Registration Dossier

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
Study period:
22NOV2012 - 10APR2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline and GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
- Name of test material (as cited in study report): C16-18, C18-unsaturated-alkyl dipropylene triamine
- Trade name: Triameen T
- CAS no.: 1219458-14-6
- Lot/batch No.: S001096
Previously this substance was identified as Tallow dipropylenetriamine with CAS number 61791-57-9
- Substance type: organic
- Physical state: white paste
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark under nitrogen
- Expiration date of the lot/batch: 29MAR2017

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 140 - 164g (males) and 96 - 135g (females)
- Housing: group housing of 5 animals per sex in Macrolon cages, individual housing in a Hi-temp polycarbonate cage during locomotor activity monitoring
- Diet: pelleted rodent diet, ad libitum (except during motor activity measurements and overnight before sacrifice)
- Water: tap water, ad libitum (except during motor activity measurements and overnight before sacrifice)
- Acclimation period: >5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22NOV2012 To: 10APR2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle:
Based on trial formulations performed at WIL Research Europe and on information provided by the sponsor.
- Concentration in vehicle:
0, 0.5, 2, 8 and 32 mg/ mL
- Amount of vehicle (if gavage):
5mL/kg

From Day 1 to Day 28 formulations (w/w) were prepared daily within 5 hours prior to dosing. From Day 29 onwards, formulations (w/w) were prepared for a maximum of 8 days prior to dosing. For formulations prepared for dosing between Days 1 and 7, the test substance was liquefied prior to weighing by heating in a water bath up to 57.6⁰C for 2 hours and 32 minutes. From Day 8 onwards, the test substance was not heated but scraped out of the container instead. Formulations were homogenized to a visually acceptable level. In order to obtain homogeneity, the formulations were heated in a water bath up to 59.8⁰C for a maximum of approximately 15 minutes. The formulations were allowed to cool down to a temperature of maximally 36.1⁰C prior to dosing. Adjustment was made for density of the test substance and specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In week 1, 26 samples were collected. In week 6, 18 samples were collected. Group 1 was the control group. Group 2, 3, 4 and 5 were dosed at 2.5, 10, 40 and 160 mg/kg bw/day, respectively. The samples of the control group, Group 3 and Group 4 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 2 and 5 were taken in duplicate from the top, middle and bottom position of the container. In addition, stability samples were taken from formulation groups 2 and 5 in week 1, in 4-fold at the middle position of the container. A pair of them were stored for 5 hours at room temperature under normal laboratory light conditions and then placed on dry ice under nitrogen, and the other pair of stability samples were stored for 8 days under nitrogen at room temperature immediately after sampling. In week 13, 16 samples were collected. Group 1 was the control group, Group 2, 3 and 4 were dosed at 2.5, 10 and 40 mg/kg bw/day, respectively. The samples of the control group and Group 3 were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 2 and 4 were taken in duplicate from the top, middle and bottom position of the container.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
once daily, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2.5, 10, 40 and 160 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 14-day dose range finding study.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, more often if required by health status.

FOOD CONSUMPTION:
- Weekly

WATER CONSUMPTION:
- Subjective appraisal (no quantitative investigation)

OPHTHALMOSCOPIC EXAMINATION: Yes
- At pretest : All animals (including spare animals)
- At Week 11: All surviving animals dosed at 160 mg/kg bw/day
- At Week 13: All animals dosed with 0or 40 mg/kg bw/day

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after necropsy (or after sacrifice in extremis)
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after necropsy (or after sacrifice in extremis)
- Animals fasted: Yes
- How many animals: All

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: after dosing at no specific time point, but within a similar time period after dosing for the respective animals (all animals dosed with 0 - 40 mg/kg bw/day in Week 12, and all surviving animals at 160 mg/kg bw/day in Week 11
- Battery of functions tested: hearing ability and locomotor activity, pupillary reflex, static righting reflex and grip strength

OTHER:
- All females had a daily vaginal lavage from Day 72 up to and including Day 92 females exposed to 0, 2.5, 10 or 40 mg/kg bw/day (or up to Day 77 for surviving females exposed to 160 mg/kg bw/day) to determine the stage of estrous.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the data:
− If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
− The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
− The Fisher Exact-test was applied to frequency data.
− The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
At 160 mg/kg bw/day, 3/10 males and 5/10 females were killed in extremis between Days 57 and 73 (with one early sacrifice on Day 18). Based on the high mortality rate and signs of deteriorated health condition of animals at 160 mg/kg bw/day, the remaining animals were sacrificed on Day 77. At 40 mg/kg bw.day, 3/10 males were killed in extremis in the last week of treatment (Day 86 or 88). At 10 mg/kg bw/day, 1 male was found dead in Week 12 (Day 84). No mortality occurred at 2.5 mg/kg. At 160 mg/kg bw/day, swelling of the snout, abdomen, forelegs and/or hindlegs was seen in most males and females from Week 9 onwards, and abdominal swelling was also noted for the male rat that died early on in the treatment phase in Week 3. All males and females showed a hunched posture from Week 2 (males) or Week 4 (females) onwards. Further signs at the high dose included breathing difficulties, and at lower incidence, uncoordinated movements, lethargy, piloerection, pallor, chromodacryorrhoea and a lean appearance (primarily in the second week of treatment). At 40 mg/kg bw/day, swelling of the hindlegs and of the area surrounding the testicles was seen at the end of treatment in 4/10 males (three of which were sacrificed) in combination with an abnormal and/or hunched posture, and abnormal gait. One female at 40 mg/kg bw/ daywas noted with swelling of the abdomen on a few days in Week 12 only, and two females displayed a hunched posture from Week 12 onwards. At 10 mg/kg bw/day, one female showed a transient swelling of the hindleg in Week 2 of treatment, and no further changes were seen in this animal. At 2.5 mg/kg bw/day, no clinical signs were noted.
Excessive salivation seen after dosing in a dose-related manner from 10 mg/kg onwards was considered to be a physiological response rather than a sign of systemic toxicity and may be related to irritancy/taste of the test substance. Other clinical signs, including scabs and alopecia, 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. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
At 160 mg/kg bw/day, body weights and body weight gain were lower than controls in males from Week 2 onwards, achieving a level of statistical significance on most occasions. Group mean body weight after 11 weeks of treatment was approximately 23% lower than control mean weight. Females showed a normal body weight development. At 40 mg/kg bw/day, a statistically significantly lower body weight (gain) was noted in males from Week 11 onwards. Group mean body weight at the end of treatment was approximately 11% lower than control mean weight. Females showed a normal body weight development. At 2.5 and 10 mg/kg bw/day, body weights were similar to controls throughout treatment. No toxicological relevance was ascribed to the slightly higher body weight or body weight gain on one occasion in females at 40 and 160 mg/kg bw/day, as these variations did not show a relation to the dose and duration of treatment.

FOOD CONSUMPTION
At 160 mg/kg bw/day, a lower food consumption was noted for males throughout treatment, achieving a level of statistical significance on most occasions. When corrected for body weight (relative food consumption), these changes were less apparent and achieved a level of statistical significance on a few occasions only. Relative food intake of females at 160 mg/kg bw/day appeared slightly lower in week 10 and 11 (without statistical significance). At 40 mg/kg bw/day, a statistically significant lower food consumption was noted for males from Week 12 onwards. At 2.5 and 10 mg/kg bw/day, food intake was similar to control levels. No toxicological relevance was ascribed to a slightly higher food consumption of females at 40 mg/kg bw/day in Week 1, as this variation did not show a relation to the dose and duration of treatment.

WATER CONSUMPTION
No unexpected observations noted.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest and/or at end of scheduled treatment consisted of (multi-)focal corneal opacity, focal corneal oedema, pallor of the retina and haemorrhage from the hyaloid vessel. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain.

HAEMATOLOGY
The following statistically significant changes in haematology parameters distinguished treated animals from control animals:
− Higher total white blood cell counts (WBC) in males and females at 40 and 160 mg/kg bw/day,
− Higher relative neutrophil counts in males at all doses and females at 10, 40 and 160 mg/kg bw/day,
− Lower relative lymphocyte counts in males and females at 10, 40 and 160 mg/kg bw/day,
− Prolonged prothrombin time (PT) in males at 160 mg/kg and females at 40 and 160 mg/kg bw/day,
− Higher platelet counts in males and females at 160 mg/kg bw/day.
Haematological changes in the male at 160 mg/kg bw/ day that was sacrificed in extremis in Week 8 primarily consisted of white blood cell changes similar to those encountered for animals that were sacrificed in Week 11 of the study.

The statistically significant changes in red blood cell parameters at 160 mg/kg bw/day in males and females were primarily due to one male and one female, and means remained (essentially) within the range considered normal for rats of this age and strain. These changes at 160 mg/kg bw/day, as well as changes in red blood cell parameters at lower dose levels were therefore considered not adverse in nature and consisted of:
− Lower red blood cell counts in females at 160 mg/kg bw/day,
− Higher reticulocyte counts in males at 160 mg/kg bw/day (mainly due to one high value for one male and not statistically significant)
− Higher red cell distribution width (RDW) in males at 160 mg/kg (mainly due to one high value for onemale),
− Lower haemoglobin and haematocrit levels in males at 160 mg/kg and in females at 10, 40 and 160 mg/kg bw/day,
− Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values in males at 40 and 160 mg/kg bw/day and in females at 40 mg/kg bw/day,
− Lower mean corpuscular haemoglobin concentration (MCHC) in males at 160 mg/kg bw/day and females at 40 and 160 mg/kg bw/day.
The lower relative monocyte, eosinophil and basophil counts in males and/or females at 40 and/or 160 mg/kg bw/day were due to the higher white blood cell counts, as absolute values remained essentially similar to control levels. The shortened activated partial thromboplastin time (APTT) in females at 160 mg/kg bw/day was considered not toxicologically relevant as the mean was only just outside the range considered normal for rats of this age and strain, and since the opposite effect (i.e. a prolonged time) would be expected in case of target organ toxicity.
Any further statistically significant changes in haematological parameters were also considered to be of no toxicological significance as they occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain. These changes consisted of a higher relative monocyte count in females at 10 mg/kg bw/day, higher reticulocyte count in females at 40 mg/kg bw/day, and higher activated partial thromboplastin time in females at 2.5 mg/kg bw/day.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
− Higher alanine aminotransferase activity in males and females at 160 mg/kg bw/day,
− Higher aspartate aminotransferase activity level in females at 160 mg/kg bw/day,
− Higher alkaline phosphatase activity level in females at 160 mg/kg bw/day,
− Lower total protein level in males at 160 mg/kg bw/day (not statistically significant) and females at 40 and 160 mg/kg bw/day,
− Lower albumin level in males and females at 40 and 160 mg/kg bw/day,
− Lower total bilirubin level in males at 40 and 160 mg/kg bw/day and females at 160 mg/kg bw/day,
− Lower urea level in females at 160 mg/kg bw/day,
− Lower creatinine level in males and females at 160 mg/kg bw/day (not statistically significant for females),
− Lower glucose level in males at 160 mg/kg bw/day,
− Lower cholesterol level in males at 40 and 160 mg/kg bw/day,
− Higher potassium level in males and females at 160 mg/kg bw/day,
− Lower chloride level in males and females at 160 mg/kg bw/day,
− Lower calcium level in females at 160 mg/kg bw/day,
− Higher inorganic phosphate level in males at 10, 40 and 160 mg/kg bw/day.
Clinical biochemistry changes in the male at 160 mg/kg that was sacrificed in extremis in Week 8 primarily consisted of higher aspartate and alanine aminotransferase activity, higher potassium and inorganic phosphate level, and lower albumin, total bilirubin and cholesterol level. Any other statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a dose-related trend-related distribution and remained within the range considered normal for rats of this age and strain. These changes included higher sodium and chloride level in males at 10 mg/kg, higher calcium level in males at 2.5 mg/kg, and lower inorganic phosphate level in females at 2.5 mg/kg.


NEUROBEHAVIOUR
Males and females at 160 mg/kg bw/day showed a reduced motor activity (only statistically significant for males). Motor activity of animals at 40 mg/kg bw/day was similar to controls and showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period. No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed among the (surviving) animals at 40 and 160 mg/kg bw/day. A delayed pupillary reflex was noted for one of the eyes of one male at 160 mg/kg bw/day (this animal also showed a delayed static righting reflex) and one female at 40 mg/kg bw/day. Since the incidence of these findings did not show an apparent relation to the dose and other animals of these dose group showed normal reflexes, this was considered not to be of toxicological relevance. Other functional observation parameters of this animal and those determined for other animals were normal.

ORGAN WEIGHTS
The following (statistically significant) changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
− Higher liver weight in females at 40 and 160 mg/kg bw/day, and higher liver to body weight ratio in males at 160 mg/kg bw/day (mainly due one animal which also showed a higher liver weight) and females at 160 mg/kg bw/day.
− Lower thymus weight in males and females at 160 mg/kg bw/day (not statistically significant for females) and for males at 40 mg/kg bw/day, and lower thymus to body weight ratio in females at 160 mg/kg bw/day and males at 40 mg/kg bw/day,
− Higher adrenal weight and adrenal to body weight ratio in males and females at 160 mg/kg bw/day,
− Higher spleen weight and spleen to body weight ratio in males at 160 mg/kg bw/day (not statistically significant for absolute weights) and in females at 40 and 160 mg/kg bw/day,
− Lower prostate weight and prostate to body weight ratio in males at 160 mg/kg bw/day,
− Lower seminal vesicle weight and seminal vesicle to body weight ratio in males at 40 and 160 mg/kg bw/day (not statistically significant for seminal vesicle to body weight ratio in males at 40 mg/kg bw/day),
− Lower uterus weight in females at 160 mg/kg (not statistically significant).
The following statistically significant changes in organ weights were considered to be related to the lower terminal body weights and occurred without correlating morphological findings: lower brain weight in males at 160 mg/kg bw/day (and higher brain to body weight ratio in males at 40 and 160 mg/kg bw/day), lower heart weight in males at 40 and 160 mg/kg bw/day, lower thyroid weight in males at 160 mg/kg bw/day, lower kidney weights and higher kidney to body weight ratio in males at 160 mg/kg bw/day, lower epididymides weight in males at 160 mg/kg bw/day, and higher testes to body weight ratio in males at 160 mg/kg bw/day. The statistically significant higher kidney weight in females at 10 mg/kg bw/day occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. None of these changes were therefore considered to be of toxicological relevance.

GROSS PATHOLOGY
Treatment-related necropsy findings consisted of:
− Emaciation in 3/10 males and 6/10 females at 160 mg/kg bw/day.
− Mesenteric lymph nodes:
- Enlarged in 3/10 males and 4/10 females at 10 mg/kg, 9/10 males and 10/10 females at 40 mg/kg bw/day, and in 8/10 males and 9/10 females at 160 mg/kg bw/day,
- Irregular surface in 2/10 females at 160 mg/kg bw/day.
− Duodenum: Thickened in 3/10 males and 3/10 females at 40 mg/kg bw/day and 9/10 males and 8/10 females at 160 mg/kg bw/day.
− Jejunum: Thickened in 2/10 males and 4/10 females at 40 mg/kg bw/day and in 9/10 males and 8/10 females at 160 mg/kg bw/day.
− Ileum: Thickened in 1/10 males at 40 mg/kg bw/day and in 5/10 males and 3/10 females at 160 mg/kg bw/day.
− Skin:
- Subcutaneous thickening of the of hind and/or fore legs in 4/10 males at 40 mg/kg bw/day and in 3/10 males and 3/10 females at 160 mg/kg bw/day.
- Thickened/stiff joint in one male at 160 mg/kg bw/day.
− Spleen:
- Nodule/focus/foci in 2/10 males at 40 mg/kg bw/day and 2/10 males and 3/10 females at 160 mg/kg bw/day,
- Enlarged/thickened in 6/10 males and 2/10 females at 40 mg/kg bw/day and 4/10 males and 5/10 females at 160 mg/kg bw/day;
- Irregular appearance in 2/10 males at 40 mg/kg bw/dayand 2/10 males at 160 mg/kg bw/day;
- Grown together with peritoneum in 1/10 males at 40 mg/kg bw/dayand 3/10 males at 160 mg/kg bw/day.
− Liver:
- Grey/white foci in 1/10 females at 40 mg/kg bw/day and in 1/10 males and 4/10 females at 160 mg/kg bw/day;
- Enlarged in 1/10 males at 40 mg/kg bw/day and in 2/10 males and 2/10 females at 160 mg/kg bw/day;
- Pale discoloration in 1/10 males at 40 mg/kg bw/day and in 1/10 males and 2/10 females at 160 mg/kg bw/day;
- Accentuated lobular pattern in 1/10 males at 40 mg/kg bw/day and 1/10 females at 160 mg/kg bw/day.
− Thymus: Reduced in size in 1/10 males at 2.5 mg/kg bw/day, 1/10 males and 1/10 females at 40 mg/kg bw/day and in 3/10 males and 6/10 females at 160 mg/kg bw/day.
− Abdominal cavity:
- Nodule on epididymal adipose tissue in 1/10 males at 40 mg/kg bw/day,
- Epididymal adipose tissue grown together with spleen in 1/10 males at 40 mg/kg bw/day,
- Gelatinous epididymal adipose tissue in 1/10 males at 160 mg/kg bw/day.
− Adrenal glands:
- Enlarged in 3/10 males and 5/10 females at 160 mg/kg bw/day,
- Pale discoloration in 1/10 males at 40 mg/kg and in 3/10 males and 3/10 females at 160 mg/kg bw/day.
− Reproductive organs (prostate/seminal vesicle and uterus): Reduced in size in 4/10 (prostate) and 5/10 (seminal vesicles) males at 160 mg/kg bw/day and in 4/10 (uterus) females at 160 mg/kg bw/day.
The incidence of other necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in the mesenteric lymph node, duodenum, jejunum and ileum, joints, spleen, heart, liver, thymus, Peyer’s Patches, bone marrow, epididymides, prostate/seminal vesicles in males and uterus and adrenal glands in females. The findings in mesenteric lymph nodes and small intestines were considered primary targets. For the findings in joints it is unclear whether it is primary or secondary. All other organs are considered to be secondary targets, resulting from the findings in mesenteric lymph nodes and small intestines. The histopathology of the animals that were killed or that died before the end of the planned treatment period was more severe but similar in nature than the histopathology of the lower dosed rats.
Primary targets
Small intestines:
Duodenum:
- Foamy macrophages in the lamina propria of 9/10 males (3 minimal, 6 slight) and 7/10 females (6 minimal, 1 slight) at 40 mg/kg bw/day and 10/10 males (2 slight, 8 moderate) and 10/10 females (2 slight, 6 moderate, 2 marked) at 160 mg/kg bw/day.
- Granulocytic inflammation in 1/10 males (slight) at 40 mg/kg bw/day and in 7/10 males (3 minimal, 4 slight) and 4/10 females (1 minimal, 3 slight) of Group 5.
Jejunum:
- Foamy macrophages in the lamina propria of 4/10 males (minimal) and 3/10 females (minimal) at 10 mg/kg bw/day , in 10/10 males (2 minimal, 5 slight, 3 moderate) and 10/10 females (3 slight, 6 moderate, 1 marked) at 40 mg/kg bw/day and 10/10 males (4 moderate, 6 marked) and 10/10 females (3 moderate, 7 marked) at 160 mg/kg bw/day .
Ileum:
- Foamy macrophages in the lamina propria of 7/10 males (6 minimal, 1 slight) and 8/10 females (6 minimal, 2 slight) at 10 mg/kg bw/ day, in 10/10 males (3 minimal, 4 slight, 3 moderate) and 10/10 females (2 minimal, 8 slight) at 40 mg/kg bw/ day and 10/10 males (2 slight, 6 moderate, 2 marked) and 10/10 females (3 slight, 6 moderate, 1 marked) at 160 mg/kg bw/ day.
Mesenteric lymph nodes:
- Granuloma(s) with central necrosis in most cases in 7/10 (1 minimal, 4 slight, 1 moderate, 1 marked) males and 6/10 females (2 slight, 4 moderate) at 10 mg/kg bw/ day, in 10/10 males (1 slight, 3 moderate, 5 marked, 1 massive) and 9/10 females (2 minimal, 3 slight, 1 moderate, 2 marked, 1 massive) at 100 mg/kg bw/ day and in 9/10 males (1 slight, 3 moderate, 4 marked, 1 massive) and 7/10 females (1 minimal, 1 moderate, 3 marked, 2 massive) at 160 mg/kg bw/ day.
- Extranodal inflammation and/or necrosis (with/without peritonitis) in 5/10 males (2 minimal, 1 slight, 2 moderate) and 2/10 females (1 minimal, 1 moderate) at 10 mg/kg bw/ day, in 9/10 males (3 slight, 4 moderate, 2 marked) and 4/10 females (2 minimal, 2 moderate) at 40 mg/kg bw/ day and in 5/10 males (1 minimal, 3 slight, 1 moderate) and 6/10 females (1 minimal, 1 moderate, 4 marked) at 160 mg/kg bw/ day.
- Foamy macrophages in 2/10 males (minimal) 1/10 females (minimal) at 2.5 mg/kg bw/ day, in 9/10 males (1 minimal, 5 slight, 3 moderate) and 9/10 females (4 minimal, 2 slight, 3 moderate) at 10 mg/kg bw/ day, 8/10 males (4 slight, 3 moderate, 1 marked) and 8/10 females (3 slight, 3 moderate, 2 marked) at 40 mg/kg bw/ day and in 9/10 males (2 slight, 6 moderate, 1 marked) and 8/10 females (5 moderate, 3 marked) at 160 mg/kg bw/ day.
- (Increased) Macrophage foci in 6/10 males (2 minimal, 3 slight, 1 moderate) and 7/10 females (3 minimal, 4 slight) at 2.5 mg/kg bw/ day, 6/10 males (1 minimal, 2 slight, 2 moderate, 1 marked) and 4/10 females (2 minimal, 1 moderate, 1 marked) at 40 mg/kg bw/ day compared to 2/10 males and 2/10 females (all minimal) in the control group.
- Lymphoid hyperplasia in 1/10 males (slight) and 2/10 females (slight) at 10 mg/kg bw/ day, in 2/10 males (1 minimal, 1 slight) and 3/10 females (2 minimal, 1 slight) at 40 mg/kg bw/ day and in 4/10 females (1 minimal, 3 slight) at 160 mg/kg bw/ day.
- Sinus ectasia in 2/10 males (1 minimal, 1 slight) at 10 mg/kg bw/ day, 3/10 males (1 minimal, 1 moderate, 1 marked) and 2/10 (1 minimal, 1 slight) at 40 mg/kg bw/ day and 5/10 males (moderate) and 1/10 females (moderate) at 160 mg/kg bw/ day.

Unclear primary/secondary targets
Joints (hind and fore legs, in two males also sternum and vertebra):
- Arthritis in 4/10 males (3 moderate, 1 massive) at 40 mg/kg bw/ day and in 3/10 males (marked) and 3/10 females (2 marked, 1 massive) at 160 mg/kg bw/ day. At 160 mg/kg bw/ day, arthritis was also observed at the vertebral joints surrounding the thoracal spinal cord (2 slight) and at the intercostal joints of the sternum (1 minimal, 1 moderate).
- Exostosis in 2/10 males (1 marked, 1 massive) at 40 mg/kg bw/ day and in 3/10 males (2 moderate, 1 marked) and 1/10 females (moderate) at 160 mg/kg bw/ day. Exostosis was also noted in 2/10 males at 160 mg/kg bw/ day in the thoracal and/or cervical vertebrae.
- Reduced bone in joint of legs in 1/10 males (marked) at 40 mg/kg bw/ day and in 1/10 males (slight) at 160 mg/kg bw/ day and in sternum in 1/10 males of Group 4 (minimal) and 5/10 males of Group 5 (3 minimal, 1 slight, 1 moderate).
Secondary targets
Spleen:
- Necrotizing inflammation, intra- and/or extra-splenic in 2/10 males (1 moderate, 1 marked) and 1/10 females (slight) at 40 mg/kg bw/ day and in 2/10 males (1 moderate, 1 marked) and 2/10 females (marked) at 160 mg/kg bw/ day.
- Inflammation of mixed inflammatory cells in surrounding tissue in 1/10 males at 100 mg/kg bw/ day (slight) and in 3/10 males (moderate) at 160 mg/kg bw/ day
- Hemopoietic foci (increased above background severity of slight) in 1/10 males (marked) and 3/10 females (2 moderate, 1 marked) at 40 mg/kg bw/ day and in 2/10 males (1 marked, 1 massive) and 4/10 females (1 moderate, 1 marked, 2 massive) at 160 mg/kg bw/ day.
Liver:
- Granulomatous lesions with central necrosis in 2/10 females (moderate) at 10 mg/kg bw/ day, in 3/10 females (1 minimal, 1 moderate, 1 marked) at 40 mg/kg bw/ day and in 3/10 males (1 minimal, 1 moderate, 1 marked) and 4/10 females (2 moderate, 1 marked, 1 massive) at 160 mg/kg bw/ day.
- Sinusoidal leukocytosis in 1/10 females at 10 mg/kg bw/ day (minimal), in 3/10 males (2 slight, 1 moderate) and 1/10 females (slight) at 40 mg/kg bw/ day and in 4/10 males (1 minimal, 2 slight, 1 moderate) and 7/10 females (1 minimal, 3 slight, 3 moderate) at 160 mg/kg bw/ day.
- Foamy macrophage foci in 3/10 females (1 minimal, 1 slight, 1 moderate) at 40 mg/kg bw/ day and 1/10 females (slight) at 160 mg/kg bw/ day.
- Increased mitosis in 1/10 males (marked) at 40 mg/kg bw/ day and in 2/10 females (moderate) at 160 mg/kg bw/ day.
- Diffuse basophilia in 1/10 males (moderate) at 40 mg/kg bw/ day and in 3/10 females (2 moderate, 1 marked) at 160 mg/kg bw/ day.
Heart:
- (Epi/myo)carditis in 1/10 males (1 moderate) at 40 mg/kg bw/ day and in 2/10 males (moderate) and 1/10 females (slight) at 160 mg/kg bw/ day.
Thymus:
- Lymphoid atrophy in 3/10 males (1 minimal, 1 slight, 1 moderate) at 40 mg/kg bw/ day and in 3/10 males (1 minimal, 1 marked, 1 massive) and 8/10 females (2 minimal, 1 slight, 2 marked, 3 massive) at 160 mg/kg bw/ day.
Bone marrow (sternum):
- Myeloid hyperplasia in 4/10 males (3 minimal, 1 slight) and 3/10 females (minimal) at 10 mg/kg bw/ day, in 9/10 males (4 slight, 4 moderate, 1 marked) and 5/10 females (3 minimal, 1 slight, 1 moderate) at 40 mg/kg bw/ day and in 10/10 males (1 minimal, 5 slight, 1 moderate, 3 marked) and 9/10 females (1 slight, 6 moderate, 2 marked) at 160 mg/kg bw/ day.
- Megakaryocytosis present (no severity given) in 1/10 males and 3/10 females at 160 mg/kg bw/ day
Peyer’s Patches:
- Lymphoid atrophy in 3/10 males (minimal) at 40 mg/kg bw/ day and in 8/10 males (3 minimal, 5 slight) and 1/8 females (minimal) at 160 mg/kg bw/ day.
Adrenal glands:
- Diffuse cortical hypertrophy in 4/10 females (1 minimal, 2 slight, 1 moderate) at 160 mg/kg bw/ day.
Reproductive organs:
Epididymides:
- Inflammation of mixed inflammatory cells/granulomatous inflammation in surrounding tissue in 3/10 males (2 minimal, 1 slight) at 10 mg/kg bw/ day, in 7/10 males (3 minimal, 2 slight, 2 moderate) at 40 mg/kg bw/ day and 3/10 males (2 moderate, 1 marked) at 160 mg/kg bw/ day.
Prostate gland:
- Reduced content in 6/10 males (1 slight, 2 moderate, 3 marked) at 160 mg/kg bw/ day.
- Acinar atrophy in 3/10 males (moderate) at 160 mg/kg bw/ day.
Seminal vesicles:
- Reduced content in 8/10 males (3 minimal, 1 slight, 4 moderate) at 160 mg/kg bw/ day.
- Glandular atrophy in 4/10 males (slight) at 160 mg/kg bw/ day.
Coagulation glands:
- Reduced content in 6/10 males (2 minimal, 4 moderate) at 160 mg/kg bw/ day.
Uterus:
- Inactive in 1/10 females at 40 mg/kg bw/ day and 7/10 females at 160 mg/kg bw/ day.
Uterine cervix:
- Inactive in 5/10 females at 160 mg/kg bw/ day.
Vagina:
- Epithelial atrophy in 9/10 females at 160 mg/kg bw/ day.


OTHER FINDINGS
No treatment-related changes in the estrous cycle length were apparent up to 40 mg/kg bw/day. At 160 mg/kg bw/day, estrous cycle length could not be determined, since the surviving females were sacrificed before more than one cycle length was completed. Estrous cycle length showed a regular pattern for all animals of all dose groups, except for one female at 40 mg/kg and one control female (resp. irregular and acyclic). The incidence of these abnormal cycle lengths was low and showed no dose-related trend, and was therefore considered to be of no toxicological relevance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Adverse effects obserevd above 2,5 mg/kg bw/ day, primarily caused by excessive aggregation of foamy macrophages in the small intestines and its draining mesenteric lymph node.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Chemical analyses showed that homogeneity of formulations was considered acceptable for the purpose of this study, and that formulations were prepared accurately, and were stable over at least 5 hours at room temperature or over at least 8 days under nitrogen at room temperature. The long term storage samples were stable at ≤-70°C for at least 38 days.

Applicant's summary and conclusion

Conclusions:
A repeated dose 90-day oral toxicity study with exposure to C16-18, C18-unsaturated-alkyl dipropylene triamine by daily gavage in the rat was performed according to OECD guideline and GLP principles. Based on the data from this study a No Observed Adverse Effect Level (NOAEL) of 2.5 mg/kg was established.
Executive summary:

A repeated dose 90-day oral toxicity study with C16-18, C18-unsaturated-alkyl dipropylene triamine was performed according to OECD guidelines and GLP principles. Male and female rats (10/sex/exposure) were exposed to 0, 2.5, 10, 40 or 160 mg/ kg bw/ day by daily gavage. Based on the high mortality rate and signs of deteriorated health condition of animals at 160 mg/kg bw/ day, this high dose group was terminated after 11 weeks of dosing.

At 40 mg/kg bw/day, 3/10 males were killed in extremis in the last week of treatment. Rats treated with 40 and 160 mg/kg bw/day showed clinical signs including a hunched posture, piloerection, pallor, and swelling of the abdomen and/or legs, and an abnormal posture or gait, as well as reduced motor activity at 160 mg/kg bw/day. A lower body weight gain and food intake was recorded for males at 40 and 160 mg/kg (body weights were approximately 11 and 23% lower than controls at the end of treatment, respectively). At 10 mg/kg bw/day one male was found dead in Week 12. This animal displayed no clinical signs prior to death and had normal body weight gain, however it is not clear if this death was substance-related. Below 40 mg/kg bw/day, no clinical signs or changes in functional behaviour were noted.

Based on histopathological findings, the general deterioration in health condition and mortality at 40 and 160 mg/kg bw/day was considered to be due to an excessive aggregation of foamy macrophages in the small intestines and its draining mesenteric lymph node, containing poorly digestable test-article related material. The degradation of those cells was considered to have caused leakage and severe (necrotizing) inflammation both inside and outside the mesenteric lymph nodes, and included peritonitis and inflammation of the serosal tissues of for example spleen and epididymides. Hematogenic and/or lymphatic dissemination of cells/material originating from these lesions also resulted in the involvement of other organs such as liver, lung, heart and spleen causing different grades of inflammatory changes and necrosis. These changes supported necropsy lesions including enlarged mesenteric lymph nodes with irregular surface, thickening of the duodenum, jejunum and ileum, nodule/foci on the spleen, with enlargement/thickening/irregular surface of the spleen, spleen grown together with peritoneum, grey/white foci on the liver, enlargement, pale discoloration, and accentuated lobular pattern of the liver, a nodule in the epididymal adipose tissue, and epididymal adipose tissue grown together with spleen and gelatinous epididymal adipose tissue. These inflammatory processes were considered to have resulted in higher neutrophil counts at 40 and 160 mg/kg bw/day (resulting in higher total white blood cell counts). Other changes in blood at 40 and/or 160 mg/kg bw/day in males and/or females included a prolonged prothrombin time and higher platelet counts, higher alanine and aspartate aminotransferase activity, higher alkaline phosphatase activity, potassium and inorganic phosphate level, and lower total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, chloride and calcium level.

At 10 mg/kg bw/ day, changes in blood were confined to a higher inorganic phosphate level, and marginally higher neutrophil counts. At this dose, adverse histopathological findings were observed in the jejunum/ileum (minimally to slight foamy macrophages in lamina propria), mesenteric lymph nodes (general minimal to moderate foamy macrophages, accompanied by cases of granulomas with central necrosis, lymphoid hyperplasia, sinus ectasia), liver (granulomatous lesion with central necrosis in 2 males) and bone marrow (minimal myeloid hyperplasia in 7/40 animals).

 

At 2.5 mg/kg bw/day, histopathology findings were confined to foamy macrophages in the mesenteric lymph node in very few (3/40) animals at minimal degree and an increased incidence and severity compared to control in macrophage foci up to moderate degree, which is also observed at 10 mg/kg bw/day, but not at all at higher dose levels . Based relatively low severity, these mesenteric lymph node findings are considered not to be adverse. No treatment-related changes occurred in any of the other examined parameters at 2.5 mg/kg bw/day. Based on these results, a No Observed Adverse Effect Level (NOAEL) for C16-18, C18- unsaturated-alkyl dipropylene triamine of 2.5 mg/kg was established.