Registration Dossier

Administrative data

Description of key information

An oral 28-day repeated dose toxicity study according to OECD 407 on Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products resulted to a NOAEL of 80 mg/kg bw/day. The 90-day NOAEL from cross-reading to AAI-DETA is 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. Other available data from the group of AAI substances, including 90 day studies in rat and dogs on a similar substance, indicate low repeated dose toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
18 November 2013 to 18 February 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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
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
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.
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.
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.
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.
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'
Critical effects observed:
not specified

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.

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:

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)Use of the results obtained from studies with AAI-DETA also represent a conservative approach in the evaluation of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA). AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects ofacute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category.

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI). Available studies are OECD Guideline compliant and performed under GLP.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

For the evaluation for repeated dose toxicity of Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA) a 28-day is available on the substance itself, as well as read-across to a 90-day study on AAI-DETA, of which the outcome can be considered a worst-case representation for AAI-AEA.

 

The available data available within the group of Amidoamines/imidazolines (AAI) substances indicate that for AAI substances based on shorter polyethyleneamines (EA), higher toxicity is observed compared to AAI based on longer EA. The forming of imidazoline itself does not seem to play a significant role. For cross-reading purposes between substances of AAI, Fatty acid reaction product with diethylene-triamine (AAI-DETA) therefore represents the worst case. In series of 28-day and combined repeated dose/reproduction screening toxicity studies (OECD 422) AAI-DETA has shown the highest level of toxicity. (See also document in support of category justification)

Use of the results obtained from studies with AAI-DETA also represents a conservative approach in the evaluation of this substance AAI-AEA: AAI-AEA differs from other substances in the AAI group that are based on fatty acids with polyamines, in that it contains a high amount of hydroxylated polyethylene amines, causing it to display more neutral OH groups, rather than basic primary amine groups. As toxicity is much related to the presence of free primary amine groups, it can be expected that in specifically in aspects ofacute toxicity related to direct chemical interactions, this substance shows less effects compared to other AAI substances, and specifically compared to AAI-DETA which represents the worst case in the AAI category.

 

Wistar rats were treated with Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA) for 28 consecutive days by daily oral gavage at dose levels 100, 300 and 1000 mg/kg followed by a 14-day treatment-free recovery period. The animals of the highest dose group were treated from Day 1 up to Day 8 at 1000 mg/kg, but following mortality, severe body weight loss and clinical signs, the dose level was lowered to 500 mg/kg from Day 9 onwards.

Three males and two females of the highest dose group were killed in extremis or found dead in the first two weeks of the study. The main causes of death or moribundity were findings in the forestomach consisting of erosions or ulcers combined with a lympho-granulocytic inflammation and hyperplasia of the squamous epithelium of the forestomach.

In the animals at 1000 mg/kg body weight loss was noted during the first week, which recovered slightly after lowering the highest dose to 500 mg/kg. Also the lower food consumption recovered after the first week.

The surviving animals in the highest dose group showed changes in haematology and clinical biochemistry. Higher red blood cell distribution width and prothrombin time were noted in males and higher white blood cell count were noted in females at the end of treatment and/or recovery. In addition, higher aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) and lower total protein and creatinine levels were noted in males and/or females at the end of treatment and/or recovery. A marked lower absolute and relative thymus weight was noted in males at 500 mg/kg. This finding was not accompanied by histopathological findings.

At histopathology examination of the surviving animals treatment-related findings were noted in the forestomach at scheduled necropsies at the end of treatment. These findings included lymphogranulocytic inflammation, minimal hyperplasia of the squamous epithelium and/or ulcer in 3 males and/or 3 females at 500 mg/kg. The 14 day treatment-free recovery period suggested incomplete recovery.

At the mid-dose of 300 mg/kg hunched posture, rales and/or piloerection were noted in males and females from week 3 onwards. No treatment-related changes were noted in the functional observations at any dose level. Only the ALAT was increased in males at 300 mg/kg at the end of treatment. Since the other liver enzymes remained within the normal range and no corroborative findings in organ weight or microscopic examination were noted, this higher level of ALAT was considered not to be toxicologically significant.

At histopathological examination lymphogranulocytic inflammation, minimal hyperplasia of the squamous epithelium and erosions were noted in one male at 300 mg/kg. Based on the finding at the highest dose level this finding is considered to be a (local) treatment related effect.

Based on the clinical signs and the effects noted in the stomach a No Observed Adverse Effect Level (NOAEL) for AAI-AEA of 80 mg/kg was established (Corrected from 100 mg/kg dose for the analytical accuracy)

 

AAI-DETA was tested in a combined repeated dose/reproduction screening toxicity study according to OECD 422. The study report concludes to a parental NOAEL of 30 mg/kg/day based on the increased incidence and severity of foamy macrophage foci in the mesenteric lymph node at the end of treatment, and in males also after the after the recovery period. However, considering that the incidence and severity of macrophage as observed at 30 mg is somewhat higher than at 10 mg and the control group, the NOAEL could also have been set at 10 mg/kg bw.

A subsequent 90-day (OECD 408, GLP) study was performed applying the same dose levels of 0, 10, 30 and 100 mg/kg/day AAI-DETA to groups of 10 animals/dose/sex. The results from this study are comparable to those obtained from the earlier OECD 422 study: The first effect 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.

Comparing the effects of foamy macrophages between the OECD 422 and the 90-day study, show that the NOAEL level around 10 is rather comparable between the two studies, but that with increase of the duration in the 90-day study, an increase in this response is seen at 30 and 100 mg/kg bw/d.

 

Other available data from the group of AAI substances, including 90-day studies in rat and dogs on a similar substance, indicate low toxicity.

 

Results used for repeated dose evaluation:

AAI-AEA: 28-day NOAEL = 80 mg/kg bw

AAI-DETA: 28-day NOAEL = 10 mg/kg bw; 90-day NOAEL = 10 mg/kg bw.

The use of the results from the 90-day study on AAI-DETA is considered to represent a conservative approach in theevaluation for repeated dose toxicity of AAI-AEA.Comparison of the NOAELs from the studies on AAI-DETA show that the threshold for toxicity does not change much upon increasing the study duration from 28-day 90-days.

 

Dermal:

For dermal exposure no good overall NOAEL can be established as effects are rather characterized by local irritating effects that are related to duration, quantity and concentration, than by systemic toxicity due to dermal uptake. The mode of action of for AAI follows from its structure, consisting of an apolar fatty acid chain and a polar end of a primary amine from the polyethyleneamine. The structure can disrupt the cytoplasmatic membrane, leading to lyses of the cell content and consequently the death of the cell.

The AAI are protonated under environmental conditions which causes them to strongly adsorb to organic matter. This all leads to a low dermal absorption.

 

Inhalation:

Physical-chemical properties of polyamines indicate a low likelihood for exposure via inhalation, with a boiling point > 300 °C and low vapour pressure (8.4x10-8Pa at 25°C). Any inhalation exposures would therefore only be possible in the form of aerosol, consisting of larger droplets depositing in upper airways which could result to local irritation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Recent study of hight quality and of longest duration representing most conservative approach.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Likelihood of exposures via inhalation is low considering the high boiling point (> 300 °C) and very low vapour pressure (8.4 x 10-8 Pa at 25°C). Any inhalation exposures would therefore only be possible in the form of aerosol, consisting of larger droplets depositing in upper airways which could result to local irritation. Uses are limited to industrial and professional users with low possibility of exposure to aerosols or droplets of an inhalable size.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Lack of exposures

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The substance is irritating to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Lack of exposures: use is limited to industrial and professional users where following its irritating properties to skin and corrosive properties to eyes will provide for sufficient protection measures to prevent dermal exposure. Besides, being irritating to the skin, local effects of irritation are to be expected and no further studies are indicated.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: other

Justification for classification or non-classification

Classification for STOT-RE Cat. 2 is required in case of significant toxic effects at levels≤100 mg/kgbw/d in case of standard 90-day study. In case of 28-day studies this can be multiplied by 3.

 

An oral 28-day repeated dose toxicity study according to OECD 407 with Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products (AAI-AEA) resulted to a NOAEL of 80 mg/kg bw/day but at next dose level of 300 mg/kg toxicity was very minimal (consisting only of a marginal increase observed in ALAT and a (local) treatment related effect in the stomach in one male), and it can be argued whether the 300 mg/kgbw should be selected as NAOEL in this study.

Also the available 90 -day study on AAI-DETA did not indicate severe toxicity at 100 mg/kg/day.

 

Both studies, as well as other available data, do not suggest severe toxicity at levels requiring consideration for classification for STOTS-RE.