Registration Dossier

Administrative data

Description of key information

An oral 28-day repeated dose toxicity study according to OECD 407 with Amides, fatty acids C18 unsat, reaction products with polyethylene amines resulted to a NOAEL of 100 mg/kg bw/day.  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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 August 2009 - 01 October 2009
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.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050
Deviations:
no
Qualifier:
according to
Guideline:
other: - Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No. 2) and ME (No. 031121002).
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 6 weeks
- Weight at study initiation: mean ranges day 1: males: 187-189 gr, females: 141-145 gr.
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring.
Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

Identification Earmark and tattoo.
Randomization By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Health inspection Prior to commencement of treatment to ensure that the animals were in a good state of health.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): actual range: 19.8 - 21.5ºC
- Humidity (%): actual range: 40 - 78%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 August 2009 to 01 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 homogenized to visually acceptable levels. At pre-weighing the test substance was heated up to maximally 68°C maximally 2 hours and 10 minutes. The formulation was heated up to maximally 45°C for maximally 15 minutes. The formulation was cooled down to maximally 40°C before dosing.
Adjustment was made for specific gravity of the test substance (mean 973 kg/m3) and vehicle. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information from the sponsor.
- Concentration in vehicle: 20, 60, 120 mg/mL.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed on a single occasion during the in-life phase 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 analytical method used was validated under NOTOX project 491585.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions, or 85-115% for suspensions. 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 (including 600 and 1000 mg/kg/day) were in agreement with target concentrations (i.e. Mean accuracies between 90% and 110%). No test substance was detected in the group 1 formulations. The formulations of group 2 and group 4 (only 1000 mg/kg/day) 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:
At least 28 days. Main animals were dosed up to the day prior to necropsy, and Recovery animals were dosed up to the day prior to start of the recovery period.
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. Animals were dosed up to the day prior to scheduled necropsy.
Remarks:
Doses / Concentrations:
100, 300, 600 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels are based on results of a 10-day dose range finding study with Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) (NOTOX project 491580). A summary of the results is included (see attachment).
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, 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.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly.

FOOD CONSUMPTION: Yes
Weekly.

FOOD EFFICIENCY:
(Average food consumption (per animal per day)/average body weight per cage) X 1000

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
- 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.
- Anaesthetic used for blood collection: Yes, iso-flurane
- Animals fasted: Yes, but water was available
- How many animals: 5 animals/sex/group.
- 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: during Week 4 of treatment. Since the these measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

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 (including optic nerve 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, (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions

* 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). Tissues were transferred to formalin after fixation for at least 24 hours.

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

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

ORGAN WEIGHTS:

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

Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate1 , Liver Seminal vesicles including coagulating glands 1, Ovaries, Thyroid including parathyroid1

1 weighed when fixed for at least 24 hours.


HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all tissues from animals of the highest dose groups which were terminated in extremis,
- all gross lesions.

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 analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (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-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 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 may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

1 C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
2 R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
3 R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY

One male and one female (Day 15/Day 9, receiving 1000 or 600 mg/kg) were sacrificed for ethical reasons due to poor health.

For the majority of animals at the highest dose group one or more of the following signs were noted most of the days during the observation period: Hunched posture, lethargy, laboured or shallow respiration, rales, piloerection,and/or chromodacryorrhoea.
The animals, which were sacrificed for ethical reasons showed in addition, swelling of the abdomen, diarrhoea and/or lean appearance.

There were no clinical signs noted during the recovery period.

No toxicological relevant clinical signs were noted in control animals and animals at 100 and 300 mg/kg.

All animals at 300 mg/kg showed salivation from Day 19 onwards (males) and from Day 26 onwards (females). All animals at 600 mg/kg showed salivation during Day 5 up to the end of treatment. No salivation was noted during the recovery period. Salivation is often noted in rats of this age and strain following oral gavage and may be related to chemical properties of the test substance. Therefore salivation was not considered to be of toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN

Body weight and body weight gain of males at 600 mg/kg were lower after start of the study until the end of treatment. The body weights remained lower during recovery, although the relative body weight gain seemed to be omewhat higher compared to controls. Body weight and body weight gain of the females at 600 mg/kg was lower after the first week only and recovered thereafter. These females showed even a higher body weight compared to controls on Day 1 of the recovery period.

Body weights and body weight gain of the other animals at 100 and 300 mg/kg remained in the same range as controls over the study period.

FOOD CONSUMPTION

Food consumption before or after allowance for body weight was lower in males at 600 mg/kg during the first week compared to control animals and recovered thereafter.
Food consumption before or after allowance for body weight was lower in females at 600 only during the first week of recovery.

No toxicologically significant changes in food consumption before or after allowance for body weight were noted in the females at 600 (only during treatment period) and the animals at 100 and 300 mg/kg.

HAEMATOLOGY

Males at 600 mg/kg showed slightly higher white blood cell counts and eosinophils level, which was normalized at the end of the recovery period.

Activated partial thromboplastin time (APTT) was lower in all female treatment groups. Since these values remained within the range considered normal for rats of this age and strain and were considered to have arisen as a result of a slightly high control value, this finding was considered to be of no toxicological significance.

Minor statistically significant changes at 100, 300 and/or 600 mg/kg were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain and/or occurred only at the end of recovery. These findings included changes in red blood cell distribution width (RDW), prothrombin time (PT), activated partial thromboplastin time (APTT), mean corpuscular volume (MCV) and reticulocyte counts.

CLINICAL CHEMISTRY

Males and females at 300 and 600 mg/kg showed higher alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels.
Additional changes are noted in animals at 600 mg/kg as follows:
• lower total protein level (males at the end of treatment),
• lower albumin level (males at the end of treatment),
• lower bile acid level (males at the end of treatment),
• higher potassium level (males at the end of treatment and females at the end of recovery),
• higher urea level (males at the end of recovery),
• lower urea level (females at the end of treatment).

No changes were noted in animals at 100 mg/kg compared to control animals.

GROSS PATHOLOGY

One male and one female were sacrificed in extremis. One or both animals showed a gastrous-intestinal tract distended with gas, emaciated appearance, reduced size of testes, epididymides, prostate, seminal vesicles and thymus, incomplete exsanguinations and gelatinous caecum.

Findings in males at 600 mg/kg at macroscopic examination included a single sided reduced size testis with flaccid appearance with a reduced size of the epididymides in one animal.

Incidental findings among control and treated animals included diaphragmatic hernia of the liver, foci in the kidney and clitoral glands, fluid in the uterus, nodules in the epididymides. 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.

ORGAN WEIGHTS

In females at 600 mg/kg a slightly lower spleen weight was noted at the end of treatment (only statistically significant after allowance for body weight). At the end of recovery a slightly lower thymus weight was noted after allowance for body weight.

At the end of recovery the males at 600 mg/kg showed lower weight of heart, liver and spleen. These organ weight changes were consistent with the expected physiological adaptive changes associated with reduced body weight gain at the end of recovery and no morphological changes were noted. Therefore these changes in organ weights are considered to be of no toxicological significance. Also, the higher brain weight after allowance for body weight was attributed to retarded body weight gain and of no toxicological significance.

No changes in organ weights were noted in males at 600 mg at the end of treatment and animals at 100 and 300 mg/kg compared to the control animals.

HISTOPATHOLOGY: NON-NEOPLASTIC

There were two unscheduled deaths in the highest dose group. These animals were emaciated and showed distension of the small intestines at necropsy. Main microscopic findings were small/immature reproductive organs and atrophy of the thymus (both animals) and atrophy of the spleen (only in the female). In the stomach minimal hyperplasia of the squamous epithelium of the forestomach was recorded in the male and minimal lymphogranulocytic inflammation of the forestomach in the female was seen.

All microscopic findings recorded in the animals surviving up to the scheduled necropsies were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.


Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Based on the mortality, clinical signs and body weight changes at 600 mg/kg and the changes noted for clinical biochemistry parameters at 300 mg/kg a No Observed Adverse Effect Level (NOAEL) for Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) of 100 mg/kg was established.
Executive summary:

Wistar rats were treated with Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) 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 7 at 1000 mg/kg. Based on the severe body weight loss and clinical signs the dose level was lowered to 600 mg/kg from Day 8 onwards. The animals of the highest dose group are described as “treated at 600 mg/kg” in this report.

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

One male and one female (600 mg/kg) were killed in extremis due to poor health. Several clinical signs were noted including swelling of the abdomen, diarrhoea, changed respiration and/or lean appearance. At macroscopic examination, the animals showed a gastrous-intestinal tract distended with gas, emaciated appearance, reduced size of testes, epididymides, prostate, seminal vesicles and thymus, incomplete exsanguinations and gelatinous caecum. Main microscopic findings were small/immature reproductive organs and atrophy of the spleen and thymus. In the stomach minimal hyperplasia of the squamous epithelium of the forestomach was recorded and minimal lymphogranulocytic inflammation of the forestomach was seen.

The cause of demise of the high dose male and female necropsied during the treatment phase could not be determined with any certainty. The microscopic findings indicated a more gradual decline in health status rather than a sudden acute event, but there was no morphological evidence of any significant target organ toxicity to account for morbidity. Although there was no conclusive evidence, the losses were restricted to the high dose group and therefore the presumption is that the morbidity and mortality was related to treatment.

The surviving animals at 600 mg/kg showed hunched posture, lethargy, labored or shallow respiration, rales, piloerection and/or chromodacryorrhoea during the observation period.

In these animals poor body weight gain was noted at the end of the first week, which was accompanied by low food consumption in the males. The body weight gain remained low during treatment and recovery in males and recovered in the females. Although food consumption appeared to be normal in females during the treatment period, low food consumption was noted during the first week of the recovery period.

No toxicologically significant changes in functional observations were noted in all treated animals.

The animals at the highest dose level showed some changes in the parameters of haematology and clinical biochemistry. The findings included slightly higher white blood cell count and eosinophils level and higher alanine aminotransferase and aspartate aminotransferase levels and lower total protein, albumin and bile acid level, higher potassium level and changes in urea level.

At the mid dose of 300 mg/kg males and females showed higher alanine aminotransferase and aspartate aminotransferase. The high dose animals recovered to normal levels at the end of the recovery and no morphological changes are noted at histopatology examination.

One of the surviving males at 600 mg/kg showed unilateral flaccid testes of reduced size and recuced size of the epididymides at macroscopic examination.

In females at 600 mg/kg a slightly lower spleen weight was noted at the end of treatment and a slightly lower thymus weight was noted after allowance for body weight at the end of recovery.

Based on the mortality, clinical signs and body weight changes at 600 mg/kg and the changes noted for clinical biochemistry parameters at 300 mg/kg a No Observed Adverse Effect Level (NOAEL) for Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) of 100 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 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

Oral: Wistar rats were treated with Fatty acid reaction product with polyethylene-polyamines (Amidoamine) (AAI-PolyEA Amide) 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.

Due to severe toxicity observed, the 1000 mg/kg does was lowered from day 8 to 600 mg/kg the animals of the highest dose group are described as “treated at 600 mg/kg”. One male and one female (600 mg/kg) were killed in extremis due to poor health.

The surviving animals at 600 mg/kg showed hunched posture, lethargy, laboured or shallow respiration, rales, piloerection and/or chromodacryorrhoea during the observation period.

In these animals poor body weight gain was noted at the end of the first week, which was accompanied by low food consumption in the males. The body weight gain remained low during treatment and recovery in males and recovered in the females. Although food consumption appeared to be normal in females during the treatment period, low food consumption was noted during the first week of the recovery period.

No toxicologically significant changes in functional observations were noted in all treated animals.

The animals at the highest dose level showed some changes in the parameters of haematology and clinical biochemistry. The findings included slightly higher white blood cell count and eosinophils level and higher alanine aminotransferase and aspartate aminotransferase levels and lower total protein, albumin and bile acid level, higher potassium level and changes in urea level.

At the mid dose of 300 mg/kg males and females showed higher alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). The high dose animals recovered to normal levels at the end of the recovery and no morphological changes are noted at histopathology examination.

One of the surviving males at 600 mg/kg showed unilateral flaccid testes of reduced size and reduced size of the epididymides at macroscopic examination.

In females at 600 mg/kg a slightly lower spleen weight was noted at the end of treatment and a slightly lower thymus weight was noted after allowance for body weight at the end of recovery.

Based on the mortality, clinical signs and body weight changes at 600 mg/kg and the changes noted for clinical biochemistry parameters at 300 mg/kg, a No Observed Adverse Effect Level (NOAEL) for Fatty acid reaction product with polyethylene-polyamines (Amidoamine) of 100 mg/kg was established. However, as the observed increase of ALAT and ASAT at 300 mg/kg was limited, it can be discussed whether the 300 mg/kg dose should be regarded as NOAEL level for this study.

 

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.

 

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

  

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.

An oral 28-day repeated dose toxicity study according to OECD 407 with Amides, fatty acids C18 unsat, reaction products with polyethylene amines resulted to a NOAEL of 100 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 ASAT), and it can be argued whether the 300 mg/kgbw should be selected as NOAEL in this study.

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