected in Group 1 formulation for use in Week 6.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
225 mg/kg bw/day (nominal)
Remarks:
Females were dosed at 150 mg/kg/day from Day 50 of treatment onwards, due to premature deaths in this group.
No. of animals per sex per dose:
10 males and 10 females. Additional 5 males and 5 females for each recovery group (control and high dose)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 28 day repeated dose toxicity study with oral exposure of Tri-n-butylamine in rats and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Fasting period before blood sampling for clinical biochemistry: Overnight with a maximum of 24 hours

- Rationale for selecting satellite groups: Control group and high dose recovery groups were included to provide the possibility to check for the reversibility of the effects on liver weight observed in the DRF

- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily; from Day 1 to Day 48 at 0 to 1-hour post-dose during dosing. Starting from Day 49 at least twice daily at 0 to 1-hour post-dose and 2.5 to 3.5 hours post-dose

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly; from at least Day 1 and throughout the study

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during pretreatment period and once during week 13.
- Dose groups that were examined: All animals, including spares, during pretreatment period. Control and high dose group and the respective recovery groups during week 13. No examination at the end of the recovery period as no test item-related effects were seen at the End of Treatment.

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood: On days of scheduled or unscheduled necropsy
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes, overnight
- How many animals: All animals from main study and recovery phase were examined
- Parameters: White blood cell Count (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute) , Large unstained cells (LUC) (absolute), Red blood cell count (RBC) Reticulocytes (absolute), Red blood cell distribution width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH) , Mean corpuscular hemoglobin concentration (MCHC), Platelets


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On days of scheduled or unscheduled necropsy
- Animals fasted: Yes, overnight
- How many animals: All animals from main study and recovery phase were examined
- Parameters: Prothrombin time (PT), Fibrinogen, Activated partial thromboplastin time (APTT), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline Phosphatase (ALP), Total protein, Albumin, Bile Acids , Total bilirubin, Urea, Creatinine, Glucose, Cholesterol, HDL and LDL Cholesterol , Sodium, Potassium, Chloride, Calcium, Inorganic phosphate (Inorg. Phos), Triiodothyronine (T3) , Thyroxine (T4) , Thyroid-stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period. The first 5 animals per sex per group during Week 12. These tests were performed after clinical observations. No test item-related findings were noted in Week 12 and therefore assessment in Week 17 for all Recovery animals was not performed.
- Dose groups that were examined: All groups.
- Battery of functions tested:
hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
fore- and hind-limb grip strength were recorded as the mean of three measurements.
locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER:
- Estrous stage determination: on the day of necropsy, a vaginal smear was taken to determine the stage of estrous from all Main Study and Recovery females that survived until the scheduled necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Main study and Recovery animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Organs weighed at necropsy: Brain; Epididymis; Gland, adrenala; Gland, Pituitary; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Heart; Kidney; Liver; Ovary; Spleen; Testis; Thymus; Uterus/cervix


HISTOPATHOLOGY: Yes
Fixed tissues: Animal identification, Artery, aorta; Body cavity, nasal; Bone, femur; Bone marrow; Bone, sternum; Brain; Cervix; Epididymisa; Esophagus; Eyea; Gland, adrenal; Gland, clitoral; Gland, harderiana; Gland, lacrimal; Gland, mammary ; Gland, parathyroid; Gland, pituitary; Gland, preputial; Gland, prostate; Gland, salivary; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue; Heart; Kidney;
Large intestine, cecum ; Large intestine, colon; Large intestine, rectum; Larynx; Liver; Lung; Lymph node, mandibular; Lymph node, mesenteric; Muscle, skeletal; Nerve, optic; Nerve, sciatic; Nerve, tibial; Ovary; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Testisa; Thymus; Tongue; Trachea; Urinary bladder; Uterus; Vagina

Microscopy evaluation: Fixed tissues (except animal identification, nasal body cavity, femur bone, clitoral gland, harderian gland, lacrimal gland, preputial gland, salivary gland parotid, larynx, tibial nerve and tongue) were microscopically evaluated. A peer review on the histopathology data was performed by a second pathologist.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
The following tests were performed :

Parametric/Non-Parametric Tests (Body Weight; Body Weight Gains; Hematology Variables; Coagulation Variables; Clinical Chemistry Variables; FOB Quantitative Variables; Organ Weights; Organ Weight relative to Body Weight):
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

For parametric tests with data from arena observations, functional observations:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-parametric tests with data from arena observations, functional observations:
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

Incidence (FOB Qualitative Variables): Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg/day, two males showed erected fur on Days 48, 63 and/or 64. In addition, four males treated at 225 mg/kg/day showed incidental erected fur between Days 50 and 60.
In six females at 225 mg/kg/day hunched posture was observed on Day 14. Following the dose reduction (150 mg/kg/day), hunched posture was noted in two females on Days 51 and 52.

Salivation was seen in all test item groups in a dose-related response starting on Day 4. The salivation was considered not toxicologically relevant, considering the nature and severity of the effect and its time in occurrence (i.e. after dosing). This sign was considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity.
In one female at 225 mg/kg/day (No. 86), abnormal breathing sounds were observed on Day 14. As this only occurred on one day, it was considered to be not test item-related and likely caused by the method of dosing. Any other clinical signs (skin lesions, scabs, swollen mouth) noted during the dosing period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

No clinical signs were observed during the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 225 mg/kg/day, four females (Animal Nos. 87, 98, 94 and 90) and one male (Animal No. 47) were found dead on Days 6, 15, 44, 45 and 82 of dosing, respectively.
Clinical signs observed consisted of hunched posture in Female No. 98 on Day 14 and in Male No. 47 on Days 14 and 15. Furthermore, slight to severe salivation was seen in these animals directly after dosing, with exception of Female No. 87. The salivation was considered not toxicologically relevant, considering the nature and severity of the effect and its time in occurrence (i.e. after dosing). This sign was considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity.
For body weights, no clear test item-related findings were observed in any of the premature deaths.
At necropsy and histopathological assessment, no cause of death could be found in any of these animals. In Male No. 47, red/purple foci in the mesenteric adipose tissue and thymus (also in Female No. 94), red discolored pancreas, small preputial gland and the lungs failed to collapse were observed. These findings were considered to be not test item related. Furthermore, autolysis and/or cannibalism was seen in Female Nos. 87, 90 and 98.

Based on the observed mortality in females at 225 mg/kg/day, the dose level for the female group was lowered to 150 mg/kg/day from Day 50 of treatment onwards.
No mortality occurred during the study period for the remaining dose levels.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistical significant effects on body weight and body weight gain was observed in males up to 75 mg/kg/day and in females up to 225 mg/kg/day.
Slightly lower body weight gain was observed in males treated at 225 mg/kg/day throughout the dosing period (4% less body weight than control at the end of the Dosing period). Mean body weight gains were 19%, 41% and 61% lower between Days 1-8, 57-64 and 71-78 compared to control (statistically significant), no statistically significant effects were observed during the other observation windows. No effects on body weight gain were seen during the recovery period. Effects on body weight gain did not result in statistically significant effects on body weight.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No clear effects on food consumption were observed in males up to 75 mg/kg/day and females up to 225 mg/kg/day.
Incidentally, statistically significant lower food consumption was observed in males and females between Days 1 and 8 (9% and 17% less than control respectively) and/or Days 64 and 71 (9% less than control in males) at 225 mg/kg/day and in multiple weeks in females at 75 mg/kg/day. As the food consumption was comparable with the control group for the majority of the dosing period and/or lacked a clear dose-related response, these incidental decreases in food consumption were considered to be not toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test item.
The nature and incidence of ophthalmology findings noted during the pre-treatment period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects on hematology parameters were noted in males and females up to 75 mg/kg/day.
In males at 225 mg/kg/day, a slight but statistically significant increase in red blood cells (1.07x of controls; 9.146e12/L vs. 8.509e12/L in Group 4 males and control males, respectively) and red blood cell distribution width (RDWG; 1.05x of controls; 12.65 vs. 12.07% in Group 4 males and control males, respectively) was observed. In females at 225/150 mg/kg/day, a slight decrease in mean corpuscular hemoglobin concentration was noted (MCHC; 0.98x of control, 330.0 vs. 226.6 g/L in Group 4 females and control males, respectively). These changes remained within the historical control values for this age and strain and were recovered at the end of the recovery period and were therefore considered to be not adverse and even not toxicologically relevant at the severity observed.
Remaining differences in hematology parameters, regardless of statistical significance, were considered to be not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
For details see tables below.
In males and females, higher alanine aminotransferase (ALT) activity was seen in all test-item treated groups, which were also outside the historical control range. The ALT activity remained slightly higher in males at 225 mg/kg/day at the end of the recovery period but recovered in females at 225/150 mg/kg/day. In females at 225/150 mg/kg/day, an increase in alkaline phosphatase activity was observed at the end of the dosing Period, which was also outside the historical control range, but not statistically significant. The ALP levels returned to values comparable with the vehicle group at the end of the recovery period.
In addition, a statistically significant increase in calcium concentrations was seen in males and females at 75 and 225/150 mg/kg/day. Calcium concentrations were outside historical control values for this rat strain. An increase in bile acids was observed in females at 225/150 mg/kg/day, which was outside historical control range. The changes returned to values comparable to the historical control at the end of the recovery period.
The statistically significant decrease in chloride concentration in males at all test item-groups and females at 225/150 mg/kg/day was caused by high control values and were therefore considered to be not test item-related.
The statistically significant increase in thyroxine (T4) level in males at 225 mg/kg/day at the end of the recovery period was caused by low control values and was therefore considered to be not test item related. Thyroid stimulating hormone (TSH) concentrations were lower in males and females at 25 and/or 75 mg/kg/day. These results remained well within the historical control values, lacked a dose-related response and were also not statistically significant. Therefore, these TSH changes were considered to be not test item-related.


No effects on coagulation parameters were noted in males up to 75 mg/kg/day and females up to 225/150 mg/kg/day.
The statistically significant lower prothrombin time (PT) in males at 225 mg/kg/day was considered to be of no toxicological relevance as the opposite effect would be expected in a toxicology study, because an increase in PT would relate to blood clotting issues.
Any other statistically significant changes in coagulation parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males, a higher liver weight was observed starting at 25 mg/kg/day (absolute and/or relative to body weight). After a recovery period of 28 days, the liver weights (absolute and relative to body weight) remained higher, but at a lower magnitude suggesting partial recovery. The microscopic correlate in the main males was centrilobular hepatocellular hypertrophy, there was no microscopic correlate in the recovery animals.

In females, higher liver and kidney weight were observed at 225/150 mg/kg/day. For both organ weight changes full recovery was noted after 28 days. The microscopic correlate for the increased liver weight was centrilobular hepatocellular hypertrophy. There was no microscopic correlate for the increased kidney weight.

Some organ weight differences were statistically significant when compared to the control group (Males: relative kidney, adrenal gland and testes weight Main Group; absolute heart weight Recovery Group at 225 mg/kg/day), but were considered to be the result of a test item-related effect on final body weight.

Any differences, including those that reached statistical significance were considered not to be test item-related due to the lack of dose-related pattern (Males: absolute pituitary and seminal vesicles weight Main Group at 75 mg/kg/day) or difference in estrous cycle-related stage (Females: absolute and relative ovary weight Recovery Group at 225/150 mg/kg/day).
There were no other test item-related organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Tri-n-butylamine were noted in the liver of the males starting at 25 mg/kg/day and in the liver of a single female at 225/150 mg/kg/day.
In males, a dose-dependent increased incidence in minimal centrilobular hepatocellular hypertrophy was observed starting at 25 mg/kg/day, which showed full recovery after a treatment-free period of 28 days.
In females, minimal centrilobular hepatocellular hypertrophy was observed in a single female treated with 225/150 mg/kg/day. No hepatocellular hypertrophy was observed after the recovery period of 28 days.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
See also attached documents with summary tables of results for additional details.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

The lack of relevant toxicological effects in the 5 necropsied animals which died during the dosing phase is in agreement with the findings of the DRF and MTD phase of the study. A 28 day repeated dose toxicity DRF study with oral exposure to 0, 50, 150 and 300 mg/kg/day has been performed where no mortality occurred in males, whereas one female at 300 mg/kg/day was found dead on Day 19. During the MTD phase (7 day oral exposure of female rats to 120 or 200 mg/kg/day Tri-n-butylamine) no mortality and only a slight decrease in body weight were observed. One female of the MTD high dose group revealed slight tremors on Day 3 at 1-hour post dose, but not at any other timepoint. Results summary tables and figures are included in the attachment. Main clinical findings are compiles in the tables below.

Summary Of Mean Clinical Chemistry Results And Percentage Of Change Of Control– Males

 

Main

Recovery

Dose level (mg/kg/day):

0

25

75

225

0

225

 

 

 

 

 

 

 

ALT (U/L)

65.2

83.5* (28%)

79.2 (22%)

92.3* (41%)

65.3

70.6 (8%)

Calcium (mmol/L)

2.623

2.639 (1%)

2.772* (6%)

2.885** (10%)

2.588

2.608 (1%)

Chloride (mmol/L)

107.5

105.8*(-2%)

105.4** (-2%)

103.4** (-4%)

109.6

107.6 (-2%)

T4 (ng/mL)

38.78

38.01 (-2%)

38.77 (0%)

37.33 (-4%)

33.48

39.24* (+17%)

TSH (mU/L)

0.2042

0.0946 (-54%)

0.0608 (-70%)

0.1340 (-34%)

0.1510

0.2390 (58%)

*: P<0.05, **: P<0.01

 

Summary Of Mean Clinical Chemistry Results And Percentage Of Change Of Control– Females

 

Main

Recovery

Dose level (mg/kg/day):

0

25

75

225/150

0

225/150

 

 

 

 

 

 

 

ALT (U/L)

61.3

70.5 (15%)

88.2* (44%)

86.9* (42%)

81.8

67.5 (-17)

ALP (U/L)

67.0

65.6 (-2%)

83.4 (25%)

107.2 (60%)

82.3

81.3 (-1%)

Calcium (mmol/L)

2.610

2.663 (2%)

2.746* (+5%)

2.757** (6%)

2.574

2.578 (0%)

Bile acids (umol/L)

28.21

32.58 (15%)

37.77 (34%)

88.45* (314%)

39.04

49.18 (-21%)

Chloride (mmol/L)

107.0

105.6 (-1%)

105.4 (-2%)

104.1** (-3%)

108.0

107.5 (0%)

TSH(mU/L)

0.0552

0.0483 (-12%)

0.0249 (-55%)

0.0827 (50%)

0.1274

0.0450 (-65%)

*: P<0.05, **: P<0.01

 

 


Historical Control Data Clinical Chemistry – Wistar Han

Parameter

Sex

Mean

P5

P95

Number of animals

ALT (U/L)

Males

41.4

28.6

62.3

132

Females

37.2

22.6

62.1

134

ALP (U/L)

Females

58

30

102

134

Bile acids (umol/L)

Females

19.8

8.4

39.5

123

Chloride (mmol/L)

Males

103

100

105

132

Females

104

101

1074

134

Calcium (mmol/L)

Males

2.56

2.42

2.67

132

Females

2.61

2.47

2.72

134

T4(ng/mL)

Males

51.1

34.4

71.0

182

TSH (mU/L)

Males

0.184

0.035

0.541

182

Females

0.089

0.010

0.269

174

Applicant's summary and conclusion

Conclusions:
Administration of Tri-n-butylamine by once daily oral gavage was well tolerated in Wistar Han rats at levels of 75 mg/kg/day in males and 150 mg/kg/day in females. Test item-related mortalities were present in one male and four females at 225 mg/kg/day, which were considered to be adverse. The other observed findings were considered to be not adverse.
Based on the adverse effect (early death) observed in animals treated at 225/150 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for Tri-n-butylamine was established to be 75 mg/kg/ day.
Executive summary:

The objective of this study was to determine the potential toxicity of Tri-n-butylamine, when given orally by gavage 90 days to Wistar Han rats and to evaluate the potential reversibility of any finding. In addition, a No Observed Adverse Effect Level (NOAEL) was evaluated.

The study design was as follows:

Group No.

Test Item Id.

Dose Level

(mg/kg/day)

 

Dose Volume (mL/kg)

Dose Concentration (mg/mL)

Subsets

Number of Animals

Males

Females

1

Control

0 (Vehicle)

5

-

Main

10

10

Rec.

5

5

2

Tri-n-butylamine

25

5

5

Main

10

10

3

75

5

15

Main

10

10

4M

225

5

45

Main

10

-

Rec

5

-

4F

225/1501

5

45/301

Main

-

10

Rec.

-

5

Id. = Identification; Rec. = Recovery

Note: Dose levels were based on the results of the dose range finding study (Test Facility Study No. 20209563).

1Females were dosed at 150 mg/kg/day from Day 50 of treatment onwards, due to premature death in the treatment group.

Chemical analyses of formulations were conducted in Weeks 1, 6 and 13 to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observation tests, body weights, food consumption, ophthalmology, estrus stage determination, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.

Test formulations prepared were considered homogenous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

At 25 and 75 mg/kg/day, a test item-related non-adverse centrilobular hepatocellular hypertrophy in the liver in males, withcorrelatingincreased liver weights (absolute liver weights 118% and 110% and relative liver weight 113% and 115% of control respectively) and alanine aminotransferase activity (ALT, 128% and 122% of control respectively), was observed. The increase ALT activity was only to a small extent and not dose dependent. In addition, at 75 mg/kg/day, a test item-related non-adverse increase in calcium concentration was seen in females (105% of control).

At 225 mg/kg/day, four females and one male were found dead during the study period. Except for hunched posture in two of these animals, no clear signs of systemic toxicity were observed prior to their death. Despite of the absence of any relevant toxicological effects prior to mortality, these spontaneous deaths were considered to be test item-related and adverse. After lowering the dose level in females to 150 mg/kg/day, no test item-related mortality occurred.
Slightly lower test item-related body weight gain was observed in males throughout the dosing period only (4% lessbody weightthan the control group at the end of the Dosing period), which was considered to be not adverse at the slight severity observed.
At clinical chemistry, a non-adverse test item-related increase in alkaline phosphatase activity in females (160% of control, but not statistically significant), calcium concentration in males and females (110% and 106% of control, respectively) and bile acids concentration in females (314% of control) were observed. These changes fully recovered at the end of the recovery period. In absence of any histopathological correlation and as these changes fully recovered at the end of the recovery period, they were considered to be not adverse.

At histopathological examination, atest item-related non-adverse centrilobular hepatocellular hypertrophy in males and in a single female with correlating higher liver weights (absolute liver weight 120% and 119% and relative liver weights 131% and 116% of control respectively) and ALT activity (142% and 142% of control, respectively) was observed. For males, partial recovery was seen for liver weight (absolute and relative liver weights119%and 114% of control, respectively) and ALT activity (108% of control) as well as total recovery of the centrilobular hepatocellular hypertrophy at the end of the recovery period, while for females, full recovery of liver effects was observed. Based on the minimal degree and absence of any degenerative or inflammatory changes and only very small (not dose-dependent) correlating increase in ALT, the hypertrophy was considered to be not adverse. In addition, a test item-related and non-adverse higher kidney weight was observed in females (absolute and relative kidney weights 120% and 117% of control, respectively), which recovered fully at the end of the recovery period and was without a histopathological correlation.

There were no relevant test item-related or toxicologically significant changes noted in any of the remaining parameters investigated in this study (i.e. clinical signs, food consumption, functional observations, ophthalmoscopy, hematology, coagulation parameters and gross pathology).

In conclusion, administration of Tri-n-butylamine by once daily oral gavage was well tolerated in Wistar Han rats at levels of 75 mg/kg/day in males and 150 mg/kg/day in females. Testitem-related mortalities were present in one male and four females at 225 mg/kg/day, which were considered to be adverse. The other observed findings were considered to be not adverse.

Based on the adverse effect (early death) observed in animals treated at 225/150 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for Tri-n-butylamine was established to be 75 mg/kg/day.