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EC number: 701-131-2 | CAS number: -
- Life Cycle description
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- Endpoint summary
- Appearance / physical state / colour
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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- Repeated dose toxicity
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- Additional toxicological data
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Repeated dose toxicity: oral
Administrative data
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 November 2013 to 18 February 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Fatty acids, C18 unsat, reaction products with diethylenetriamine
- EC Number:
- 629-715-1
- Cas Number:
- 1226892-43-8
- Molecular formula:
- UVCB substance - not applicable
- IUPAC Name:
- Fatty acids, C18 unsat, reaction products with diethylenetriamine
- Test material form:
- other: Clear slightly viscous amber liquid
- Details on test material:
- - Name of test material (as cited in study report): Fatty acids, C18 unsat, reaction products with diethylenetriamine
- Substance type: Clear slightly viscous amber liquid
- Physical state: liquid
- Lot/batch No.: S000922
- Expiration date of the lot/batch: 02 July 2017
- Storage condition of test material: At room temperature in the dark under nitrogen
- Purity/composition correction factor required: No
- Hygroscopic: Not indicated
- Volatile: No
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Specific Gravity / Density: 0.926
- pH: 10-12 at concentration of 75%
- Stability in Propylene glycol: Stability for at least 6 hours at room temperature is confirmed over the concentration range 2 to 20 mg/mL, (WIL Research Europe B.V. project 491556) and 2 to 30 mg/mL (WIL Research Europe B.V. project 503870) Stability for at least 8 days in the refrigerator under nitrogen is confirmed over the concentration range 2 to 20 mg/mL, WIL Research Europe B.V. project 503866
- Solubility in Propylene glycol: Up to 200 mg/g
Constituent 1
Test animals
- Species:
- rat
- Strain:
- other: Wistar (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- - Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old).
- Weight at study initiation: mean weight range at start of treatment was 147-148 gr (males) or 122-125 gr (females).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
IN-LIFE DATES: From: 18 November 2013 to 18 February 2014
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- propylene glycol
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
VEHICLE
- Justification for use and choice of vehicle: The same vehicle was used under project 491556 (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Tall oil diethylenetriamine imidazoline in rats by oral gavage, followed by a 14-Day recovery period).
DOSE VOLUME:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Analyses were conducted during the treatment phase. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 8 days in the refrigerator under nitrogen was also determined (highest and lowest concentration, in Week 1). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. 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:
- 90 days
- Frequency of treatment:
- Once daily, 7 d/w.
Doses / concentrations
- Remarks:
- Doses / Concentrations:
0, 10, 30, 100 mg/kg bw/day
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 combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Tall oil diethylenetriamine imidazoline in rats by oral gavage, followed by a 14-Day recovery period.
Note: “Tall oil diethylenetriamine imidazoline” is the same test substance as “Fatty acids, C18 unsat, reaction products with diethylenetriamine”.
In this study rats were dosed by oral gavage at dose levels of 10, 30 and 100 mg/kg. At 100 mg/kg, histopathology revealed foamy macrophage foci in the ileum of all selected males and females, a slightly increased incidence and degree of macrophage foci in the mesenteric lymph node of both sexes, and increased incidence of lymphoid atrophy in the thymus of females. At the end of the 14-day recovery period for males, foamy macrophage foci in the ileum had completely resolved, whilst macrophage foci in the mesenteric lymph node persisted at higher severity than observed at the end of treatment. Given the persistence of this finding it was considered to be of an adverse nature. However, it was considered that a dose of 100 mg/kg would be tolerated in a 90-day study. No toxicologically relevant changes were noted at 10 and 30 mg/kg. - Positive control:
- Not required.
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards in all animals between 1-2 hours after dosing.
BODY WEIGHT:
- Time schedule for examinations: Weekly.
FOOD CONSUMPTION
- Time schedule for examinations: Weekly.
WATER CONSUMPTION: No quantitative investigation (subjective appraisal).
OPHTHALMOSCOPIC EXAMINATION
- Time schedule for examinations: at pretest : All animals (including spare animals), at week 13 : control and high dose.
HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes (maximally 24 hours)
- How many animals: all animals
- Parameters checked: According to test guidelines
CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes (maximally 24 hours)
- How many animals: all animals
- Parameters checked: According to test guidelines
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: During Week 12 of treatment, between 1 and 3 hours after dosing.
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, locomotor activity test.
ESTROUS CYCLE DETERMINATION
All females had a daily lavage from Day 72 up to and including Day 92 to determine the stage of estrous. - Sacrifice and pathology:
- GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines
ORGAN WEIGHTS:
Organs checked according to test guidelines
HISTOPATHOLOGY:
According to test guidelines - 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.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Based on subjective appraisal.
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Urinalysis findings:
- not specified
- Behaviour (functional findings):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period. No clinical signs of toxicity or abnormalities during weekly arena observations were noted during the observation period.
BODY WEIGHT AND WEIGHT GAIN
Males at 30 and 100 mg/kg showed a lower mean body weight and body weight gain from Week 5 of the treatment period onwards, being statistically significant at 100 mg/kg. The lower mean body weight at 100 mg/kg was considered to be primarily due to a lower weight gain of two males. Mean body weight and body weight gain of females at 100 mg/kg also appeared slightly lower than controls during the last weeks of treatment.
FOOD CONSUMPTION
Males at 30 and 100 mg/kg showed a slightly lower food intake during the larger part of the treatment period. After correction for body weight, food consumption of these males was similar to control levels. Food consumption before or after correction for body weight for females remained similar to control means over the study period.
OPHTHALMOSCOPIC EXAMINATION
No ophthalmology findings were noted that were considered to be related to treatment.
HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.
CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase activity (ALAT) in males and females (most notably for one female) at 100 mg/kg,
- Higher aspartate aminotransferase activity (ASAT) in males (most notably for one male) and females at 100 mg/kg,
- Lower total protein levels in males (most notably for one male) and females at 100 mg/kg,
- Lower albumin levels in males (most notably for one male) and females at 100 mg/kg,
- Higher glucose levels in males at 100 mg/kg (upon excluding one male),
- Lower urea levels in males at 100 mg/kg (a trend towards an increase was also apparent among female dose groups),
- Higher bile acid levels in females at 100 mg/kg,
- Lower calcium levels in females at 100 mg/kg.
One male at 100 mg/kg also showed a lower glucose value and a higher total bilirubin, bile acid and inorganic phosphate level.
NEUROBEHAVIOUR
Males at 100 mg/kg showed a statistically significantly lower motor activity (based on counts for total movements). Females also showed a trend towards lower motor activity at 30 and 100 mg/kg, but means did not achieve a level of statistical significance.
ESTROUS CYCLE DETERMINATION
No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 72 up to and including Day 92). One control female, and three females at 30 mg/kg showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.
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:
- Lower liver weights in males at 100 mg/kg (with a decreasing trend for absolute liver weights across other male groups),
- Lower thymus weights in males at 100 mg/kg (with a decreasing trend for absolute thymus weights across other male groups),
- Lower spleen weights in males at 100 mg/kg (with a decreasing trend for absolute spleen weights across other male groups).
- Lower prostate weights at 100 mg/kg.
GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
HISTOPATHOLOGY
The following microscopic findings were considered to be related to treatment:
- Lung (alveolar (mainly foamy) macrophage aggregations):
At 100 mg/kg: increased incidence and severity in females (6/10 females, 4 minimal, 2 slight).
- Small intestines (foamy macrophages in the lamina propria):
At 10 mg/kg: in jejunum in 1/10 males and 3/10 females and in ileum in 3/10 males and 3/9 females (all minimal).
At 30 mg/kg: in duodenum in 1/10 males (slight), in jejunum in 1/10 males (minimal) and 4/10 females (minimal) and in ileum in 8/10 males (6: minimal, 2: slight) and females 10/10 females (5: minimal, 5: slight).
At 100 mg/kg: in duodenum in 8/10 males (7: minimal, 1:slight) and in 5/10 females (4: minimal, 1: slight), in jejunum in 10/10 males (2: minimal, 8: slight) and 8/10 females (6: minimal, 2: slight) and in ileum in 10/10 males (1: minimal, 7: slight, 2: moderate) and in 10/10 females (8: slight, 2: moderate).
- Kidneys (foamy macrophages in the glomeruli):
At 100 mg/kg: in 7/10 males (6: minimal, 1: slight) and in 9/10 females (minimal).
- Mesenteric lymph node (foamy macrophages):
At 10 mg/kg: in 1/10 female (slight),
At 30 mg/kg: in 2/10 males (minimal) and in 3/10 females (minimal)
At 100 mg/kg: in 10/10 males (7: slight, 2: moderate, 1: marked) and in 8/10 females (1: slight, 7: moderate).
- Mesenteric lymph node (pigmented macrophage foci):
At 30 mg/kg: slightly increased incidence and/or severity in 9/10 males (4: minimal, 5: slight) and in 9/10 females (7: minimal, 2: slight).
At 100 mg/kg: slightly increased incidence and/or severity in 10/10 males (5: minimal, 5: slight) and in 7/10 females (3: minimal, 4 slight).
The spermatogenic staging profiles were normal for all males of the control group and the 100 mg/kg group, except for one male at 100 mg/kg (all stages missing). The macroscopic and microscopic findings recorded for this male at 100 mg/kg included effects in the reproductive organs (flaccid and reduced size of testes, microscopic correlate: undeveloped testes; reduced size of epididymides, microscopic correlate: reduced sperm; reduced size of prostate gland, microscopic correlate: immature) and liver (reduced size, no microscopic correlate and accentuated lobular pattern, microscopic correlate: difference in cell size periportal-centrilobular). These findings for this single male at 100 mg/kg were considered to be incidental findings and unrelated to treatment.
Effect levels
- Dose descriptor:
- NOAEL
- Effect level:
- ca. 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Accuracy of preparation: The concentrations analysed in the formulations for the 10, 30 and 100 mg/kg dose groups were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). A small response at the retention time of the test substance was observed in the chromatograms of the control formulations. It was considered to derive from carry over since a similar response was obtained in the analytical blanks.
Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation = 10%).
Stability: Formulations at the entire range were stable when stored under nitrogen in a refrigerator for at least 8 days.
Applicant's summary and conclusion
- Conclusions:
- The 90-day NOAEL is considered to be 10 mg/kg bw/d. At higher dose levels an increase of the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes is observed, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.
- Executive summary:
Tall oil diethylenetriamine imidazoline was administered by daily oral gavage to groups of 10 male and 10 female Wistar Han rats for 90 days at dose levels of 0, 10, 30 and 100 mg/kg/day. The study was performed under GLP and based on OECD TG 408.
Evaluated parameters:
Chemical analyses of formulations preparations; clinical signs daily; functional observation tests in Week 12; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; estrous cycle determination; clinical pathology and macroscopy at termination; organ weights and histopathology (including spermatogenesis staging) on a selection of tissues.
Results
Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 8 days.
All animals survived up until scheduled termination. No toxicologically significant clinical signs were noted during the observation period.
The treatment-related lower motor activity of males at 100 mg/kg, and a trend towards lower motor activity for females at 30 and 100 mg/kg were considered not to represent an adverse effect on neurobehaviour. These results were not supported by clinical observations or other functional observation tests, were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.
Males at 30 and 100 mg/kg showed a lower mean body weight and body weight gain with lower food intake, essentially during the second half of the treatment period, with a slightly lower body weight and body weight gain of females at 100 mg/kg during the last week of treatment.
No treatment-related ophthalmology findings, changes in haematological parameters or macroscopic findings were noted.
Test item-related microscopic findings consisted of:
- foamy macrophages in the lamina propria of the small intestines (males and females starting at 10 mg/kg/day),
- foamy macrophages in the mesenteric lymph nodes (females starting at 10 mg/kg/day, males starting at 30 mg/kg/day),
- increased incidence and severity of pigmented macrophage foci in the mesenteric lymph nodes (males and females starting at 30 mg/kg/day),
- increased incidence and severity of alveolar (mainly foamy) macrophage aggregations in the lung (females at 100 mg/kg/day),
- foamy macrophages in the glomeruli of the kidneys (males and females at 100 mg/kg/day).
The findings in the lamina propria of the small intestines were considered to have caused reduced protein uptake, and to correlate with lower total protein and albumin levels in males and females at 100 mg/kg.
Other treatment-related changes in clinical biochemistry parameters at 100 mg/kg consisted of higher alanine and aspartate aminotransferase activity in males and females, higher total bilirubin and glucose levels in males, lower urea levels in males (with a trend towards an increase among female dose groups), higher bile acid levels in females, and lower calcium levels in females at 100 mg/kg (possibly secondary to the lower albumin levels).
The lower liver, thymus and spleen weights in males at 100 mg/kg (with a decreasing trend across other male groups) had no histopathological correlates, and were therefore ascribed to the lower terminal body weights for these males. As such, these changes were considered to be of no toxicological relevance.
One male at 100 mg/kg showed various microscopic (and correlating macroscopic and organ weight) changes in reproductive organs including undeveloped testes (correlating to absence of all spermatogenesis stages) with reduced sperm content in the epididymides and immature prostate, and a difference in cell size in the periportal-centrilobular area of the liver. In addition, this animal showed a lower weight gain during treatment, and blood analyses showed a lower glucose value and a higher bile acid and inorganic phosphate level. Since these findings were confined to this single animal, these were considered unrelated to treatment with the test substance.
There were no indications of possible reproductive toxicity based on the parameters determined in this study. No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 72 up to and including Day 92), and histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions.
Conclusion
The results from this study are comparable to those obtained from the earlier OECD 422 study.
The first effects to occur is the presence of foamy macrophages in the lamina propria of the small intestines and mesenteric lymph nodes. These effects are considered to represent a local, porte d’entrée related effect due to the route of application, rather than a systemic effect.
The magnitude of these effects as observed at the lowest dose level of 10 mg/kg, is often also observed in control groups in general, and was also seen in the control group of the OECD 422 study performed before on the same substance. These effects are therefore not considered adverse. As no other effects were observed, this dose level is considered to represent the NOAEL. At higher dose levels an increase in foamy macrophages is observed beyond levels that can occur in control groups, as well as lower mean body weight and body weight gain, especially in the males, with lower food intake, essentially during the second half of the treatment period.
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