N-[3-(dimethylamino)propyl]docosanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 2016 - 08 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

September 1998

Deviations:

yes

Remarks:

Although sensory activity observations were performed, they were not recorded. This is not considered to have an effect on the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

yes

Remarks:

Although sensory activity observations were performed, they were not recorded. This is not considered to have an effect on the validity of the study.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 3 November 2015

Limit test:

no

Test material

Test material information

1

Reference substance name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

EC Number:

932-121-8

Cas Number:

1147459-12-8

Molecular formula:

UVCB substance not applicable

IUPAC Name:

N-[3-(dimethylamino)propyl] C 12-C18 alkylamide

Test material form:

other: Paste

Details on test material:

- State of aggregation: paste
- Density: 0.885 g/cm3 at 20°C (solid)
- Analytical purity: 100% (UVCB)
- Color gardner 2: 7
- Purity test date: 18 May 2016
- Lot/batch No.: BS-160515001-001
- Expiration date of the lot/batch: 15 May 2021
- Storage condition of test material: At room temperature container flushed with nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stable as solution in corn oil for at least 5 hours at room temperature and 11 days in the refrigerator (confirmed over the concentration range 1 to 200 mg/mL)
OTHER SPECIFICS: no correction factor applied for purity; pH 11 at 1%.
Mean mol weight ~ 296 Range 228- 368

batch BS-160515001-001

Test animals

Species:

rat

Strain:

other: Crl:WI (Han)

Details on species / strain selection:

Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

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: approximately 6 weeks
- Weight at study initiation: males 143-178 g, females 120-141 g,
- Fasting period before study: no
- Housing: group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring,
animals were housed individually in a Hi-temp polycarbonate cages 948.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 19.0-21.3
- Humidity (%): 51-60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 September 2016 To: 08 December 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

This study should provide a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible 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 (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity/density of the test item and vehicle. No correction was made for purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the Sponsor.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations) in Week 1, 6 and 13. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

At least 90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Concurrent vehicle controls, Group 1

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for this 90-day oral gavage study were selected by the Sponsor at 0, 15, 30 and 60 mg/kg and agreed based on available information of the Sponsor.

Positive control:

Not included.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately (0 - 15 minutes) after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest and at week 13
- Dose groups that were examined: at pretest: all animals, at week 13: controls and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: yes, overnight for a maximum of 24 hours
- How many animals: all animals
- Parameters examined: white blood cells, differential leucocyte count, red blood cells, reticulocytes, red blood cells distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 a.m. at the end of the treatment
- Animals fasted: yes, overnight for a maximum of 24 hours
- How many animals: all
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, bile acids, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13
- Dose groups that were examined: all groups, first 5 animals/sex/group
- Battery of functions tested: grip strength (fore and hind limb), motor activity. The hearing ability, pupillary reflex and static righting reflex were not recorded.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. The following organ weights were recorded: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus including cervix, prostate, seminal vesicles including coagulating glands, thyroid including parathyroid.

HISTOPATHOLOGY: Yes

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the study period and no additional findings were noted during the arena observations.
Salivation seen after dosing among 30 and 60 mg/kg bw/day animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.
One 30 mg/kg bw/day female displayed piloerection on a few occasions, however at the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight remained similar to the control level over the study period.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The nature and incidence of ophthalmology findings noted was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly higher relative eosinophil counts were recorded for 60 mg/kg bw/day females.
Other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. These included changes in mean corpuscular haemoglobin (MCH) (30 mg/kg bw/day males), mean corpuscular haemoglobin concentration (MCHC) (30 and 60 mg/kg bw/day males), neutrophil and lymphocyte (30 mg/kg bw/day females) levels.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters were considered unaffected by treatment.
Any statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. This included changes in sodium and chloride levels in 15 and 30 mg/kg bw/day males and females, in albumin and inorganic phosphate levels of 30 mg/kg bw/day males, and total bilirubin levels of 15 mg/kg bw/day females.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Grip strength and motor activity were similar between treated and control groups. Regarding motor activity, all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing, pupil reflex (left), pupil reflex (right), and static reflex were not recorded. However, it was considered unlikely that treatment related changes had occurred in these parameters since there were no signs of toxicity in related observations, such as clinical signs, arena observations, ophthalmoscopy, grip strength, and motor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.
All organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the test item were noted in the nonglandular stomach of 15, 30, and 60 mg/kg bw/day males and females.
A dose-dependent increase in incidence and severity of hyperplasia, squamous cell was recorded in the non-glandular stomach (forestomach) in males and females of 15, 30 and 60 mg/kg bw/day animals from a minimal to slight level, and to a moderate level in 60 mg/kg bw/day males. Related to this finding, hyperkeratosis and undulations at the basement membrane zone were often observed concomitantly. In two males treated with 30 mg/kg bw/day, and one male and one female treated with 60 mg/kg bw/day, signs of slight cellular atypia were noted. There were no other test item-related histological changes observed. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Oral administration of the test item resulted in local proliferative lesions in the non-glandular stomach (the forestomach) in all treatment groups, with an increasing incidence and severity (see Table 1 in "Any other information on results including tables" for incidence and severity) (20, 75 and 90% of the animals affected at 15, 30 and 60 mg/kg bw/day respectively). The lesions were characterized by areas of a thickened epithelial layer, frequently accompanied by hyperkeratosis (5, 60 and 70% of the animals affected at 15, 30 and 60 mg/kg bw/day, respectively) and undulations at the basement membrane zone (10, 55 and 70% of the animals affected at 15, 30 and 60 mg/kg bw/day, respectively). Undulations are believed to be caused by the space occupying expansion of the epithelial layer of the forestomach and considered a secondary
change. In a single male treated with 60 mg/kg bw/day the lesion became slight papillary. Signs of slight cellular atypia were noted in few males treated with 30 mg/kg bw/day and in a male and female treated with 60 mg/kg bw/day. The finding of obvious squamous cell hyperplasia (with early signs of atypia in some cases) in the non-glandular stomach of rats treated with 30 or 60 mg/kg bw/day is considered an adverse finding. Lesion in the non-glandular stomach of rodents however are considered clinically irrelevant since humans do not have a forestomach equivalent.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

For local effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

These effects are considered to represent local effects in the non-glandular stomach.

2

Key result

Dose descriptor:

NOAEL

Remarks:

For systemic toxicity

Effect level:

>= 60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

As no signs of systemic toxicity were observed, the NOAEL for systemic toxicity is 60 mg/kg bw, the highest dose tested in this study.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table 1. Summary of the test item-related findings at scheduled euthanasia (days 91 -92):

	Males				Females
Dose level (mg/kg/day)	0	15	30	60	0	15	30	60
NON-GLANDULAR STOMACH^a	10	10	10	10	10	10	10	10
Hyperplasia, Squamous cell
Minimal	-	2	4	1	-	2	3	5
Slight	-	-	5	6	-	-	3	4
Moderate	-	-	-	2	-	-	-	-
Hyperkeratosis
Minimal	-	1	3	5	-	-	3	2
Slight	-	-	4	4	-	-	2	3
Moderate	-	-	-	-	-	-	-	-
Undulations at basement membrane
Present	-	2	7	8	-	-	4	6

^a = Number of tissues examined from each group.

Results of the formulation analysis:

No test item was detected in the Group 1 formulation (week 1, week 6 and week 13). The concentrations analysed in the formulations of Group 2, 3 and 4 (week 1, week 6 and week 13)

were in agreement with the target concentrations (i.e. respective mean accuracies of 98.8% (n =6), 100.2% (n = 2) and 101.0 ( n = 6) in week 1; 101.1% (n = 6), 100.3% (n = 2) and 99.1% (n = 6) in week 6 and 102.7% (n = 6), 100.85 (n = 2) and 100.0% (n = 3) in week 13).

The formulations of Group 2 and Group 4 prepared (week 1, week 6 and week 13) were homogeneous (i.e. coefficient of variation 0.5 and 0.5 in week 1, 0.6 and 0.5 in week 6 and 1.0 and 1.1 in week 13).

Applicant's summary and conclusion

Conclusions:

In a GLP-compliant OECD guideline 408 study, the NOAEL for local toxicity was set at 15 mg/kg bw/day, based on the local proliferative lesions (including squamous cell hyperplasia with early signs of atypia in some cases) in the forestomach. As no signs of systemic toxicity were observed, the NOAEL for systemic toxicity is 60 mg/kg bw, the highest dose tested in this study.

Executive summary:

In a GLP-compliant OECD guideline 408 study, groups of 10 male and female Crl:WI (Han) rats were administered the test substance in corn oil by oral gavage at dose levels of 0, 15, 30 and 60 mg/kg bw/day for 90 days. There were no mortalities or relevant clinical signs. Body weights, body weight gains and food consumption were unaffected. There were no adverse changes on ophthalmological, haematological or clinical chemistry parameters and no macroscopic changes or organ weight changes observed at gross necropsy. Histopathological examination revealed the presence of local proliferative lesions in the forestomach in all treatment groups, with an increasing incidence and severity (see attached illustration). The lesions were characterized by areas of a thickened epithelial layer, frequently accompanied by hyperkeratosis and undulations at the basement membrane zone. Undulations are believed to be caused by the space occupying expansion of the epithelial layer of the forestomach and considered a secondary change. Signs of slight cellular atypia were noted in few males treated with 30 mg/kg/day bw/day and in a male and female treated with 60 mg/kg bw/day. The finding of obvious squamous cell hyperplasia (with early signs of atypia in some cases) in the non-glandular stomach of rats treated with 30 or 60 mg/kg bw/day is considered an adverse finding. Lesion in the non-glandular stomach of rodents however are considered clinically irrelevant since humans do not have a forestomach equivalent. Based on the results of the study, the NOAEL was set to 15 mg/kg bw/day.

As no signs of systemic toxicity were observed, the NOAEL for systemic toxicity is 60 mg/kg bw, the highest dose tested in this study.