2-Propenoic acid, heptadecyl ester, branched

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

heptadecyl (branched) acrylate

EC Number:

810-816-6

Cas Number:

1473386-36-5

Molecular formula:

C20H38O2

IUPAC Name:

heptadecyl (branched) acrylate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Housing: individually (polycarbonate cages type III)
- Diet (e.g. ad libitum): ad libitum (ground Kliba maintenance die mouse/rat "GLP" meal)
- Water (e.g. ad libitum): ad libitum (drinking water)
- Acclimation period: 8 day prior substance administration

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionized water containing 0.5% sodium carboxymethyl cellulose with 50 mg/L Cremophor

Details on oral exposure:

Heptadecyl Acrylate (C17A) was applied as an emulsion. To prepare this emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water containing 0.5% sodium carboxymethyl cellulose with 50 mg/L Cremophor was filled up to the desired volume, subsequently released with an Ultraturrax. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced once weekly. The administration volume was 10 mL/kg body weight.Heptadecyl Acrylate (C17A) was applied as an emulsion. To prepare this emulsion, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water containing 0.5% sodium carboxymethyl cellulose with 50 mg/L Cremophor was filled up to the desired volume, subsequently released with an Ultraturrax. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced once weekly. The administration volume was 10 mL/kg body weight.

Details on analytical verification of doses or concentrations:

The stability of Heptadecyl Acrylate (C17A) in deionized water containing 0.5% sodium carboxymethyl cellulose with 50 mg/L Cremophor at room temperature for a period of 7 days was proven before the start of the administration period.

Duration of treatment / exposure:

28 days (+ 14 days recovery)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (dose group 50 and 150 mg/kg bw/day)
10 (control group and 750 mg/kg bw/day)

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration (3 times a day). Abnormalities and changes were documented for each animal.

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observation battery: all animals at the end of the administration (home cage observation and open field observation)
Motor activity assessment: all animals at the end of the administration

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals (main [F1] and recovery [R1] groups) were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Organ weights: anesthetized animals (final body weight), adrenal glands (fixed), brain, epididymides, heart, kidneys, liver, ovaries (fixed), prostate (dorsolateral and ventral part together; fixed), seminal vesicles with coagulating glands (fixed), spleen, testes, thymus (fixed), thyroid glands (with parathyroid glands; fixed), uterus with cervix
Organ/tissue fixation: he following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution (main and recovery groups):
all gross lesions; Adrenal glands; Aorta; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Epididymides; Esophagus; Extraorbital lacrimal glands; Eyes with optic nerve (modified Davidson’s solution); Femur with knee joint; Harderian glands; Heart; Ileum; Jejunum (with Peyer’s patches); Kidneys; Larynx; Liver; Lung; Lymph nodes (mesenteric and axillary lymph nodes); Mammary gland (male and female); Nose (nasal cavity); Ovaries; Oviducts; Pancreas; Parathyroid glands; Pharynx; Pituitary gland; Prostate; Rectum; Salivary glands (mandibular and sublingual glands); Sciatic nerve; Seminal vesicles; Skeletal muscle; Skin; Spinal cord (cervical, thoracic and lumbar cord); Spleen; Sternum with marrow; Stomach (forestomach and glandular stomach); Testes (modified Davidson’s solution); Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina

HISTOPATHOLOGY: Yes
Main groups: All gross lesions; Adrenal glands; Bone marrow (femur); Brain; Cecum; Cervix; Coagulating glands; Colon; Duodenum; Epididymides; Eyes with optic nerve; Heart; Ileum; Jejunum; Kidneys; Liver;
Lung; Lymph nodes (mesenteric and axillary lymph nodes); Ovaries; Peyer’s patches; Pituitary gland; Prostate; Rectum; Sciatic nerve; Seminal vesicles; Skeletal muscle; Spleen; Spinal cord; cervical, thoracic and lumbar cord); Sternum with marrow; Stomach (forestomach and glandular stomach); Testes; Thymus; Thyroid glands; Trachea; Urinary bladder; Uterus; Vagina
Recovery groups:

Statistics:

Blood parameters: KRUSKAL-WALLIS test and WILCOXON-test
Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: WILCOXON-test
Urine pH, volume and specific gravity: KRUSKAL-WALLIS test and WILCOXON-test
fWeight parameters: KRUSKAL-WALLIS test and WILCOXON-test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical findings were observed for male and female animals of all test groups (50, 150 and 750 mg/kg bw/d).

Eight male and all female animals of test group 3 showed slight to moderate salivation directly after treatment on several days of the study, observed for the first time in male animals No. 24 on study day 7 and female animal Nos. 52 and 55 on study day 6. Salivation was also observed in 1 female animal of test group 2 (150 mg/kg bw/d) starting on study day 15.
From the temporary, short appearance immediately after dosing (or shortly before) it was concluded the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related changes of mean body weights and mean body weight change values in were observed for male animals and female animals of test groups 1-3 (50, 150 and 750 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes with regard to food consumption were observed over the entire administration period.

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

no effects observed

Description (incidence and severity):

No test substance-related, adverse changes with regard to water consumption were observed over the entire administration period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
After four weeks of administration, in female rats of test group 3 (750 mg/kg bw/d) absolute and relative basophil cell counts were significantly higher compared to controls, but the values were within historical control ranges (females, absolute basophils 0.00-0.03 Giga/L; relative basophils 0.1-0.7%). Therefore, these alterations were regarded as incidental and not treatment-related.
After the two-week recovery period, in females of test group 3 (750 mg/kg bw/d) relative neutrophil counts were significantly lower whereas relative lymphocyte counts were significantly higher compared to controls. The values were slightly below (neutrophils) and above (lymphocytes) historical control ranges (females, relative neutrophils 9.5-19.1%; relative lymphocytes 70.2-86.7%). However, neither total white blood cell (WBS) counts nor absolute neutrophil and lymphocyte counts were significantly changed. Additionally, absolute and relative eosinophil and large unstained cell (LUC) counts were significantly increased, but the values were within historical control ranges (females, absolute eosinophils 0.05-0.15 Giga/L; relative eosinophils 1.5-3.6%; absolute LUC 0.00-0.03 Giga/L; relative LUC 0.2-0.7%). Therefore, the mentioned alterations among the differential blood cell counts were regarded to be incidental and not treatment-related.
After the recovery period, in males of test group 3 (750 mg/kg bw/d) red blood cell (RBC) counts were significantly lower whereas mean corpuscular volume (MCV) and mean corpuscular hemoglobin content were significantly higher compared to controls. However, all values were within historical control ranges (males, RBC 7.50-8.51 Tera/L, MCV 50.8-55.5 fL, MCH 1.05-1.18 fmol). Therefore, these changes were regarded to be incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
After the administration period, in rats of both sexes of test groups 1, 2 and 3 (50, 150 and 750 mg/kg bw/d) total bilirubin levels were significantly decreased. In absence of any signs of anemia, these changes were most probably due to an increased conjugation rate of bilirubin followed by an accelerated excretion via the bile because of liver enzyme induction. This alteration was regarded as adaptive and non-adverse. Concomitantly with bilirubin, bile acids were also conjugated and excreted with the bile. Therefore, total bile acids were also significantly decreased at least in females of test groups 2 and 3 (150 and 750 mg/kg bw/d). Because of this coincidence, total bile acid decrease was also regarded as adaptive and non-adverse.
The following parameters were not dose-dependently changed and, therefore, regarded as incidental and not treatment-related: decreased total protein, albumin and globulin levels in males of test group 2 (150 mg/kg bw/d); decreased triglyceride levels in females of test groups 1 and 2 (50 and 150 mg/kg bw/d).
After the two-week recovery period, in females of test group 3 (750 mg/kg b/d), total bile acid levels were significantly increased, but the mean was within the historical control range (females, total bile acids 14.8-55.5 µmol/L). Therefore, this alteration was regarded as incidental and not treatment-related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among urinalysis parameters were observed.
After the administration period in rats of both sexes of test groups 2 and 3 (150 and 750 mg/kg bw/d) urine volume was significantly decreased and urine specific gravity was significantly increased. In males, these alterations did not occur in a dose-dependent manner. Additionally, in males of test group 3 (750 mg/kg bw/d) urine pH values were significantly decreased. The mentioned alterations could have been related to treatment, but lower urine volume combined with higher specific gravity in the urine reflects the physiological concentration capacity of the kidneys. Therefore, the mentioned changes were regarded as adaptive and non-adverse.
In males of test group 3 (750 mg/kg bw/d) significant higher incidences of granulated and epithelial cell cast occurred in the urine sediment. As histopathological findings were not observed in the kidneys, this isolated change was regarded as potentially treatment-related, but non-adverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related, adverse effects were observed.
Slight salivation was observed in male animal No. 30 and female animal Nos. 53 and 54 of test group 3 (750 mg/kg bw/d). Additionally, male animal No. 1 of test group 0 (0 mg/kg bw/d) showed reduced exploration of the area. These observations were considered to be incidental.
Sensorimotor tests/reflexes:No test substance-related effects were observed.
Quantitative parameters:No test substance-related effects were observed.

Motor activity measurement:
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals of any test group.

Comparing the single intervals with the control groups, significantly increased values were measured for female animals of test group 2 (150 mg/kg bw/d) and test group 3 (750 mg/kg bw/d) at interval 3. The changes were regarded to be incidental and not related to treatment as only one single interval was changed, and the overall motor activity was not affected.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The increase of absolute and relative liver weight in males and females of test group 3 (750 mg/kg bw/d; main group) was regarded to be treatment-related. The increase of relative heart weight in males of test group 3 (750 mg/kg bw/d) was slightly above historical control values, but as no histopathologic finding was observed and the absolute weight was not significantly changed it was considered to be incidental and not related to treatment. The decreased absolute seminal vesicles’ weight in test group 2 (150 mg/kg bw/d) males and the increased relative adrenal glands’ weight of test group 1 (50 mg/kg bw/d) males were regarded to be incidental due to a missing dose-response relationship. The reduced ovary weight in test group 3 (750 mg/kg bw/d; the recovery group) was still within historical control data and therefore regarded to be incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred either individually or in low incidences or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males and females of test group 3 (750 mg/kg bw/d; main group) showed a diffuse increase in hepatocellular size diagnosed as hypertrophy. One male of test group 2 (150 mg/kg bw/d; main group) also revealed a minimal diffuse hypertrophy. One male of the recovery group showed a minimal diffuse hypertrophy. All these findings were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: increase liver weight

Organ:

liver

Treatment related:

yes

Any other information on results incl. tables

Absolute organ weights:

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Main groups

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (50)	2 (150)	3 (750)	1 (50)	2 (150)	3 (750)
Liver	+6.2%	+6.9%	+30.9%**	+3.8%	+7.0%	+28.0%**
Seminal vesicle	-9.4%	-19.5%*	+3.1%

*p≤0.05; **p≤0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Recovery groups

	Female animals
Test group (mg/kg bw/d)	3 (750)
Ovaries	-16.4%**

*p≤0.05; **p≤0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights:

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Main groups

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (50)	2 (150)	3 (750)	1 (50)	2 (150)	3 (750)
Adrenal glands	+18.9%**	+11.4%	+24.6%
Heart	+0.9%	+0.5%	+10.6%**
Liver	+6.7%	+7.7%	+35.2%**	+4.1%	+3.9%	+30.7%**

*p≤0.05; **p≤0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Recovery groups

All mean relative weight parameters did not show significant differences when compared to the control group 0.

Histopathology:

Treatment-related findings were observed in the following organ with incidences and grading according to the tables below:

Main groups

Liver	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (50)	2 (150)	3 (750)	0 (0)	1 (50)	2 (150)	3 (750)
No. of animals	5	5	5	5	5	5	5	5
Hypertrophy, diffuse	0	0	1	4	0	0	0	2
Grade 1				2
Grade 2			1	2				2

Recovery groups

Liver	Male animals		Female animals
Test group (mg/kg bw/d)	0 (0)	3 (750)	0 (0)	3 (750)
No. of animals	5	5	5	5
Hypertrophy, diffuse	0	1	0	0
Grade 1		1

Applicant's summary and conclusion

Conclusions:

The administration of Heptadecyl Acrylate (C17A) by gavage to male and female Wistar rats for 4 weeks with 2 weeks recovery period caused test substance-related, adverse findings at a dose level of 750 mg/kg bw/d taking liver weight changes and diffuse hepatocellular hypertrophy into account. These changes were not observable any more after a 2-weeks recovery period. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) for this test compound in male and female Wistar rats of both sexes was 150 mg/kg bw/d.