Reaction products of fatty acids, tall oil and fatty acids, C18 unsaturated, trimers and fatty acids, C18 unsaturated, dimers with (9Z)-octadec-9-en-1-amine

Endpoint summary

Description of key information

RA to oleylamine: 28-day, rat, oral: NOAEL 12.5mg/kg (OECD 407, GLP, Clariant 2003)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted July 27th, 1995

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

adopted July 30th, 1996

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: OPPTS 870.3050, Health Effects Test Guidelines: Repeated dose 28-day oral toxicity study in rodents, July 2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: app. 6 weeks
- Weight at study initiation: app. 126g (males), 123g (females)
- Fasting period before study: no
- Housing: 5 animals per cage in Makrolon type IV cages with soft wood granulate
- Diet (e.g. ad libitum): "ssniff" ad lib., except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water ad lib. except for the period in which the animals were kept in diuresis cages
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20%
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved daily in sesamy oil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 0.325 - 5 mg/mL
Volume of vehicle + test substance: 10ml/kg b.w.

Duration of treatment / exposure:

28days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
3.25, 12.5, 50mg/kg b.w.
Basis:
actual ingested

No. of animals per sex per dose:

5 (+ 5 per sex per recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a dose range finding study, 3 males and 3 females received 25, 100, and 400mg/kg of the test substance for 14 days. One high dose male and female died on days 4 and 7, respectively. The other animals of this dose group were killed for animal welfare reasons. Necropsy revealed changes in the the stomach and intestinal mucosa, probably due to the irritant properties of the test substance. Mid dose animals showed impairments of motility and respiration, with males being clearly more sensitive. Body weight gain was also impaired, and necropsy revealed reddening of the stomach mucosa. Except for one female, that showed uncoordinated gait on study day 2 only, no signs of toxicity were observed in the low dose group.
Based on the results, dose levels of 3.25, 12.50 and 50.00 mg/kg body weight per day were selected for the 4-week repeat dose gavage study.

- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, salivation, nasal discharge, piloerection, pupil size, and unusual respiratory pattern. Changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, tremor, and any other abnormal motor movements (such as excessive grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded. In addition, defecation and urination were evaluated.

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

FOOD CONSUMPTION:
- evaluated twice weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during detailed clinical examinations
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes (Ketamine-Hydrochloride + Xylazine)
- Animals fasted: No
- How many animals: all
- The following parameters were examined: erythrocyte count, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoblobin concentration, reticulocyte counts, hemoglobin, hematocrit, Heinz Body counts (only high dose and control), differential leukocyte counts, clotting time, thrombocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No
- How many animals: all
- The following parameters were examined: GGT, ALAT, albumin, albumin/globulin ratio, alkaline phosphatase, ASAT, bilirubin, Ca, Cl, cholesterol, creatinine, globulin, glucose, inorganic phosphorous, K, Na, total protein, triglycerides, urea, uric acid

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from days 25 to 26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: appearance, bilirubin, blood, color, glucose, ketone bodies, microscopic examination (control and high dose only), pH, protein, urobilinogen, specific weight

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at study termination
- Dose groups that were examined: all
- Battery of functions tested: sensory reactivity incl. startle response, response to approach, righting reflex, pupillary constriction, motor activity, forelimb and hindlimb grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: skin, orifices, eyes, teeth, oral mucosa, internal organs
HISTOPATHOLOGY: Yes
organ weights: adrenals, heart, spleen, testes, kidneys, brain, epididymides, liver, thymus
organs processed for histpathological investigations: adrenals, lung, stomach, bone marrow, lymph nodes (mandibular, iliac), testes, thymus, brain, epididymides, sciatic nere, thyroid gland, heart, ovaries trachea, small and large intestine, prostate, seminal vesicle, urinary bladder, uterus, kidneys, spinal cord, nasal cavities, liver, spleen, all gross lesions.

The lungs (intratracheally) and the nasoturbinates (in skull) were instilled/infused with formaldehyde (4 %). Following completion of the fixation, the lungs and nasoturbinates were fixed, together with the other organs, in formalin solution (4% neutral buffered formaldehyde).

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths throughout the study.
Clinical observations remained unaffected by the administration of the test compound in the low and intermediate dose groups.
In the high-dose group impairments of motility from the 2nd or 3rd week onwards (stilted or uncoordinated gait) were observed in 2 males and 1 female of the main group animals up to the end of the treatment period. These observations were also noted in 4 females in the high dose recovery group starting at the end of treatment (2nd or 3rd week), with subsequent recovery during the recovery period. Respiratory sounds were noted in one female of the high-dose group only on day 13 of the study.
No changes of the oral mucosa, or impairment of dental growth was observed in all groups.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was not influenced by treatment in low dose animals and mid dose females. The mean body weight was significantly decreased in high dose males and females (app. -10% for both genders) at the end of treatment, with clear subsequent recovery. Mean body weight was slightly but statistically significantly decreased for mid-dose males from study day 22 onwards until end of treatment. This finding was marginal, i.e., less than 5 % as compared to the control. Moreover, it was not observed for the mid-dose females. Hence, it was considered not toxicologically significant.

FOOD CONSUMPTION
unaffected by test substance

OPHTHALMOSCOPIC EXAMINATION
No opacity of the refracting media of the eyes was observed in all groups.

HAEMATOLOGY
- Slightly increased hematocrit values and slightly decreased reticulocyte counts for high-dose males (a similar tendency without statistical significance was noted for high-dose females), subsequent recovery
- Slightly increased white blood cell (WBC) counts for high-dose males and females (not statistically significant, however, for males outside the range of the historical control data), with a shift towards increased neutrophils in both genders, with subsequent recovery

CLINICAL CHEMISTRY
All observed differences to control animals were either within historical control data or showed no dose response relationship.

URINALYSIS
unaffected by test substance

NEUROBEHAVIOUR
Neurological measurements were unaffected by treatment.

ORGAN WEIGHTS
unaffected by test substance

GROSS PATHOLOGY
There were no gross pathology findings, which could be related to administration of the test compound.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no histopathological findings, which could be related to administration of the test compound.

Dose descriptor:

NOAEL

Effect level:

12.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weight

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

12.5 mg/kg bw/day

Additional information

No data are available for the Reaction products of Fatty acids, tall-oil, compds. with oleylamine and Fatty acids, C18-unsatd., trimers, compds. with oleylamine. But since the substance is a salt, it is expected to dissociate into fatty acids C18 -unsatd., trimers, fatty acids, tall oil, and oleylamine. So the test substance was assessed based on it ions, following the approach outlined below. A detailed justification for the performed read across is attached in IUCLID chapter 13.

Fatty acids, C18-unsatd., trimers, CAS 68937-90-6, has already been registered during the first phase of REACH. Extensive data exist either for fatty acids, C18-unsatd., trimers, or for the dimeric form, which has been used as a read across substance in the dossier. After acute oral exposure, no clinical signs or mortality was observed up to 5000mg/kg b.w. The substance did not cause skin or eye irritation, skin sensitization, or gene mutation. After repeated exposure via diet for 13 weeks, some changes in histopathology were observed at a dose just below 4000mg/kg, which is considerably higher than the limit dose of 1000mg/kg used in current protocols. No effects were observed in low dose animals, that received app. 800mg/kg. No effects on reproductive organs, fertility or developmental toxicity were observed in an OECD 421 screening study.

The main component of fatty acids, tall-oil is oleic acid (CAS 112-80-1), which is a natural constituent of vegetable and animal fat, e.g., 70-75% of olive oil are oleic acid dimers and trimers. In it monomeric form, oleic acid causes skin and eye irritation, is almost not toxic after acute exposure (LD50 app. 4200mg/kg, LD0 app. 2850mg/kg), and did not cause mutations in salmonella cells (NTP).

Because of the obvious non-toxicity of fatty acids, tall-oil and fatty acids, C18-unsatd., trimers, oleylamine was identified as the hazard inducing component and used for hazard assessment as a worst case approach. This substance is part of an EU defined category for primary alkyl amines (EU risk assessment for primary alkyl amines, 2008; CLH report for coco alkyl amine, 2010). Read across was performed to several members of this category, depending on availability of reliable studies. According to the CLH Dossier, which was adopted by the RAC in 2011, primary alkyl amines are classified to cause damage to the eyes, skin corrosion or irritation and respiratory tract irritation. In addition, organ toxicity (liver, GI-tract, immune-system) was observed, presumably secondary to the local irritant properties of the alkyl amine. Though the reaction mass of fatty acids, tall-oil, compds. with oleylamine and fatty acids, C18-unsatd., trimers, compds. with oleylamine is not corrosive but causes only skin irritation, similar effects as described below for the repeated dose study for oleylamine were also observed in the acute oral toxicity study at 2000mg/kg, i.e.,swollen lymph nodes and peyer's plaques in region of the small intestine and red discoloration of the small intestine and corresponding severe weight loss.

Oleylamine, CAS 112 -90 -3, OECD 407

Groups of five male and female SD rats received oleylamine in sesame oil by oral gavage at dose levels of 0, 3.25, 12.5, or 50 mg/kg bw/d for a period of 28 days (Clariant, 2003). On day 29, animals were necropsied. In the control and high dose groups, additional five male and females were examined and necropsied after a recovery period of 14 days.

Throughout the main study, treatment did not result in unscheduled deaths. Behaviour and state of health remained unaffected by the administration of the test compound in the low and mid dose groups. Clinical findings in the high dose group (2 males and 5 females out of 10) from the 2nd or 3rd week onwards comprised impairment of motility (stilted and/or uncoordinated gait) and lasted

until the end of treatment, with subsequent recovery, but neurobehavior was unaffected in any group. Mean body weight was significantly lower for high dose males from study day 11 and for high dose females from day 22 until the end of treatment (-10% for males and females), and remained different from that of the controls at the end of recovery period, though a tendency to recover was observed for females. Also mid-dose male body weights were slightly but statistically significantly decreased compared to the controls from study day 22 until the end of treatment (-7.5%). No significant changes in food consumption, heamatology, blood or urine chemistry, and histopathology were noted in all groups. Because the effect on body weight was only slight the no observed adverse effect level was set to 12.5mg/kg b.w.

Further studies

The summary is based on the data provided in theEU risk assessment for primary alkyl amines, 2008.

In a subacute study by APAG (2000) with Tallow alkyl amine (CAS 61790 -33 -8) according to GLP and OECD 407 (no neurotoxicity testing was perfomed), the test substance was administered in sesame oil to Sprague Dawley rats at dosages of 0, 12.5, 50, and 150 mg/kg bw/d once a day for 4 consecutive weeks.

At 150 mg/kg/day, the test compound induced salivation after treatment, piloerection, hunched posture, fur loss and soft stools variously associated, and a marked decrease in body weight (-21 to -25%, f/m) and food consumption. Two males and three females died between day 8 and 27. Histopathology revealed erosions in the gastric and small intestinal mucosa and abnormalities in the lungs (probably due to inhalation of test substance particals during gavage procedure), which were consistent with inflammations caused by the strong irritative properties of the test compound. These findings were considered local rather than a systemic effects. These local effects on the gastro-intestinal or the respiratory system can be considered as the cause of death for the 5 animals. In the surviving animals gastro-intestinal changes resulted in emaciation due to malabsorption. Single cell necrosis was observed in the liver, which fits increased values for ALAT and ASAT. This finding was associated with inflammation and histiocytic accumulation in the mesenteric lymph nodes and the lamina propria of the small intestine. Histiocytosis of the small intestine and mesenteric lymph-nodes can be associated with the general picture of the known "accumulation enteropathies", which involve the absorption and accumulation in the small intestine of a variety of orally administered compounds or their products. Presumably as a response to stress, thymus weights were decreased, while adrenal weights were increased in this group. Further decreases in absolute or relative organ weight are considered to be related to general growth depression in this group.

At 50 mg/kg/day one female died on day 11, showing lung changes as previously described and considered the cause of death. Body weights were reduced by 5% and 12% for males and females, respectively. Food consumption was also reduced. Significant decreased liver and kidney weights are most likely related to general body weight loss. Increased leukocytes, neutrophils, and platelets are considered to be related to inflammation in the GI tract due to the corrosive properties of the amine. Besides minor changes in blood chemistry, moderate histiocytic accumulations were observed in the mesenteric lymph nodes and the lamina propria. Reduced thymus weight was confined to one male and one female.

At the low dose, 12. 5 mg/k/day, treatment-related changes were limited to slight histiocytic vacuolation of mesenteric lymph nodes and small intestine, without abnormal histiocytic accumulation.

One 209 -day study, and one 2 -year study in rats exist for octadecylamine (CAS 124 -30 -1). It was also administered for 1 year to dogs (Deichmann, 1958). All studies are less reliable due to outdated protocols, missing control groups, and high incidences of pulmonary infections in all dose groups including controls. The main findings were reduced body weight and histiocytic accumulations in mesenteric lymph nodes, lamina propria and liver at 88mg/kg after 209 days. After two years, neither these findings nor any other differences were observed at a lower dose of 28mg/kg in rats. At 15mg/kg one dog died due to gastro-intestinal irritation, while some histiocytes were observed in the mesenteric lymph nodes of the two surviving animals. No effects were observed at 8mg/kg.

These finding basically confirm the results from the reliable guideline studies, i.e., the primary effect is local irritation of the gastro-intestinal tract, which is accompanied by "accumulation enteropathies" and inflammation, and causes malabsorbtion and weight loss. Since the no effect level is comparable to the one observed in the subacute studies, no bioaccumulation and worsening of effects is expected after chronic treatment.

Justification for classification or non-classification

The effects of the primary alkyl amines (oleylamine and tallow alkyl amine) are comparable. The NOAEL was set to 12.5mg/kg, based on reduced body weight. In the study using tallow alkyl amine, histiocytic accumulations in the mesenteric lymph nodes were additionally observed (these lymph nodes were not examined in the study using oleylamine). This finding can be associated with the general picture of the known "accumulation enteropathies", which involve the absorption and accumulation in the small intestine of a variety of orally administered compounds or their products, and is especially frequently observed in studies using oil as a vehicle. Accompanying inflammation and malabsorbtion is probably due to the irritating properties of the test substance in the gastro-intestinal tract. Effects observed on other organs like liver, thymus, spleen, and adrenals at exceedingly toxic doses, i.e., more than half of the animals died, are likely due to stress and poor general state of the animals. At these dosages, severe local irritation and cachexia were observed, which are assumed to be the cause for the poor general state, since no other causes were found. Many of the effects observed might not be observed with the reaction mass of fatty acids, tall-oil, compds. with oleylamine and fatty acids, C18-unsatd., trimers, compds. with oleylamine, since the substance is not corrosive, but causes only skin irritation.

But following a worst case approach, since systemic effects cannot be excluded without doubt without further data, the reaction mass of fatty acids, tall-oil, compds. with oleylamine and fatty acids, C18-unsatd., trimers, compds. with oleylamine will be labelled in agreement with the EU-CLH dossier for primary alkylamines with R48/22 according to 67/548/EEC and as STOT RE cat. 2 (may damage the gastro-intestinal tract, the liver, and the immune system) according to CLP/EU-GHS.