Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Endpoint summary

Description of key information

A short-term repeated-dose toxicity study was conducted on the registered substance in accordance with the OECD Testing Guideline 407. Based on the results presented in this report a NOAEL of 100 mg/kg bw/day was established.
Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters belongs to the Primary Fatty Amines (PFAs) category, and consistent effects were identified during the short-term repeated-dose toxicity study on the substance, when compared to other members belonging to this category, thus supporting a category approach from PFAs to assess the toxicity of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters following a repeated exposure.

Taking into account a category approach strategy, the most sensitive NOAEL of PFA category member was used, which is a NOAEL of 3.25 mg/kg bw/d obtained during a GLP-compliant short-term repeated-dose toxicity study conducted on (Z)-octadec-9-enylamines (CAS 112-90-3). This value corresponds to 9.2 mg/kg body weight per day of Amines, C16-18 (even numbered)-alkyl, salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters.

Repeated exposure to PFAs induces principally local inflammatory effects. Therefore, no increase of the toxicity is presumed with the extension of exposure duration. This is supported by the data of chronic feeding studies (2 years) performed in dogs and rats with Octadecylamines (CAS 124-30-1) for which the NOAEL was established at 10 mg/kg/day. Consequently, experimental data from short-term repeated-dose toxicity studies are deemed as suitable and sufficient to assess the toxicity of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters following a repeated exposure.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept 2020 - Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

At this time, studies in laboratory animals provide the best available basis for extrapolation to
humans and are required to support regulatory submissions. Acceptable models which do not
use live animals currently do not exist.
The rat was chosen as the animal model for this study as it is an accepted rodent species for
preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to
properly characterize the effects of the test item. This study has been designed such that it did
not require an unnecessary number of animals to accomplish its objectives.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 142-176 g for males and 133-153 g for females
- Fasting period before study: no
- Housing: Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
Up to 5 animals of the same sex and same dosing group together.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (ad libitum): SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water (ad libitum): municipal tap water
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Analyses were available. It is considered that there were no known contaminants in the food or water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 38-58
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Oct 2020 To: 11 Nov 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is the intended route of human exposure
during manufacture, handling or use of the test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were heated to a maximum temperature of 80°C for to obtain visual homogeneity. Formulations were released for dosing when they obtained a temperature of 40°C or lower.
Test item dosing formulations (w/w) were homogenized by mortar and pestle and stirring to
visually acceptable levels at appropriate concentrations to meet dose level requirements. The
dosing formulations were prepared weekly as a suspension, filled out in daily portions and
stored in the refrigerator protected from light. The dosing formulations were removed from
the refrigerator and stirred for at least 30 minutes before dosing. On the day of use, formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
For groups 2-4 one formulation of 100 mg/mL was prepared for each dosing day.
No adjustment was made for specific gravity of the test item. No adjustment was made for the
purity/composition of the test item. An adjustment was made for the specific gravity of the vehicle.

VEHICLE
- Justification for use and choice of vehicle: based on trial preparations
- Concentration in vehicle: 100 mg/mL
- Amount of vehicle (if gavage): up to 10 mL/kg
- Lot/batch no. (if required): MKCK6411 and MKCM3364 (Sigma-Aldrich, Steinheim, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis of concentration in all groups from middle of dosing container, and for homogeneity from top, middle and bottom of 100 mg/mL test item containing formulation in week 1 and 4.
Analysis was performed using a validated analytical procedure (Test Facility Study No. 20246316).
Acceptance criteria: For concentration: mean sample concentration results within or equal to
± 15% suspensions of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of = 10% for each group.

Stability analyses performed previously in conjunction with the method development and
validation study (Test Facility Study No. 20246316) demonstrated that the test item is stable
in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

group 2

Dose / conc.:

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

Remarks:

group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 4

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of the dose range finding study were selected based on results of an Acute Toxicity study with oral exposure of Amines, C16-18 (even numbered)-alkyl, salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters in rats (Test Facility Study No. 20246314). The dose levels of the main study were based on the results of the dose range finding study (Test Facility Study No. 20246317) and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: At least twice daily beginning upon arrival through termination. Except on days of receipt and necropsy where frequency was at least once daily.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 at 0 to 1 hour postdose

DETAILED CLINICAL OBSERVATIONS: Yes, outside the cage
- Time schedule: Weekly; from Week 1 and throughout the study, and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Day 1 and throughout the study. Fasted weight was determined on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, quantitatively measured per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes, monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes (o/n)
- How many animals: 5/sex/dose
- Parameters examined:
White blood cells (WBC)
Neutrophils (absolute)
Lymphocytes (absolute)
Monocytes (absolute)
Eosinophils (absolute)
Basophils (absolute)
Large unstained cells (LUC) (absolute)
Red blood cells (RBC)
Reticulocytes (absolute)
Red blood cell distribution width (RDW)
Hemoglobin
Hematocrit
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelets
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes (o/n)
- How many animals: 5/sex/dose
- Parameters examined:
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
Total protein
Albumin
Bile acids
Total bilirubin
Urea
Creatinine
Glucose
Cholesterol
Triglycerides
Sodium
Potassium
Chloride
Calcium
Inorganic phosphate (Inorg. Phos)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the dosing period
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex, fore- and hind-limb grip strength and locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

Study animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.
Study animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

ORGAN WEIGHTS:
Brain
Epididymisa
Gland, adrenala
Gland, parathyroid
Gland, prostate
Gland, seminal vesiclea
Gland, thyroid
Heart
Kidneya
Liver
Ovarya
Spleen
Testisa
Thymus
Uterus/cervix

HISTOPATHOLOGY:
Bone, femur
Bone marrow
Bone, sternum
Brain [cerebellum, mid-brain, cortex] (8 levels)
Epididymis
Eye
Gland, adrenal
Gland, parathyroid
Gland, prostate
Gland, salivary
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Liver
Lung
Lymph node, mandibular
Lymph node, mesenteric
Muscle, skeletal
Nerve, optic
Nerve, sciatic
Ovary
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testis
Thymus
Trachea
Urinary bladder
Uterus
Vagina

Statistics:

DATA COLLECTION IN PROVANTIS
Any data collected during the Pretreatment Period are tabulated, summarized or statistically analyzed. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter.

Inferential statistical methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric/Non-parametric (body weight (gain), hematology, coagulation, clinical chemistry, FOB quantitative variables, organ weight (relative to body weight):
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

ANCOVA:
The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test.

Incidence (FOB qualitative variables):
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

For data collected in Toxdata, see 'Any other information on materials and methods".

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted in this study.
Scabs and a thin fur cover were noted in individual animals (including one control male) during the Dosing Period, which occurred within the range of background findings to be expected for rats of this age and strain and housed and treated under the conditions and also lacked a dose-related trend. These findings were considered to be unrelated to treatment with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 300 and 1000 mg/kg bw/day, body weight gain was slightly decreased from Day 15-22 onwards, resulting in a decreased body weight on Day 28 of 0.97x control at both doses. In addition, body weight gain was also slightly decreased in females at 300 and 1000 mg/kg bw/day, from Day 15-22 onwards, resulting in a decreased body weight on Day 28 of 0.98 and 0.95x, respectively.
A lower body weight and body weight gain was observed in one control male when compared to the other control animals. As this animal does not show any other notable findings it can be concluded that this animal is still suitable as control animal.
A statistically significantly lower body weight in females at 100 mg/kg bw/day (0.94x of control) was observed on Day 22. In absence of a clear dose-related response and as a higher increase in body weight was seen on Day 28 compared to the control group, this finding was considered to be not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No effects on food consumption were noted in males and females up to a 1000 mg/kg bw/day.
Food consumption was slightly increased in males and females dosed at 100 and 300 mg/kg bw/day, which was considered to be not toxicologically relevant.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No effects on hematology parameters were noted in males and females up to 300 mg/kg bw/day.
An increase in neutrophils and monocytes was seen in males and females at 1000 mg/kg bw/day (males: 2.52x and 2.40x of control, females; 2.81x and 4.37x of control).
One female at 100 mg/kg/day showed a clear increase in white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells, which had a large effect on the group mean. As the results of the other females at 100 mg/kg bw/day were comparable with the control group, the findings observed in this particular animal were considered not to be caused by the test item.
Remaining differences in hematology parameters were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
No effects on coagulation parameters were noted in males up to 1000 mg/kg bw/day and in females up to 300 mg/kg bw/day.
An increase in prothrombin time was observed in females dosed at 1000 mg/kg bw/day (1.07x of control). This finding remained within the historical control data and also lacked a histopathological correlation and was therefore considered to be not toxicologically relevant ( historical control data (132 animals): mean 16.7; P5-95: 14.5-18.7).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See also table under "any other information on results".
No effects on clinical chemistry parameters were noted in males up to 1000 mg/kg bw/day and in females up 300 mg/kg bw/day.
Increased bile acid concentration was noted in females dosed at 1000 mg/kg bw/day (1.41x of control).
A decreased alkaline phosphatase activity was seen in males and females dosed at 100, 300 and 1000 mg/kg bw/day, which lacked a dose response and remained within the historical control data (see table below for the mean values and the historical control values). Therefore, this finding was considered to be not test item-related.
Other values achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly high or low control values (see table below for the mean values and the historical control values), occurred in the absence of a dose-related distribution and/or were, given the magnitude of change/an opposite effect would be expected, considered to be of no toxicological significance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Motor activity was similar between treated and control groups, with exception of one male at 1000 mg/kg bw/day. This animal had lower number of total movements and ambulations. The total movements result for this animal was also outside the historical control data (see table below for the historical control data). As this only occurs at a single animal and the remainder of the group showed results comparable with control, these findings were considered to be not toxicologically relevant. See also table under "any other information on results".
All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See also tables under "any other information on results".
Test item related lower thymus weights (absolute and relative to body weights) were noted in males at 1000 mg/kg bw/day. The microscopic correlate for this lower weight was decreased lymphoid cellularity.
Test item related higher spleen weights (absolute and relative to body weights) were noted in females at 1000 mg/kg bw/day. There was no microscopic correlate for this higher weight.
There were no other test item-related organ weight changes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, jejunum, ileum and mesenteric lymph nodes of males and females up to 1000 mg/kg bw/day group and in the thymus and heart of males at 1000 mg/kg bw/day (see tables below under "any other information on results".).

In the liver, periportal hepatocellular vacuolation at a minimal to mild degree was seen in 3/5 females at 1000 mg/kg bw/day. Furthermore, multifocal mononuclear cell infiltration at a mild to moderate degree in 4/5 males and 5/5 females and hepatocellular single cell necrosis at a minimal to mild degree in 4/5 males and 5/5 females was observed in the liver at 1000 mg/kg bw/day. Multifocal granulomatous inflammation in the liver at a mild to moderate degree was seen in 5/5 males and 5/5 females at 1000 mg/kg bw/day. The incidences and severities recorded of periportal vacuolation and mononuclear cell infiltrates in the remaining dose groups including controls were considered within background.
In the jejunum, accumulation of vacuolated macrophages in the lamina propria at a minimal degree was seen in 5/5 males and 5/5 females at 1000 mg/kg bw/day. In the ileum, accumulation of vacuolated macrophages in the lamina propria was seen at a minimal to mild degree in 5/5 males and 5/5 females at 1000 mg/kg bw/day, in 5/5 males (minimal to mild degree) and 5/5 females (minimal degree) at 300 mg/kg bw/day and in 2/5 males and 1/5 females at minimal degree at 100 mg/kg bw/day.
In the mesenteric lymph node, diffuse sinus histiocytosis was seen in 5/5 males (moderate to marked degree) and 5/5 females (mild to marked degree) at 1000 mg/kg bw/day, in 5/5 males (minimal to moderate degree) and 5/5 females (minimal to moderate degree) at 300 mg/kg bw/day and in 5/5 males (minimal degree) and 5/5 females (minimal to mild degree) at 100 mg/kg bw/day. Macrophage aggregates in the mesenteric lymph node were seen at mild to moderate degree in 4/5 males and 4/5 females at 1000 mg/kg bw/day, in 3/5 males (mild to moderate degree) and 5/5 females (minimal to moderate degree) at 300 mg/kg bw/day and in 2/5 females (minimal degree) at 100 mg/kg bw/day.
In the thymus, decreased lymphoid cellularity at a minimal degree was seen in 5/5 males at 1000 mg/kg bw/day.
In the heart, mild mononuclear cell infiltrate was seen in the right ventricle of the heart in 1/5 males at 1000 mg/kg bw/day. Although this an isolated finding, it cannot be excluded that the mild mononuclear cell infiltrate in the right ventricle of the heart in one male at 1000 mg/kg bw/day was test item-related.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

immunology

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

DOSE FORMULATION ANALYSIS

Accuracy
The concentrations analyzed in the formulations of Groups 2-4 (combined formulation) in Weeks 1 and 4 were in agreement with target concentrations (i.e. mean accuracy 90 and 96%, respectively).
No test item was detected in the Group 1 formulation prepared for use in Week 1. A small response at the retention time of the test item was observed in chromatograms of the Group 1 formulation prepared for use in Week 4. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

Homogeneity
The formulations of Groups 2-4 (combined formulation) in Weeks 1 and 4 were homogeneous (i.e. coefficient of variation 1.9 and 2.1%, respectively).

 

Historical control data motor activity - Wistar Han

Parameter	Sex	Mean	P5	P95	Number of animals
Total movement	Males	3807	2118	6244	405
	Females	5377	2564	8967	399
Ambulations	Males	866	381	1523	405
	Females	1557	651	2705	399

Mean results and HCD clinical chemistry - Wistar Han

Parameter	Sex	Dose Level (mg/kg bw/day)				Historical control data
						Mean	P5	P95	Number of animals
		0	100	300	1000
ALP (U/L)	Males	203.7	185.1 (0.91x)	155.7 (0.76x)	149.7 (0.74x)	191	124	273	83
	Females	103.4	75.6* (0.73x)	67.1** (0.65x)	75.4* (0.73x)	100	57	157	83
ALT (U/L)	Males	69.5	43.5* (0.63x)	46.5* (0.67x)	86.4 (1.24x)	38.1	23.1	68.0	83
Urea (mmol/L)	Males	4.60	6.86** (1.49x)	6.11 (1.33x)	5.46 (1.19x)	5.8	3.2	9.4	83
Potassium (mmol/L)	Males	3.98	3.76 (0.95x)	3.56* (0.89x)	4.30 (1.08x)	3.90	3.48	4.44	80
Cholesterol (mmol/L)	Females	1.364	1.220 (0.89x)	1.202 (0.88x)	1.002** (0.73x)	1.35	0.95	1.80	80

*: P<0.05, **: P<0.01. Ratio difference compared to the control group is presented between brackets

Mean percent thymus weight differences from control group

	Males
Dose level (mg/kg bw/day):	100		300		1000
THYMUS
Absolute	-5		-14		-28
Relative to body weight	-4		-10		-26*
*: P<0.05

 

Mean percent spleen weight differences from control group

	Females
Dose level (mg/kg bw/day):	100		300		1000
SPLEEN
Absolute	11		-7		33*
Relative to body weight	20		-1		40*
*: P<0.05

Summary test item-related microscopic findings at scheduled euthanasia (liver, jejunum, ilieum and lymph node, mesenteric)

	Males				Females
Dose level (mg/kg bw/day):	0	100	300	1000	0	100	300	1000
LIVER ^a	5	5	5	5	5	5	5	5
Vacuolation, hepatocellular,     periportal
Minimal	1	-	-	-	1	-	-	2
Mild	-	-	-	-	-	-	-	1
Infiltration, mononuclear cell
Minimal	4	2	4	1	3	3	5	-
Mild	-	-	-	4	-	-	-	3
Moderate	-	-	-	-	-	-	-	2
Single cell necrosis
Minimal	-	-	-	4	-	-	-	4
Mild	-	-	-	-	-	-	-	1
Inflammation, granulomatous
Mild	-	-	-	4	-	-	-	4
Moderate	-	-	-	1	-	-	-	1
JEJUNUM ^a	5	5	5	5	5	5	5	5
Accumulation vacuolated     macrophages         Minimal   ILEUM ^a     Accumulation vacuolated     macrophages         Minimal         Mild   LYMPH NODE, MESENTERIC ^a      Sinus histiocytosis          Minimal          Mild          Moderate          Marked        Aggregate, macrophage          Minimal          Mild          Moderate	-   5     - -   5   - - - -     - - -	-   5     2 -   5   5 - - -     - - -	-   5     3 2   5   1 2 2 -     - 2 1	5   5     4 1   5   - - 3 2     - 1 3	-   5     - -   5   - - - -     - - -	-   5     1 0   5   1 4 - -     2 - -	-   5     5 0   5   1 3 1 -     1 2 2	5   5     2 3   5   - 3 1 1     - 1 3

^a  =  Number of tissues examined from each group.

Summary test item-related microscopic findings at scheduled euthanasia (thymus, heart)

	Males
Dose level (mg/kg bw/day):	0	100	300	1000
THYMUS ^a	5	5	5	5
Decreased cellularity, lymphoid
Minimal	1	-	-	5
HEART ^a	5	5	5	5
Infiltration, mononuclear cell
Mild	-	-	-	1

^a  =  Number of tissues examined from each group.

 

 

Conclusions:

Administration of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters by once daily oral gavage for 28 days was well tolerated in Wistar Han rats at levels of 1000 mg/kg bw/day. Morphological adverse alterations were recorded in the mesenteric lymph node (from 300 mg/kg bw/day, both sexes) with correlating higher monocyte counts, liver (at 1000 mg/kg bw/day, both sexes) and heart (at 1000 mg/kg bw/day, one male). No adverse effects were seen at 100 mg/kg bw/day.
Based on the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters of 100 mg/kg bw/day was established.

Executive summary:

A 28-day repeated dose toxicity study in rats was performed according to guidelines and GLP principles. Wistar Han rats (5/sex/dose) were given 0, 100, 300 or 1000 mg test item/kg bw/day for 28 days by oral gavage.

The following parameters and end points were evaluated in this study: clinical signs, functional observation tests, body weights, food consumption, clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy findings, organ weights, and histopathologic examinations.
Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.
At 100 mg/kg bw/day, a test item-related non-adverse diffuse sinus histiocytosis in the mesenteric lymph node was seen in males and females at a minimal to mild degree. Furthermore, a test item-related non-adverse accumulation of vacuolated macrophages in the lamina propria in the ileum was observed in males and females at a minimal degree.
At 300 mg/kg bw/day, a test item-related adverse diffuse sinus histiocytosis in the mesenteric lymph node was seen in males and females at a minimal to moderate degree. Furthermore, a test item-related non-adverse accumulation of vacuolated macrophages in the lamina propria in the ileum was observed in males and females at a minimal to mild degree.
At 1000 mg/kg bw/day, a test item-related non-adverse increase in neutrophil count in males and females and bile acid concentration in females was observed.
At organ weights a higher spleen weights was noted in females, which was probably test item-related but no significant histopathological findings were recorded in this organ. Therefore, this finding was considered to be not adverse.
At histopathological examination, test item-related microscopic findings were noted in the liver, mesenteric lymph nodes, heart, small intestines (jejunum and ileum) and thymus.
In the liver, periportal hepatocellular vacuolation, multifocal mononuclear cell infiltration, hepatocellular single cell necrosis and multifocal granulomatous inflammation in males and/or females was observed. These correlated with increases in neutrophil and monocyte counts in males and females. At the severity observed, these findings were considered to be adverse.
In the mesenteric lymph node, diffuse sinus histiocytosis and macrophage aggregates were noted in males and females at mild to marked degree, which correlated with increased neutrophil and monocyte count in males and females. At the severity observed this finding was considered to be adverse.
In the heart, mild mononuclear cell infiltrate in the right ventricle of the heart in one male was observed, which was an isolated finding, but it cannot be excluded that this finding was test item-related. This finding was considered to be adverse.
In the small intestines, minimal to mild accumulation of vacuolated macrophages in the lamina propria of the jejunum and ileum in males and females was seen. These findings correlated with increased neutrophil and monocyte count and at the severity observed, they were considered to be not adverse.
In the thymus, decreased lymphoid cellularity in the males was observed with correlating lower thymus weight, which was probably test item-related but regarded to be non-adverse.
No mortality and no test item-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, coagulation and macroscopic examination).
In conclusion, administration of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters by once daily oral gavage for 28 days was well tolerated in Wistar Han rats at levels of 1000 mg/kg bw/day. Morphological adverse alterations were recorded in the mesenteric lymph node (from 300 mg/kg bw/day, both sexes) with correlating higher monocyte counts, liver (at 1000 mg/kg bw/day, both sexes) and heart (at 1000 mg/kg bw/day, one male). No adverse effects were seen at 100 mg/kg bw/day.
Based on the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters of 100 mg/kg bw/day was established.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

9.2 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant studies support a category approach to assess the repeated-dose toxicity of the substance

Organ:

other: gastrointestinal-tract, liver, immune system

Repeated dose toxicity: inhalation - systemic 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 an alkyl phosphate and alkyl amine salts and is mainly composed by primary alkylamines, monoalkyl dihydrogenophosphate and dialkyl hydrogenophosphate salts. As the primary alkyl amine part is much more toxic than the alkyl di/hydrogenophosphates part, it is expected that the toxicological behavior of the substance will not be far different from that of the primary alkylamine compounds or at least not more severe than the latter in view of its low release. Thus, a read-across from the primary aliphatic amines category to the registered substance is considered pertinent.

The term 'primary alkylamines' stands for a group of substances which share essential chemical key aspects. From a toxicological point of view, this category of chemicals exhibites a closely related effect spectrum not only related to qualitative but also quantitative aspects. On that basis, read across of data from one primary alkylamine to another is broadly accepted and has been used in the existing EU risk assessment and Committee for Risk Assessment (RAC) Opinions on primary alkylamines. Hence, the same principles are applied here.

There are no human data available on repeated dose toxicity of hydrogenated tallow alkylamines as for the whole class of primary alkylamines. For the oral route of exposure information from guideline-compliant subacute 28-day studies are available for (Z)-octadec-9 -enylamine (CAS 112-90-3) and tallow alkylamines (CAS 61790-33-8) which demonstrated a closely related toxicity profile. Additional data in form of a chronic feeding studies in rats and dogs exist for octadecylamine (CAS 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 poor 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 repeated dose toxicity is used for the assessment which was derived from the valid oral 28-day study on (Z)-octadec-9 -enylamine (CAS 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 for the route-to-route evaluations with regard to the inhalative and dermal exposure patterns. This approach is supported by findings from a 2-year chronic toxicity study in rats and a 1-year chronic feeding study in dogs which yielded in oral NOAELs of about 10 and 3 mg/kg body weight per day respectively. Based hereupon, additional testing was not considered necessary in the existing EU risk assessment on primary alkylamines.

This corresponded to 9.2 mg/kg body weight per day of Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters, as (Z)-octadec-9-enylamines has a MW of 267 and the registered substance a MW of 757. 3.25x757/267 = 9.2 mg/kg b/d.

Justification for classification or non-classification

Read across is made with hydrogenated tallow alkylamines inside the primary alkylamines category, an approach supported by the results of a short-term repeated-dose toxicity study conducted on the registered substance.

The category approach for primary alkylamines is also applied for classification considerations, and a Cat. 2 for STOT-RE, H373: may cause damage to organs through prolonged or repeated exposure is proposed for hydrogenated tallow alkylamines which is in line with the proposed classification from the EU risk assessment on primary alkylamines and the RAC. 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.