Registration Dossier

Administrative data

Description of key information

A combined repeated dose/reproduction screening toxicity study according to OECD 422 with Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA) resulted to a NOAEL of 300 mg/kg bw/day being the highest tested dose level. All already available data from the group of Amidoamine/imidazolines AAI substances, including 90 -day studies in rat and dogs on a similar substance, also 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:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2009 - 19 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD 422 guidelines and GLP principles.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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 12 weeks old instead of approximately 10 weeks. A slight deviation in age does not affect the study integrity. Mating started shortly after the animals had attained full sexual maturity according to the OECD 422 guideline.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This also accounts for the Recovery males for the complete treatment period.
Mating: Females were caged together with Main males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Main males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during overnight activity monitoring.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 22.0°C
- Humidity (%): 31 - 79%
Temporary deviations from the minimum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room.

On Day 3 or 4 of lactation, the maximum allowed deviation from the dark period of 1 hour was exceeded with a maximum of approximately 9 minutes for the 5 selected females with live pups per group. These temporary deviations from the light/dark cycle were considered not to have affected the study outcome.

IN-LIFE DATES: From: 25 August 2009 To: 19 October 2009
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 homogenised to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of vehicle (1.036).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 6, 20 and 60mg/mL

- 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 on a single occasion during treatment phase according to a validated method (NOTOX project 491638). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

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%.

RESULTS:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature for at least 6 hours.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination or up to the day prior to start of the recovery period for Recovery males. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Female no. 73 was not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on the results of a 10-day pilot study (NOTOX Project 491888; see attached file) and taking into account the results of a MTD-study with Tall oil diethylenetriamine imidazoline (NOTOX Project 491555; see endpoint study record 7.5.1: repeated dose toxicity: oral.rat_NOTOX 491555 ) and a 10-day pilot study with Tall oil reaction products with tetraethylene-pentamine (NOTOX Project 491569; see endpoint study record 7.5.1: repeated dose toxicity: oral.NOTOX 491568).
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards (immediately after dosing from Days 1-9, between approximately 1 and 2 hours after dosing from Day 10 onwards, and once daily during the recovery period) detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period, except for Recovery males. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: Yes
(average food consumption [per animal per day]/average body weight per cage)x1000.

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes, iso-flurane
- How many animals: 5 animals/sex/group (females with live offspring only).
- Parameters examined were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.
Sacrifice and pathology:
All animals were fasted overnight (with a maximum of approximately 21 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

Necropsy was conducted on the following days:
Females which delivered: Lactation Day 5-7.
Females which failed to deliver: Post-coitum Day 26-27 (females with evidence of mating)
Males (Main): Following completion of the mating period (a minimum of 28 days of dose administration).
Males (Recovery): After a recovery phase of 14 days.
Female sacrificed in extremis during littering: Day 1 of lactation.

Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of 1 hour and 12 minutes. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.

GROSS PATHOLOGY: Yes
All parental animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Selected 5 animals/sex/group, all Recovery males and the animal that was killed in extremis@: Identification marks: not processed, Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides*, Seminal vesicles including coagulating gland, Eyes with optic nerve (if detectable) and Harderian gland*, Skeletal muscle, (Skin), (Female mammary gland area), Spinal cord (cervical, midthoracic, lumbar), Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes*, Kidneys, Thymus, (Lacrimal gland, exorbital), Thyroid including parathyroid (if detectable), (Larynx), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions.

All remaining animals and females which failed to deliver #: Identification marks: not processed, Prostate gland, Cervix, Seminal vesicles including coagulating glands, Clitoral gland, Testes*, Epididymides*, Uterus, Ovaries, Vagina, Preputial gland, All gross lesions.

@: Recognizable fetuses were examined externally, sexed, euthanized by decapitation (if necessary) and preserved in 10% neutral-buffered formalin.
*: Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
#: In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group and all Recovery males: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate*, Liver, Seminal vesicles including coagulating glands*, Ovaries, Thyroid including parathyroid*.

* weighed when fixed for at least 24 hours.

All remaining males: Epididymides, Testes.

No thyroid was weighed from one animal (Group 1). This was a non-pregnant female, and hence organ weights of this animal cannot be used to compare with pregnant animals. Therefore, this deviation does not adversely affect the interpretation of the results.

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males/group of the control and high dose Main group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 Main animals/sex of Groups 1 and 4.
- The adrenal glands from the selected 5 Main animals from Groups 2 and 3 and from Recovery animals of Groups 1 and 4*.
- The additional slides of the testes of the selected 5 Main males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of the animal (Group 3) that was killed in extremis.
- The reproductive organs# of all Main animals that failed to conceive, or deliver healthy pups:
Group 1: One male and one female (failed to conceive)
Group 3: One male (one female was killed in extremis during parturition)
Group 4: One male and one female (failed to conceive)
- All gross lesions of all animals (all dose groups).

* Based on possible treatment-related histopathological changes in high dose Main animals.
# Reproductive organs included cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

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 might 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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One female at 100 mg/kg/day was killed in extremis due to complications during parturition. No further mortality occurred.

There were no clinical signs of toxicity noted during the observation period.

Incidental findings included rales, salivation, diarrhoea and alopecia. The nature and/or incidence of these findings was considered to be within the normal range of biological variation for rats of this age and strain. No clinical signs were noted among control males.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were observed over the study period.

The statistically significant lower body weight gain of males at 300 mg/kg/day during the premating and Repro period was considered to be of no toxicological relevance since these variations were minor in nature, and body weight gain was similar to control values from Day 1 of the recovery phase onwards.

FOOD CONSUMPTION:
No toxicologically significant changes in food consumption before or after allowance for body weight were noted.

The statistically significant changes in (relative) food consumption among females at 300 mg/kg/day were incidentally noted during the premating and post-coitum phase and were slight in nature. Therefore, these variations were not considered to be of toxicological relevance.

HAEMATOLOGY
No toxicologically significant changes in haematology parameters were noted.

The statistically significant higher prothrombin time (PT) and mean corpuscular volume (MCV) in males at 300 mg/kg/day at the end of treatment and recovery phases respectively, were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution, were absent at the end of the treatment phase and/or remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals at the end of the treatment phase:
- Higher alanine aminotransferase activity (ALAT) and aspartate aminotransferase activity (ASAT) in males and females at 300 mg/kg/day at the end of treatment.
- Lower total protein and higher total bilirubin levels in males at 300 mg/kg/day at the end of treatment (within normal range for rats of this age and strain).
- Higher inorganic phosphate level in females at 300 mg/kg/day.
By the end of the treatment period these changes had returned to values comparable to controls.

Other statistically significant changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution, remained within the range considered normal for rats of this age and strain and/or were absent at the end of the treatment phase. These changes included higher sodium and/or chloride levels in males at 30, 100 and 300 mg/kg/day at the end of treatment, lower glucose and calcium, and higher inorganic phosphate levels in males at 300 mg/kg/day at the end of recovery, and higher bile acid levels in females at 100 mg/kg/day.

NEUROBEHAVIOUR
Motor activity of females at 300 mg/kg/day appeared higher than controls, and achieved a level of statistical significance for low sensor counts.

No toxicologically significant changes in hearing ability, pupillary reflex, static righting reflex and grip strength were noted among the animals. Motor activity of males (all dose levels) and females (30 and 100 mg/kg/day) remained similar to control levels.

One animal at 30 mg/kg/day showed no grip reflex, and one male at 100 mg/kg/day did not show a hearing reflex. Given the incidental nature of these findings and absence of a treatment-related incidence, these were considered to be of no toxicological significance.

ORGAN WEIGHTS
A lower heart weight and heart to body weight ratio was recorded for males and females at 300 mg/kg/day at the end of treatment, achieving a level of statistical significance (except for relative heart weight of males). These changes were absent at the end of the recovery phase for males.

No further changes in organ weights were noted that were considered to be of toxicological significance. The statistically significant lower liver weight of males at 300 mg/kg/day at the end of the treatment phase was considered to be related to the lower terminal body weight (after correction for terminal body weight, liver weights were similar to those of control animals). The statistically significant lower epididymides weight, epididymides to body weight ratio and/or seminal vesicle to body weight ratio of males at 30, 100 and 300 mg/kg/day at the end of treatment did not show a dose-related trend. These changes were within the range considered normal for rats of this age and strain and were absent at the end of the recovery phase.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

Incidental necropsy findings at the end of the treatment period consisted of a nodule on the epididymides, pelvic dilation of the kidneys, red foci on the thymus or kidneys, reduced size of the thymus, a red-brown focus on the clitoral glands, enlarged clitoral glands or spleen, red discolouration of the mesenteric or mandibular lymph nodes, alopecia and fluid in the uterus.
The incidence of these findings 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 significance.

HISTOPATHOLOGY:
A morphological alteration in the adrenal gland consisting of a slight, not statistically significant, increase in incidence and severity to moderate, of multifocal vacuolation of the zona glomerulosa in animals of both sexes was noted at 300 mg/kg/day. Following a recovery period of at least two weeks for males this finding had reverted to a background incidence and severity of minimal.

No alterations in spermatogenesis staging were recorded.

There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.

The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.
Dose descriptor:
NOAEL
Remarks:
(F0)
Effect level:
>= 300 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions.
Critical effects observed:
not specified
Conclusions:
Overall, all parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions. Therefore, the parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was derived.
Executive summary:

Tall oil fatty acids, reaction products with polyethylenepolyamines was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). A total of 5 animals each of Groups 1 and 4 were allowed a 2-week recovery period. The females were exposed for at least 42 days, i.e. 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

Formulation analysis showed that the formulations were prepared accurately, were homogeneous and were stable for at least 6 hours at room temperature.

Parental findings:

Treatment up to 300 mg/kg/day was well tolerated and did not result in toxicologically relevant clinical signs or changes in body weight and food intake. A higher motor activity was recorded for females at 300 mg/kg/day, although this was not accompanied by supportive clinical signs (e.g. hyperactivity) or changes in functional observation battery parameters. Therefore, this change was not considered to be of toxicological relevance.

Clinical biochemistry measurements at the end of treatment at 300 mg/kg/day showed notably higher alanine and aspartate aminotransferase activity in both sexes (but without corroborative histopathological findings, e.g. liver damage), along with lower total protein and higher total bilirubin levels in males (within normal range for rats of this age and strain) and higher inorganic phosphate level in females. These changes had resolved to values comparable to controls at the end of the recovery period for males. No (further) changes were noted in clinical biochemistry and haematology parameters at any dose level.

Macroscopic examination at the end of treatment revealed a lower absolute and relative heart weight for both sexes at 300 mg/kg/day. No histopathological correlate was found and no such change was apparent at the end of the recovery period for males. No (further) macroscopic abnormalities or organ weight changes were observed at any dose level.

Histopathology showed a slight, statistically non-significant, increase in incidence and severity of multifocal vacuolation of the zona glomerulosa of the adrenal glands in both sexes at 300 mg/kg/day. Following the recovery period this finding had reverted to background incidence and severity.

No signs of parental toxicity were observed at 30 and 100 mg/kg/day.

Reproductive/Developmental findings:

No reproductive/developmental toxicity was observed at any dose level.

Overall, all parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions. Therefore, the parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was derived.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Consistent results from all studies within the whole group of Amidoamine/imidazolines (AAI), indicating a low level of toxicity. (See also document in support of category justification).

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

A combined repeated dose/reproduction screening toxicity study according to OECD 422 has been performed with Fatty acid reaction product with pentaethylene-hexamine (AAI-PEHA). A 9-day rangefinder study with 500 and 1000 mg resulted to mortality of 1/3 animals. Although 500 mg seemed to show only limited toxicity, experience on other running studies indicated that a 9-day range finding was possibly too short a period for full development of toxicity.

In the subsequent full study, AAI-PEHA was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. Tall oil fatty acids, reaction products with polyethylenepolyamines (Amidoamine/Imidazoline) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg/day. The males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). A total of 5 animals each of Groups 1 and 4 were allowed a 2-week recovery period. The females were exposed for at least 42 days, i.e. 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation.

Treatment up to 300 mg/kg/day was well tolerated and did not result in toxicologically relevant clinical signs or changes in body weight and food intake. There was a small change in motor activity observed in females at 300 mg/kg/day, but this was not considered to be of toxicological relevance.

Clinical biochemistry measurements at the end of treatment at 300 mg/kg/day showed notably higher alanine and aspartate aminotransferase activity in both sexes (but without corroborative histopathological findings, e.g. liver damage), along with lower total protein and higher total bilirubin levels in males (within normal range for rats of this age and strain) and higher inorganic phosphate level in females. These changes had resolved to values comparable to controls at the end of the recovery period for males.

Macroscopic examination at the end of treatment revealed a lower absolute and relative heart weight for both sexes at 300 mg/kg/day. No histopathological correlate was found and no such change was apparent at the end of the recovery period for males. No (further) macroscopic abnormalities or organ weight changes were observed at any dose level.

Histopathology showed a slight, statistically non-significant, increase in incidence and severity of multifocal vacuolation of the zona glomerulosa of the adrenal glands in both sexes at 300 mg/kg/day. Following the recovery period this finding had reverted to background incidence and severity.

No signs of parental toxicity were observed at 30 and 100 mg/kg/day.

No reproductive/developmental toxicity was observed at any dose level.

Overall, all parental findings observed at the end of treatment at 300 mg/kg/day were not considered to be adverse since these changes were generally slight and reversible in nature, and occurred in the absence of supportive histopathological lesions. Therefore, the parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg/day was derived.

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

 

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 in general 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).

To set the NOAEL for repeated dose more accurately, read across to a planned full 90-day study (OECD 408) is proposed for AAI-DETA.

 

For dermal exposure no good overall NOAEL can be established as effects are rather characterized by local corrosive 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 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 (< 0.00017 mPa at 25°C).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Although not the study of longest duration, it is the study of highest reliability

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 (< 0.00017 mPa at 25°C). The potential for inhalation is not significant to justify this study. Furthermore, as the substance is classified as corrosive, such testing should normally not be conducted.

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:
All substances from the group of Amidoamine/imidazolines (AAI) are corrosive to the skin and are 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 severe corrosive properties will provide for sufficient protection measures to prevent dermal exposure.
Besides, being corrosive to the skin, local effects of irritation or corrosion are to be expected and no further studies are indicated.

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.

A combined repeated dose/reproduction screening toxicity study according to OECD 422 with AAI-PEHA resulted to a NOAEL of 300 mg/kg bw/day, the highest dose tested. Also available data from the group of Amidoamine/Imidazoline (AAI) substances, including 90-day studies in rat and dogs on a similar substance, indicate very low toxicity.

Consequently, serious toxicity is not observed at levels requiring consideration classification for STOTS-RE.