C16-18-(even numbered)-alkyl fatty acid, compound with C16-18-(even numbered)-alkylamine

Endpoint summary

Description of key information

For the registered salt:
- 14d range-finder (OECD 407): NOAEL 150 mg/kg bw/day 
Z-octadec-9-enylamines (CAS: 112-90-3)
- 14d range-finder (OECD 407): NOAEL 25 mg/kg bw/day 
- Key study 28d (OECD 407, GLP): NOAEL 3.25 mg/kg bw/day (corresponding to 6.7 mg/kg bw/day of the registered salt.
Tallow alkylamine (CAS: 61790-33-8)
- 14d range-finder (OECD 407): NOAEL 62.5 mg/kg bw/day 
- 28d (OECD 407, GLP): NOAEL 12.5 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 August 2012 - 27 November 2012

Reliability:

other: Klimisch scale not applicable

Rationale for reliability incl. deficiencies:

other: range-finding study: complies neither with GLP nor with a testing guideline.

Qualifier:

no guideline followed

Principles of method if other than guideline:

range-finding study: does not comply with a testing guideline

GLP compliance:

no

Remarks:

not required for a range-finding study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 6 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 188 g (range: 178 g to 202 g) and the females had a mean body weight of 165 g (range: 152 g to 187 g)
- Fasting period before study: no
- Housing: the animals were housed by four, in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 29 August 2012 to 12 September 2012.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed progressively with the required quantity of vehicle. The dose formulations were then kept under magnetic stirring for at least 30 minutes, in a bath water at approximately 40°C.
The dose formulations were prepared daily, stored at room temperature and delivered to the study room in brown flasks.

VEHICLE
The vehicle used in this study was selected from the results of solubility assays performed by the CiToxLAB France Pharmacy, where a homogenous suspension was obtained in corn oil at the concentration of 90 mg/mL.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
50, 150 and 450 mg/kg/d
Basis:
actual ingested

No. of animals per sex per dose:

4 males and 4 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor based on the results of repeated toxicity studies performed with primary alkylamines.
The substance ACAA was a salt composed of primary hydrogenated alkylamine and N-hydrogenated tallow fatty acid. It was assumed that the acid part had no toxicological impact. Consequently, the toxicity of the substance could be attributed to the primary alkylamines part. The aim of this study was to demonstrate that the test item could be integrated in the current approach of grouping of primary alkylamines substances. In a preliminary study, rats received orally Z-octadec-9-enylamines (CAS: 112-90-3) considered as the worst case of the category approach, at dose-levels of 25, 100 and 400 mg/kg/day over a period of 14 days. Mortality and premature sacrifices occurred in the high-dose-group. The animals of the 100 mg/kg/day group (especially the males) showed clinical signs of impaired motility and respiration. After administration of 25 mg/kg/day no symptoms were observed except of one female rat, which showed uncoordinated gait at study day 2. Necropsy of the dead and prior killed animals showed changes in the stomach and intestinal mucosa. The animals of 100 mg/kg/day showed reddening of the stomach mucosa. In the main study, treatments of 3.25, 12.5 and 50 mg/kg/day for 28 consecutive days resulted in no unscheduled deaths but clinical signs were noted in the high-dose group from the second week until the end of the study. Mean body weight gain was significantly lower at the end of treatment for high-dose males (-19%), high-dose females (-20%) and for mid-dose males (-15%) compared to control animals. Some slight and reversible hemato-biochemical changes were noted in the high-dose group including ASAT and ALAT activity in the liver of high-dose males. No anatomic pathology correlates (organ weights, macroscopy and microscopy) of toxicological significance were detected.Tallow alkylamine (CAS: 61790-33-8) given to rats by oral gavage in a 14-day dos range finding study at doses of 62.5, 250 and 1000 mg/kg/day, appeared to be satisfactorily tolerated only at the lowest dose-level, inducing only mild clinical effects and slight dilation of the intestinal tract. At higher doses the compound was poorly tolerated causing body weight depression, moderate to severe impairment of clinical conditions and dilation of the stomach and/or intestinal tract. At 1000 mg/kg/day these changes led to death of all the animals. In the main study, tallow alkyl amine when given orally at 0, 12.5, 50 and 150 mg/kg/day for 4 consecutive weeks to rats was found to be well tolerated only at the lowest dose-level which did not cause any relevant effects. At the two higher doses, the compound appeared poorly tolerated due to its strong irritative properties which caused local gastro-intestinal modifications and lung inflammatory changes, the
latter possibly as a consequence to the inhalation of foamy saliva occurring after gavaging (a well known phenomenon for this type of materials). The gastro-intestinal and lung alterations were the cause of some premature deaths. In addition the involvement of the digestive system resulted in malabsorption mainly evident at 150 mg/kg/day.

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day from the start of the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: once a day.

BODY WEIGHT:
- Time schedule for examinations: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, and over periods of 3 or 4 days including the eve of sacrifice.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded on same intervals that the body weight.
Food consumption was calculated per animal and per day.

HAEMATOLOGY:
- Time schedule: at the end of the treatment period.

BLOOD BIOCHEMISTRY:
- Time schedule: at the end of the treatment period.

Sacrifice and pathology:

ORGAN WEIGHTS:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, testes, thymus, thyroids with parathyroids and uterus.

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all animals.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

only at 450 mg/kg/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

only at 450 mg/kg/day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

only at 450 mg/kg/day

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

only at 450 mg/kg/day

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
No unscheduled deaths occurred during the study.

CLINICAL SIGNS:
Piloerection and hunched posture were observed on the last day of treatment in 2/4 females given 450 mg/kg/day.
Ptyalism was observed at the end of the treatment period in 4/4 males given 450 mg/kg/day and 1/4 males given 150 mg/kg/day. This finding, commonly observed when a test item is administered as a suspension by the oral route, was attributed to the test item treatment but was considered not to be an adverse effect.

BODY WEIGHT:
When compared to controls, males and females treated at 450 mg/kg/day showed a lower mean body weight gain between days 1 and 14, that was more pronounced in females.
This effect on body weight was considered to be treatment-related and adverse at the high dose-level.
No relevant differences were observed between controls and animals given 50 or 150 mg/kg/day.

FOOD CONSUMPTION:
When compared to controls, lower mean food consumption was observed over the treatment period and more markedly during the last days, in males and females given 450 mg/kg/day. This change, correlating with lower body weight in these animals, was attributed to the test item treatment and was considered to be adverse at the high dose-level.
No relevant differences were observed between controls and animals given 50 or 150 mg/kg/day.

HAEMATOLOGY:
When compared to controls, higher mean white blood cell counts (generally including neutrophils, basophils, lymphocytes and monocytes counts) were observed at the end of the treatment period in males (with statistical significance) and females given 450 mg/kg/day.
Statistically significantly prolonged mean prothrombin time was observed in high-dose females. The same trend was observed in males without statistical significance.
Lower mean cell volume was observed in all test item-treated groups of both sexes. In the absence of other relevant variations among red blood cell parameters, this change was considered to be of no toxicological importance.

BLOOD BIOCHEMISTRY:
When compared to control values, higher mean aspartate and alanine aminotransferase activities were observed in females given 450 mg/kg/day. The same trend, although of small magnitude, was observed in males given the same dose-level. This was accompanied in females by lower mean alkaline phosphatase activity.
Lower mean proteins, albumin and cholesterol levels were also observed in high-dose animals.

ORGAN WEIGHTS:
Higher relative-to-body adrenal weights and lower absolute and relative-to-body thymus weights were noted in females treated at 450 mg/kg/day. These weight differences correlated with minimal thymus atrophy in two females. A relationship to treatment with ACAA could not be excluded, most likely as a result of stress.
Lower absolute and/or relative weights of the liver, heart, prostate, and testes were noted in males and higher spleen weights in females treated at 450 mg/kg/day. These weight differences did not correlate with any macroscopic or microscopic findings and therefore, a relationship to treatment was considered to be unlikely.
Other weight differences were minimal, not dose-related or did not correlate with macroscopic or microscopic findings and therefore a relationship to treatment was considered to be unlikely.

GROSS PATHOLOGY:
No macroscopic findings were attributed to treatment with the test item.
The gross findings were of those commonly recorded in the rat or were also observed in controls and therefore a relationship to treatment was considered to be unlikely.

MICROSCOPIC EXAMINATION:
Treatment-related microscopic findings were observed in the mesenteric lymph nodes at all dose levels, in the liver, jejunum and ileum in males and/or females at 150 and/or 450 mg/kg/day.
Foamy macrophages, characterized by expanded and clear cytoplasm, were noted in the medulla of mesenteric lymph nodes and in the lamina propria of jejunum/ileum.
Minimal thymus atrophy was noted in 2/4 females treated at 450 mg/kg/day. As this finding correlated with decreased thymus weights and was also associated with higher adrenal weights, a relationship to treatment could not be excluded, most likely as a result of stress.
Other microscopic findings were of those commonly reported in the rat and therefore were considered not to be treatment-related.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: does not cover guideline endpoints (range-finding study)

Critical effects observed:

not specified

Conclusions:

The toxicity of the test item was evaluated in Sprague-Dawley rats following daily oral administration (gavage) for 2 weeks at dose-levels of 50, 150 and 450 mg/kg/day.
At 450 mg/kg/day in both sexes, the test item induced lower body weight gain and changes in clinical pathology parameters consisting of higher white blood cell count, prolonged prothrombin time and higher aspartate/alanine aminotransferase activities, with lower proteins, albumin and cholesterol levels. Higher adrenal and lower thymus weights were noted in females treated at 450 mg/kg/day. Foamy macrophages were observed in the mesenteric lymph nodes, jejunum and ileum (females only), together with increased inflammatory cell foci in the liver.
Except for foamy macrophages in the mesenteric lymph nodes, no relevant effects were observed at 150 or 50 mg/kg/day.
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 14 days.

 

Methods

Three groups of four male and four female Sprague-Dawley rats received the test item daily by gavage at dose-levels of 50, 150 and 450 mg/kg/day for 2 weeks. A further group of four males and four females received the vehicle alone (corn oil) under the same experimental conditions, and acted as a control group. The dosage forms were administered under a constant dosage-volume of 5 mL/kg/day.

 

The animals were checked daily for mortality and clinical signs. Body weight was recorded once during the pre-treatment period, on the first day of treatment and then on days 4, 8, 11 and 14. Food consumption was recorded twice a week during the study. Hematology and blood biochemistry investigations were performed at the end of the treatment period (day 15).

On completion of the treatment period, the animals were sacrificed and submitted for a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from all animals.

 

Results

No unscheduled deaths occurred during the study.

 

Piloerection and hunched posture were observed on the last day of treatment in 2/4 females given 450 mg/kg/day. Ptyalism was observed at the end of the treatment period in 4/4 males given 450 mg/kg/day and 1/4 males given 150 mg/kg/day.

 

Lower body weight, correlating with lower food consumption, was observed throughout the treatment period in males and females given 450 mg/kg/day.

 

At hematology, higher white blood cell counts (generally including neutrophils, basophils, lymphocytes and monocytes counts) and prolonged prothrombin time (more marked in females) were observed in males and females given 450 mg/kg/day.

 

At blood biochemistry, higher aspartate and alanine aminotransferase activities were observed in females, and to a lesser extent, in males given 450 mg/kg/day. This was accompanied in females by lower alkaline phosphatase activity. Lower proteins, albumin and cholesterol levels were also observed in high-dose animals.

At pathology, higher relative-to-body adrenal weights and lower absolute and relative-to-body thymus weights were noted in females treated at 450 mg/kg/day. These weight differences correlated with minimal thymus atrophy in two females. A relationship to treatment with the test item could not be excluded, most likely as a result of stress.

No treatment-related macroscopic findings were observed.

Treatment-related microscopic findings were observed in the mesenteric lymph nodes at all dose levels, in the liver, jejunum and ileum in males and/or females at 150 and/or 450 mg/kg. More specifically, foamy macrophages, characterized by expanded and clear cytoplasm, were noted in the medulla of mesenteric lymph nodes and in the lamina propria of jejunum/ileum. Increased severity of inflammatory cell foci consisting primarily of macrophage aggregates was also observed in the liver parenchyma of females at 150 or 450 mg/kg/day.

 

Conclusion

The toxicity of the test item was evaluated in Sprague-Dawley rats following daily oral administration (gavage) for 2 weeks at dose-levels of 50, 150 and 450 mg/kg/day.

 

At 450 mg/kg/day in both sexes, the test item induced lower body weight gain and changes in clinical pathology parameters consisting of higher white blood cell count, prolonged prothrombin time and higher aspartate/alanine aminotransferase activities, with lower proteins, albumin and cholesterol levels.Higher adrenal and lower thymus weights were noted in females treated at 450 mg/kg/day. Foamy macrophages were observed in the mesenteric lymph nodes, jejunum and ileum (females only), together with increased inflammatory cell foci in the liver.

 

Except for foamy macrophages in the mesenteric lymph nodes, no relevant effects were observed at 150 or 50 mg/kg/day.

 

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study according to GLP but the reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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, Germany
- Age at study initiation: approx. 6 weeks
- Fasting period before study: no
- Housing: transparent macrolon cages (type IV)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 hours periodically

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 applications in 29 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0 mg/kg body weight per day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
3.25 mg/kg body weight per day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
12.5 mg/kg body weight per day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg body weight per day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary dose-range-finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery group
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment, then twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined continously two times per week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination and after recovery period
- Anaesthetic used for blood collection: Yes (ketamin-hydrochloride + xylazine)
- Animals fasted: No
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after blood sampling for haematological testing, animals were killed and exsanguinated
- Animals fasted: No
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine:urine collection overnight from day 25 to day 26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes: food and water was withdrawn during this period

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before first treatment, then once a week
- Dose groups that were examined: all
- Examinations: at all examinations: changes in appearance, occurence of secretions/excretions, autonomic activity (lacrimation, salivation, nasal discharge, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic/tonic movements, tremor, other abnormal motor movements (excessive grooming, repetitive circling, other stereotypes), bizarre behaviour, defecation, urination
- Battery of functions tested: at termination of the study:sensory activity, grip strengt, motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: macroscopic examinations: skin, orifices, eyes, teeth, oral mucosa, internal organs
HISTOPATHOLOGY: Yes (macroscopic and microscopic examinations): adrenals, bone marrow (sternum), brain with medulla oblongata, epididymides, heart, small intestine (jejunum), large intestine (colon) kidneys, liver, lungs, lymph nodes (mandibular, iliac), nerve (sciatic), ovaries with oviducts, prostate, seminal vesicle, spinal cord (cervical) spleen, stomach, testes, thymus, thyroid gland with parathyroids, trachea with larynx, urinary bladder, uterus, nasal cavities

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

stilted gait in high-dose animals, no unscheduled deaths

Mortality:

mortality observed, treatment-related

Description (incidence):

stilted gait in high-dose animals, no unscheduled deaths

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased in high-dose males and females, and mid-dose males

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

slightly increased neutrophils in high-dose animals

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

slightly increased ASAT and ALAT activity in high-dose males

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- no unscheduled deaths throughout the study
- 50 mg/kg/day: motility impairment from 2nd/3rd week in 2 males, 1 female (main group) and in 4 females (with recovery) in the recovery group;
respiratory sounds (1 female, day 13)
- 12.5 mg/kg/day: no changes
- 3.25 mg/kg/day: no changes


BODY WEIGHT AND WEIGHT GAIN
- 50 mg/kg/day: approx. 10% decrease in body weight in males from day 11 onwards for about; the effect showed a tendency of recovery. Mean body weight was decreased in females from day 22 onwards, with clear subsequent recovery. Mean overall body weight gain over the study period was moderately affected.
- 12.5 mg/kg/day: Slight decrease (<5%) of mean body weight and mean overall body weight gain in males from day 22 onwards; the effects were considered not to be toxicologically relevant, since they were marginal and not recorded in females.
- 3.25 mg/kg/day: no significant changes


FOOD CONSUMPTION AND COMPOUND INTAKE
remained unaffected in all groups


HAEMATOLOGY
- 50 mg/kg/day: males: slightly increased haematocrit, slightly decreased reticulocyte counts, subsequent recovery; females: similar tendency without statistical significance. Slightly increased white blood cell counts with shift towards neutrophils in males and females (not stat. significant but
outside range of historical controls). All findings being reversible.
- 12.5 mg/kg/day: no significant changes
- 3.25 mg/kg/day: no significant changes


CLINICAL CHEMISTRY
50 mg/kg/day: increased total bilirubin in males and females, slightly increased urea nitrogen in females and ASAT and ALAT activity in males.
12.5 mg/kg/day: increased total bilirubin and slightly increased urea nitrogen in females.
3.25 mg/kg/day: increased total bilirubin in females

URINALYSIS
remained unaffected in all groups

NEUROBEHAVIOUR
- Open field observations, assessment of sensory function, and forelimb and hindlimb grip strength not influenced in all groups.
- Negative trend in number of movements in males, but not significant and not present in females. Effect within the range of historical (inhouse)
controls, and thus, this was not considered to be related to treatment.

ORGAN WEIGHTS
- 50 mg/kg/day: Statistically significantly decrease of heart and brain weights in females and tendencially in males. Increase in relative adrenal weight (main group) and relative spleen weight (recovery group) in males. Since the effects were related to decreased terminal body weight and no histologically correlate was found, they are regarded to be no organ specific effects.
- 12.5 mg/kg/day: no changes
- 3.25 mg/kg/day: no changes

GROSS PATHOLOGY
No gross pathology findings attributable to administration of test compound in all dose groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
No histopathological findings which could be related to the administration of the test compound in all test groups. Sporadically observed changes occurred at an incidence and severity historically seen for rats of this age and strain.

HISTOPATHOLOGY: NEOPLASTIC
No neoplastic chages observed in any dose group.

Dose descriptor:

NOAEL

Effect level:

3.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Conclusions:

Based on the test results the study director concluded that repeated administration of genamin OL 100 D at the high dose level of 50 mg/kg body weight per day induced general clinical signs, impairment of body weight gain and mild clinical pathology findings without histopathology correlates. It is concluded that the NOAEL from this valid oral 28-day study is 3.25 mg/kg body weight per day.

Executive summary:

Groups of five male and female SD-rats received octadecenylamine (Genamin OL 100 D, vehicle sesame oil) by oral gavage at dose levels of 0,3.25,12.5 or 50 mg/kg bw/day for a period of 28 days. 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. The study design and examinations conducted were in full accordance to the EU method B.7 (OECD 407) for subacute oral toxicity (including neurobehavioral observation and functional observation battery testing). In a preliminary study on dose-range finding three males and females received the test substance at doses of 25, 100 and 400 mg/kg bw/d over a period of 14 days and surviving animals were necropsied on day 15. After administration of 400 mg/kg bw/d one male and one female died at days 4 and 7, respectively. The other animals of this dose group were killed for animal welfare reasons. The animals of the 100 mg/kg bw/d group showed clinical signs of impaired motility and respiration. The male animals were clearly more sensitive than the female animals. After administration of 25 mg/kg bw/d no symptoms were observed except of one female rat, which showed uncoordinated gait at study day 2. The body weight gains of animals exposed to doses of 25 and 100 mg/kd bw/d were impaired. Necropsy of the descendent and prior killed animals showed changes in the stomach and intestinal mucosa. The animals of 100 mg/kg bw/d showed reddening of the stomach mucosa. No macroscopically visible changes were observed in the 25 mg/kg dose groups. In the main study, treatment resulted in no unscheduled deaths throughout the study. 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 impairments of motility (stilted and/or uncoordinated gait) and lasted until the end of treatment, with subsequent recovery. In addition, respiratory sounds were noted in one high dose female only on study day 13. No opacity of the refracting media of the eyes, changes of the oral mucosa, or impairment of dental growth was observed. No abnormal neurobehavior was observed 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, and remained to be different as compared to the controlat the end of recovery period. Also mean body weights for mid dose males were significantly lower from day 22 onwards to the end of treatment. Compared to control animals, mean body weight gain was significantly lower at the end of treatment for high dose males (-19%), high dose females (-20%) and for mid dose males (-15%). A dose-dependent, small (-9%, statistically non-significant) reduction of body weight gain was also reported for the low dose males. The weight gain was normalised at the end of the recovery for high dose males or even higher in the high dose female group indicating a tendency to recovery (no recovery conducted for the mid dose group). Food consumption remained unaffected throughout the study in all dose groups. Hematology findings in high dose groups included significantly increased hematocrit and decreased reticulocyte counts (males only) and slightly increased white blood cell counts with a shift towards increased neutrophils (both genders), all findings being reversible. Clinical chemistry changes comprised significantly increased total bilirubin for the high dose group, slightly increased urea nitrogen for mid and high dose females and very slightly increased ASAT and ALAT activity in the liver of high dose males. Urinalysis remained unaffected in all dose groups. Likewise the urine sediment was unobtrusive for control and high dose group animals. No anatomic pathology correlates (organ weights, macroscopy, microscopy) of toxicological significance were detected. In conclusion, repeated administration of Genamin OL 100 D (octadecenylamine) at a dose of 50 mg/kg bw/day induced clinical signs as gait abnormalities, reduction in body weight gain and clinical pathology findings indicating mild toxic effects on the liver and kidneys. The only treatment-related effects observed at the mid-dose level were reduction in growth and increased urinary concentration of urea nitrogen. By standard requirements on screening for neurotoxicology given for this type of study, stilted gait or uncoordinated gait was not associated with any other symptom of altered neurobehavior or neurotoxicity and may be discussed as being of unspecific nature. In the preliminary study, macroscopic findings in the gastrointestinal mucosa were observed in early deaths and in animals receiving 100 mg/kg bw/d, but were absent in animals at a dosage of 25 mg/kg bw/d from the 14-day study and in dose groups receiving 50 mg/kg bw/d. Except for growth reduction, full recovery of findings was seen at the end of recovery period. Based on the significantly reduced body growth at 12.5 mg/kg bw/d observed in the present 28-day study, the NOAEL of 3.25mg/kg bw/d was derived.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Data are coming from literature referring to studies conducted prior to existing test guidelines. However, data had been reviewed based on nowadays standards and had been included in the existing EU risk assessment on primary fatty amines.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Feeding Study

GLP compliance:

not specified

Remarks:

GLP was not implemented at time of study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
20 ppm, 100 ppm, 200 ppm, 500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

12

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: not stated

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not stated
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not stated

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

accumulation of histiocytes in mucosa of small intestine and mesenteric lymph nodes

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the exisitng EU Risk Assessment on primary Alkylamines, 200 ppm (10 mg/kg bw/d) is proposed as NOAEL for the 2-year rat studies from the study of Deichmann et al. (1958)

Critical effects observed:

not specified

Conclusions:

The study director concluded a NOAEL of 500 ppm (100 mg/kg body weight per day). However, based on the available data and performed reviews, a NOAEL for this 2-year study in rats of 200 ppm (10 mg/kg body weight per day) is proposed in the existing EU Risk Assessment of primary fatty
amines.

Executive summary:

A 2-year toxicity study of octadecylamine ("stearamine") was conducted using Sprague-Dawley rats. Four groups of 24 rats (12/sex) were fed diets containing 20, 100, 200, and 500 ppm stearamine (reviewed by Pang, 1995, original dataUniversityofMiami, 1957, Deichmann et al., 1958). A control group of animals was fed the base diet alone. The organs examined microscopically included the heart, lungs, liver, spleen, kidneys, gastroenteric tract (3 levels), pancreas, lymph nodes, bone marrow, brain, pituitary, adrenals, and ovary or testis. The feed consumption, growth rate, death rate, and blood cell counts for the experimental animals were comparable to those of the control animals. The survival rate for this study was 17-33% including the control group (meaning that 2, 3 or 4 animals/dose only survived until the end of study). In both control and experimental rats there was a higher than expected rate of mortality due to respiratory infections associated with chronic pneumonia and multiple organ inflammation. One rat receiving 500 ppm showed histiocytic hyperplasia of the mesenteric lymph node. At necropsy, no significant differences were found between the experimental and control groups in the incidence and types of lesions observed.[Calculated from diet consumption/day, the mean dose was about1.05,5.79, 10.68, and 27.15 mg/kg/day for 350 g males and1.20,5.80,10.08,and 28.20 mg/kg/day for 250 g females. No other information available.]. The study director concluded a NOAEL of 500 ppm (100 mg/kg body weight per day). However, since histiocytic accumulations were also present in high dose (3000 ppm) animals from a previous range-finder study, the single case of histiocytic hyperplasia in the mesenteric lymph node of the single rat treated with 500 ppm (25 mg/kg bw/d) for 2 years seen in this study could be interpreted as a treatment-related adverse effect. Therefore a NOAEL of 200 ppm (10 mg/kg bw/d) for this 2-year rat study on dodecylamine is proposed in the existing EU risk assessment on primary fatty amines.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: Dose-Range finder

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999/2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Dose-range finding study based on OECD guideline 407 for subacute 28 day study according to GLP but the reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

14 day exposure period

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: Charles River Italia
- Age at study initiation: ca. 4 weeks
- Weight at study initiation: 75 - 85 g (males); 60 - 70 g (females)
- Fasting period before study: 5 days
- Housing: wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 hour interval

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days
7 days/week

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
62.5, 250, 1000 mg/kg bw/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

4 animals / sex / group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose range finder
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: n.a.
- Post-exposure recovery period in satellite groups: n.a.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed for their physical appearance, behaviour and general and local clinical signs daily and deviations from normality were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the animals were observed twice a day for mortality and daily for the occurence of toxicity signs (general and local clinical signs).

BODY WEIGHT: Yes
- Time schedule for examinations: prior the beginning of the treatment, twice per week throughout the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- twice a week for each cage (2 animals); individual food consumption was calculated to g/ animal/ day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 15)
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: all


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 15)
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period (day 15), pretreatment before sampling: tap water 10 mL/kg bw (gavage)
- Metabolism cages used for collection of urine: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: skin and mammary gland, urinary bladder, prostate, testes*, epididymides, seminal vesicles, vagina, uterus, ovaries*, spleen*, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), mesenteric lymph nodes, pancreas, liver*, kidneys*, adrenals*, submandibular salivary glands and lymph nodes, sternum with bone marrow, heart*, thymus, lungs, aorta, trachea, esophagus, thyroids with parathyroids, eyes, Harder´s lacrimal glands, tongue, brain*, pituitary*, skeletal muscle (biceps femoris), peripheral nerve (sciatic nerve), spinal cord (thoracic, cervical, lumbar), vertebrae, femur (including articular surface), gross lesions. Organs with * = weighed organs
HISTOPATHOLOGY: Yes: examination of liver and spleen in all animals

Statistics:

Absolute figures and mean and standard deviation were expressed.
Parameters statistically examined were: body weight, body weight gain, food consumption, hematology, blodd chemistry, urinalysis, organ weights.
Barlett´s test for homogeneity of variance:
If homogeneous: ANOVA
If not homogeneous: Kruskal-Wallis test/ANOVA

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:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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
1000 mg/kg/day: 100% lethality between days 4 and 10. Before exitus the animals showed severe impairment of clinical conditions. Main clinical changes were: hypomotility, hunched posture, piloerection, salivation after treatment, abdominal dilation and soft stools.
250 mg/kg7day: one male died on day 13. Clinical sings of this animal: piloerection, salivation, thinness and hunched posture. The surviving animals had a symptomatology similar but less severe than in the highest dosage group.
62.5 mg/kg/day and control: neither mortality nor clinical changes were observed.

BODY WEIGHT AND WEIGHT GAIN
1000 mg/kg/day: strong inhibition of body weight gain
250 mg/kg/day: body weight was markedly decreased in both sexes starting from day 7. At the end of the study, the mean body weight was -36% (males) and -26% (females) compared to controls.
62.5 mg/kg/day: a trend to decrease in mean body weight was seen in males. No changes were observed in females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
1000 and 250 mg/kg/day: marked decrease in food consumptions was observed in both sexes
62.5 mg/kg/day: slight decrease in males, but no appreciable effects in females.

HAEMATOLOGY
1000 mg/kg/day: in males a significant increase of erythrocytes, neutrophils and prothrombin time related to a decrease of lymphocytes, MCV, MCH and platelets was observed. In females, an increase of neutrophils and decrease of eosinophils, MCV and MCH was seen.
No important changes were seen in the lower dosage groups. Statistical variations including an increase in erythrocytes (males, 250 mg/kg), neutrophils (males, 250 and 62.5 mg/kg; females 250 mg/kg) an prothrombin time (males, 250 mg/kg) and a decrease in MCV and MCH (males/females 250 mg/kg) and platelets (males 250 mg/kg) were regarded as trends rather than related to treatment.

CLINICAL CHEMISTRY
250 mg/kg/day: moderate increase in urea serum level and SGOT, slight to moderate decrease in alkaline phosphatase activity, moderate to severe increase in SGPT with pathological relevance, slight decrease in total serum protein, minor variations in serum electrolytes, moderate decrease in total cholesterol (females)
62.5 mg/kg/day: slight to moderate decrease in alkaline phosphatase activity, moderate to severe increase in SGPT

URINALYSIS
no changes

ORGAN WEIGHTS
Statistically significant changes in absolute (decrease) or relative (increase) organ weights compared to controls were seen in almost all organs from the survivors dosed at 250 mg/kg/day. All were considered cinsequent to the marked decrease in final body weight observed.

GROSS PATHOLOGY
250 mg/kg/day: macroscopic: moderate dilation of the stomach and/or intestinal tract, soft stools in the large intestine; moderate decrease in size of epididymides and seminal vesicles (considered to be due to the marked decrease in body weight associated with the animals´young age.
62.5 mg/kg/day: dilation of isolated tracts of intestine at different levels, soft stools

HISTOPATHOLOGY: NON-NEOPLASTIC
250 mg/kg/day: slight or minimal hepatocellular atrophy in two rats and slight to marked follicular atrophy of the spleen were observed, related with the decreased body weight. Since splenic atrophy is regarded as non-specific reaction to stress or severe weight loss, the effects are deemed to be not related to treatment.

Dose descriptor:

LOAEL

Effect level:

62.5 other: mg/kg

Sex:

male/female

Basis for effect level:

other: mild clinical effects, slight dilation of intestinal tract

Critical effects observed:

not specified

Executive summary:

Genamin TA 100, given to rats by oral gavage in a 14 -day dose range finding study at doses of 62.5, 250 and 1000 mg/kg body weight per day, appeared to be satisfactorily tolerated only at the lowest dose, inducing only mild clinical effects and slight dilation of the intestinal tract. At higher doses the compound was poorly tolerated causing body weight depression, moderate to severe impairment of clinical conditions and dilation of the stomach and/or intestinal tract. At 1000 mg/kg body weight per day these changes led to death in 100 % of the animals.

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

1999 - 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study according to GLP but the reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

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: Charles River Italia
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 75 - 85g (male animals), 60 - 70g (female animals)
- Fasting period before study: no
- Housing: wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 hour interval

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): sesame oil (solubility reasons)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test article formulate analysis was carried out by HPLC and involved the determination and evaluation of stability and test concentrations used.

Duration of treatment / exposure:

28 days
7 days/week

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
12.5, 50, 150 mg/kg bw/day
Basis:
other: analytical verified

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on preliminary dose-range-finder study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: n.a.
- Post-exposure recovery period in satellite groups: n.a.
- Section schedule rationale (if not random): random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed for their physical appearance, behaviour and general and local clinical signs daily and deviations from normality were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the animals were observed twice a day for mortality and daily for the occurence of toxicity signs (general and local clinical signs).

BODY WEIGHT: Yes
- Time schedule for examinations: prior the beginning of the treatment, then weekly throughout the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- weekly for each cage; individual food consumption was calculated to g/ animal/ day.

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes were examined (naked and by means of an ophthalmoscope) 4-5 days before and at the end of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 29)
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 29)
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period (day 29), pretreatment before sampling: tap water 10 mL/kg bw (gavage)
- Metabolism cages used for collection of urine: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: (skin and mammary gland), urinary bladder, prostate, testes*, epididymides*, (seminal vesicles), (vagina), uterus, ovaries, spleen*, stomach, intestine (duodenum, jejunum, ileum, cecum, colon, rectum), mesenteric lymph nodes, pancreas, liver*, kidneys*, adrenals*, (submandibular salivary glands), mandibular lymph nodes, sternum with bone marrow, heart*, thymus*, lungs, (aorta), trachea, (esophagus), thyroids (with parathyroids), (eyes), (Harder´s lacrimal glands), (tongue), brain*, pituitary*, skeletal muscle (biceps femoris), peripheral nerve (sciatic nerve), spinal cord (thoracic, cervical, lumbar), vertebrae, (femur including articular surface), gross lesions. * = weighed organs; ( )=organs collected but not histologically examined

HISTOPATHOLOGY: Yes
Control/High dose group: examination of above listed unbracketed organs/tissues.
Low/intermediate dose group: examination of liver, spleen, mesenteric lymph nodes, thymus, lungs, adrenals, duodenum, jejunum, ileum

Statistics:

Absolute figures and mean and standard deviation were expressed.
Parameters statistically examined were: body weight, body weight gain, food consumption, hematology, blodd chemistry, urinalysis, organ weights.
Barlett´s test for homogeneity of variance:
If homogeneous: ANOVA
If not homogeneous: Kruskal-Wallis test/ANOVA

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:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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
150 mg/kg/day: clinical changes: salivation, piloerection, hunched posture, fur loss, soft stools. 2 males/3 females died between day 8-27
50 mg/kg/day: clinical changes: salivation, piloerection (1 female). 1 female died on day 11
12.5 mg/kg/day: no changes

BODY WEIGHT AND WEIGHT GAIN
150 mg/kg/day: decrease in males and females, resulting in - 25% (males) and -21% (females) body weight at the end of the study.
50 mg/kg/day: moderate decrease in females (-12%), very slight decrease in males (-5%)
12.5 mg/kg/day: Very slight decrease in females (-5%), no changes in males

FOOD CONSUMPTION AND COMPOUND INTAKE
150 and 50 mg/kg/day: decrease (not statistically significant, but dose-related in males)
12.5 mg/kg/day:no changes

OPHTHALMOSCOPIC EXAMINATION
No changes

HAEMATOLOGY
150 mg/kg/day: males: increase of erythrocytes, leukocytes, neutrophils, platelets, prothrombin time; decrease of lymphocytes, MCV. Females: increase of erythrocytes, platelets; decrease of MCV, MCH
50 mg/kg/day: males: increase of leukocytes, neutrophils, MCHC, prothrombin time; decrease of lymphocytes, MCV. Females: increase of platelets; decrease of MCV
12.5 mg/kg/day: females: decrease of MCV
(MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration)
CLINICAL CHEMISTRY
150 mg/kg/day: marked increase of SGOT and marked to moderate increase of SGPT (males/females), slight increase of gammaGT (females), slight to moderate decrease of total protein (females), minor percentage variations in serum protein electrophoretic pattern: decrease of albumin, increase of globulin and alpha 1 globulin (males); decrease of albumin, increase of globulin and alpha 2 and beta globulins (females), moderate decrease of total cholesterol (females), slight increase of urea (males, 1 female)
50 mg/kg/day: marked to moderate increase of SGPT (males/females), slight to moderate decrease of total protein (females), minor percentage variations in serum protein electrophoretic pattern: increase of alpha 1 globulin (males); decrease of albumin, increase of globulin and alpha 2 and beta globulins (females).

URINALYSIS
No changes

ORGAN WEIGHTS
150 mg/kg/day: Changes in absolute (decrease) and/or relative (increase) organ weight in almost all organs; most were considered indirectly related with growth arrest. Treatment related effect was considered for thymus (decreased weight) and adrenals (increased weight), presumably indirectly related with general distress.
50 mg/kg/day: males: decrease of absolute liver and kidney weight (related to body weight decrease)

GROSS PATHOLOGY
150 and 50 mg/kg/day: slight to moderate dilation and/or congestion of the intestinal tract.

HISTOPATHOLOGY: NON-NEOPLASTIC
150 mg/kg/day: slight necrosis (liver), associated with inflammation (neutrophilic infiltration), decreased hepatocellular vacuoles; slight follicle atrophy in the spleen, slight neutrophilic infiltration; moderate depletion of lymphoid elements in the thymus; moderate/marked histiocytosis in mesenteric lymph nodes with vacuolated histiocytes; slight to severe histiocytic accumulation in the lamina propria in the small intestine with vacuolated histiocytes, slight to moderate hyperplasia of the mucosa; slight hypertrophy of the adrenal glands (1 male, 1 female); slight lung inflammatory foci, bronchial oedema, fibrosis; gastric wall thinning in the stomach; some cases of lumen dilation in the small and large intestine.
50 mg/kg/day: moderate depletion of lymphoid elements in the thymus; moderate/marked histiocytosis in mesenteric lymph nodes with vacuolated histiocytes; slight to severe histiocytic accumulation in the lamina propria in the small intestine with vacuolated histiocytes, slight to moderate hyperplasia of the mucosa; slight lung inflammatory foci, bronchial oedema.
12.5 mg/kg/day: Slight vacuolation of histiocytes in mesenteric lymph nodes and the small intestine.
Findings in animals that died during the study:
150 mg/kg/day: lung modifications, atrophy of lymphoid tissue in mandibular lymph nodes and others of hair follicles of the skin, increased foci of mineralization in kidneys (1 female)
50 mg/kg/day: necrosis (liver), moderate follicle atrophy in the spleen, vacuolated histiocytes in mesenteric lymph nodes, lung modifications

Dose descriptor:

NOAEL

Effect level:

12.5 other: mg/kg

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical changes, mortality at 50 mg/kg/bw/day

Critical effects observed:

not specified

The highest dose of the test item tested induced morphological modifications to various organs and tissues; concomittant poor body conditions were also induced, as well as deaths in 50% of the animals. The changes deemed due to direct toxic effects were slight single cell necrosis in the liver, with inflammation, and marked histiocytic accumulation in mesenteric lymph nodes and in the lamina propria of the small intestine, with mucosal epithelium hyperplasia. The latter can be associated with the picture of accumulation enteropathies. Atrophy in liver, spleen, thymus and lymph nodes, and hypertrophy of adrenal glands were considered to be secondary to the poor body condition and/or to treatemnt-related general stress.

The main macroscopic finding was dilation of the intestinal tract. Gastro-intestinal modifications were considered to be related to direct local effects of the strongly irritating compound. As well, lung inflammatory changes correlated with inhalation of foamy salivation (presumably with compound) occuring just after gavage, are considered to be due to local irritating effects. Involvement of diestive and/or respiratory systems was considered to be the cause of death of premature dead animals, while gastro-intestinal changes resulted in emaciation due to malabsorption.

Conclusions:

The study director concluded that Genamin TA 100 when administered to rats by the oral route for 4 consecutive weeks at 12.5, 50 and 150 mg/kg body weight per day was found to be well tolerated only at the lowest dose which did not cause any relevant effects. At the two higher doses, the compound appeared poorly tolerated due to its strong irritative properties which caused local gastro-intestinal modifications and lung inflammatory changes, the latter possibly as a consequence to the inhalation of foamy saliva occurring after gavaging (a well known phenomenon for this type of materials). The gastro-intestinal and lung alterations were the cause of some premature deaths. In addition the involvement of the digestive system resulted in malabsorption mainly evident at 150 mg/kg body weight. At this dose evident signs of liver toxicity were also noted. On the basis of the results obtained the dose of 12.5 mg/kg body weight per day could be considered the NO(A)EL.
However, following an evaluation carried out during an EU Risk Assessment, it was concluded that a NO(A)EL could not be derived from the study and that 12.5 mg/kg body weight per day should be considered a LO(A)EL.

Executive summary:

In an oral 28-day study (gavage, SD rats) Genamin TA 100 was administered in sesame oil (vehicle) to Sprague Dawley Crl:CD rats at dosages of 0, 12.5, 50 and 150 mg/kg bw/day once a day for 4 consecutive weeks. The subacute test procedure was in accordance with OECD TG 407 except that all specific investigations on the neurofunction were lacking.

At 150 mg/kg bw/day two males and three females of the high dose group died between day 8 and 27. Animals at this dose level showed salivation after treatment, piloerection, hunched posture, fur loss and soft stools.

At 50 mg/kg bw/day salivation and piloerection were observed. One female died at day 11 showing lung changes as described for the high dose animals (see below).

At 12.5 mg/kg bw/day no toxicological relevant clinical signs occurred.

There was a dose-related body weight decrease. For males this reduction involved the intermediate and high dose group, i.e. -5% and -25% vs. controls, while in females all treated groups were effected, i.e. -5%, -12%, -21% vs. controls. Food consumption during administration period of animals treated at 50 and 150 mg/kg bw/d was generally lower than in controls. At 12.5 mg/kg bw/d food consumption was comparable to the controls.

Hematology revealed a slight increase in erythrocyte count at 150 mg/kg bw/d, which was accompanied by reduced values for MCV. A dose related moderate to marked increase in leukocyte counts and a slight to moderate increase in platelets were recorded at 50 and 150 mg/kg bw/d. The leukocyte enhancement was generally accompanied by variations in the white blood cell differential count, consisting of increases in neutrophiles percentage (i.e. at 50 mg/kg bw/d in males and at 150 mg/kg bw/day in both sexes).

Clinical-chemistry tests showed a number of alterations. Toxicologically relevant changes were markedly increased activities of ALAT and ASAT at 150 mg/kg bw/d. GGT activity was slightly increased in females of this dose group. The serum concentrations of protein and albumin and the A/G ratios were dose-dependently reduced in the 50 and 150 mg/kg bw/d groups.

Compared to control levels, a decrease in total cholesterol in high dose females and increases in the urea level were observed in both sexes of this group.

Organ weight of almost all organs of the surviving high dose group animals showed statistically significant absolute and/or relative changes (most often decreases). This was considered to be indirectly related to the growth depression in this group. There was, however, a decrease in the thymus weight and an increase in the adrenal weight indicating a direct effect of the treatment.

At the intermediate dose there were significant decreases in liver and kidney weights in males indicating again relation to general body weight loss.

Gross pathology findings were mainly slight to moderate dilations of the intestinal tract in intermediate and high dose animals.

Histopathologically, the premature descendents of the high dose group showed erosions in the gastric and small intestinal mucosa with marked accumulation of vacuolated histiocytes in mesenteric lymph nodes and lamina propria of the small intestine as well as epithelial hyperplasia in the mucosa. Histiocytic effects were more pronounced in the jejunum and ileum than in the duodenum. In the lungs abnormalities were mainly localised to the bronchi/bronchioli and consisted of inflammation associated with fibrosis and bronchial oedema.

Similar histopathological findings were observed in surviving animals of that dose group together with single cell necrosis and focal inflammation in the liver and areas of hyperplasia in the small intestinal mucosa as well as atrophy of the thymus and splenic follicle. In one male and one female there was a slight cortical hypertrophy of the adrenal glands.

At the dose of 50 mg/kg bw/d signs of atrophy of thymus were seen in one male and one female. Histiocytic involvement in small intestine and mesenteric lymph nodes was still present in moderate degree and also mucosal hyperplasia occurred in the intestine, and the lungs had similar changes compared to the high dose.

At the low dose histopathological changes were limited to slight histiocytic vacuolation in mesenteric lymph nodes and small intestine, without abnormal accumulation of histiocytes.

In conclusion, the primary adverse effects following repeated gavage administration into the stomach were gastrointestinal dilatation and erosions of the gastric and small intestine mucosa. Repeated exposures over time appears to exacerbate the gastrointestinal damage and the bad general health status and is suggested to be the cause of unscheduled deaths. Reduced food consumption, emaciation, mucosal hyperplasia and leukocytosis were related effects. Histiocytosis and histiocytic vacuolation indicated that the test substance was absorbed and accumulated intracellularly in the lymphoid tissues of the intestine. Liver cell necrosis associated with inflammatory response and increased activities of liver enzymes demonstrated that the test substance also has a hepatotoxic potential. The elevated urea concentrations might point to a minor renal toxicity. Increases in adrenal weights fit to the hyperplasia of the gland which may be a non-specific effect. Increased erythrocyte counts (hemoconcentration) might be assumed as secondary to reduced water consumption (no data) which could accompany reduced appetite due to painful lesions in the gastrointestinal tract. Bronchiolar and peribronchiolar inflammatory and fibrotic changes were interpreted to be related to the gavage application and thereby to the unintended aspiration of some material of the irritative test substance. Histiocytic vacuolation of the mesenteric lymph nodes and small intestine, observed at slight degree at 12.5 mg/kg bw/d, occurred at all dose levels and increased dose-dependently to higher severity and progressed in accumulation. On the basis of the results obtained the dose of 12.5 mg/kg body weight per day was regarded as `no observed adverse effect level` (NOAEL) by the study director. However, following an reevaluation carried out during an EU risk assessment exercise it was concluded that a NOAEL could not be derived and that 12.5 mg/kg body weight per day should be regarded to be a LOAEL .

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

6.7 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available data support the current approach of grouping of substances and read across of data.

Organ:

duodenum

ileum

jejunum

kidney

liver

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

The registered substance is a salt composed of primary hydrogenated alkylamine and N-hydrogenated tallow fatty acid. As the primary alkyl amine part is much more toxic than the tallow fatty acid part, it is expectedthat the toxicological behaviour of the substance will not be far different from that of the primary alkylamine compounds or at least not more severe. This has been demonstrated by results obtained with the registered substance in a short-term repeated toxicity study (e.g. 14 days). Thus, assessment of the registered substance can be integrated in thecurrent approach of grouping of primary alkylamines substances. Regarding the toxicity of these latter, it has been expected that the members with a shorter chain and/or a higher degree of insaturation will have a higher toxicity among the category or at least will represent the worst case. Thus, a read-across could be applied to longer chains into shorter and to saturated molecules into unsaturated ones.

 

Several studies (from short-term to subchronic administrations) are used to confirm the read-across and/or assess the toxicological properties of the substance. 

 A 14-day dose range-finding study on the registered substance itself was performed with dosegroups of 0, 50, 150 and 450 mg/kg bw/day (gavage: 5 mL/kg bw in corn oil). At 450 mg/kg bw/day in both sexes, the test item induced lower body weight gain and changes in clinical pathology parameters consisting of higher white blood cell count, prolonged prothrombin time and higher aspartate/alanine aminotransferase activities, with lower proteins, albumin and cholesterol levels. Higher adrenal and lower thymus weights were noted in females treated at 450 mg/kg/day. Foamy macrophages were observed in the mesenteric lymph nodes, jejunum and ileum (females only), together with increased inflammatory cell foci in the liver.  Except for foamy macrophages in the mesenteric lymph nodes, no relevant effects were observed at 150 or 50 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg bw/day under the experimental conditions of the study.

 

In a preliminary study, rats received orally Z-octadec-9-enylamines (CAS: 112-90-3) considered as the worst case of the category approach, at dose-levels of 25, 100 and 400 mg/kg bw/day over a period of 14 days. Mortality and premature sacrifices occurred in the high-dose-group. The animals of the 100 mg/kg bw/day group (especially the males) showed clinical signs of impaired motility and respiration. After administration of 25 mg/kg/day no symptoms were observed except of one female rat, which showed uncoordinated gait at study day 2. Necropsy of the dead and prior killed animals showed changes in the stomach and intestinal mucosa. The animals of 100 mg/kg bw/day showed reddening of the stomach mucosa. In the main study, treatments of 3.25, 12.5 and 50 mg/kg bw/day for 28 consecutive days resulted in no unscheduled deaths but clinical signs were noted in the high-dose group from the second week until the end of the study. Mean body weight gain was significantly lower at the end of treatment for high-dose males (-19%), high-dose females (-20%) and for mid-dose males (-15%) compared to control animals. Some slight and reversible hemato-biochemical changes were noted in the high-dose group including ASAT and ALAT activity in the liver of high-dose males. No anatomic pathology correlates (organ weights, macroscopy and microscopy) of toxicological significance were detected.

Tallow alkylamine (CAS: 61790-33-8) given to rats by oral gavage in a 14-day dos range finding study at doses of 62.5, 250 and 1000 mg/kg/day, appeared to be satisfactorily tolerated only at the lowest dose-level, inducing only mild clinical effects and slight dilation of the intestinal tract. At higher doses the compound was poorly tolerated causing body weight depression, moderate to severe impairment of clinical conditions and dilation of the stomach and/or intestinal tract. At 1000 mg/kg/day these changes led to death of all the animals. In the main study, tallow alkyl amine when given orally at 0, 12.5, 50 and 150 mg/kg/day for 4 consecutive weeks to rats was found to be well tolerated only at the lowest dose-level which did not cause any relevant effects. At the two higher doses, the compound appeared poorly tolerated due to its strong irritative properties which caused local gastro-intestinal modifications and lung inflammatory changes, the latter possibly as a consequence to the inhalation of foamy saliva occurring after gavaging (a well known phenomenon for this type of materials). The gastro-intestinal and lung alterations were the cause of some premature deaths. In addition the involvement of the digestive system resulted in malabsorption mainly evident at 150 mg/kg/day.

Additional data in form of a chronic feeding studies in rats and dogs exist for octadecylamine (CAS-No. 124 -30 -1). However, due to limitations in quality, these are not used as key studies but do support the derived conclusions. Based on validity considerations therefore the available data for (Z)-octadec-9 -enylamine and for tallow alkylamines are used for the assessment and read-across is applied for all primary alkylamines considered in this category approach. Leading health effects after oral exposure were mortalities at higher dose-levels associated with precedent bad general health status and gait abnormalities, erosions of the mucosa of the gastrointestinal tract, accumulation of histiocytes in the submucosa of the distal parts of the small intestine and in the mesenteric lymph nodes, and mild toxic effects in liver and kidneys. With respect to local effects, the available data clearly demonstrate that primary alkylamines cause damage along the exposure route, i.e. the mucosa of the gastrointestinal tract. The data also indicate a cytotoxic potential at any site of contact along the exposure route and can be explained by the strong dermal irritative / corrosive nature of the primary alkylamines.

Taking the approach of applying read-across implies that the most sensitive NOAEL for the endpoint ‘repeated dose toxicity` is used for the assessment which was derived from the valid oral 28 -day study on (Z)-octadec-9 -enylamine (CAS No. 112-90 -3). The NOAEL from this study was 3.25 mg/kg body weight per day which will be also used as starting point. As the registered substance is a salt of primary hydrogenated alkylamine and N-hydrogenated tallow fatty acid with a molar ratio of 1:1, the NOAEL value should be converted as follows: Mw oleylamine = 267.5, MW salt = 547.5 ; 3.25 x 547.5/267.5 = 6.7 mg/kg bw/day

Justification for classification or non-classification

The category approach for primary alkylamines is also applied for classification considerations. A Cat. 2 for STOT-RE, H373: may cause damage to organs through prolonged or repeated exposureis proposed for hydrogenated tallow alkylamines which is in line with the proposed classification from the EU risk assessment on primary alkylamines. However, it should be noted that the available data consistently points to a primarily local mode of action due to the strong irritative / corrosive properties of primary alkylamines which leads to secondary effects with relevance of systemic toxicity However, the hydrogenated tallow alkylamines induced less severe local effects and is only classified irritating to skin and the registered salt is not classified and considered as slightly irritating.