Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1, 2017 to February 15, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
DRF for 90-day study.
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April 20, 2017 to September 14, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Standard DRF study.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: Laboratory standard operating procedures
Deviations:
no
Principles of method if other than guideline:
The Institutional Animal Care and Use Committee (IACUC) of Smithers Avanza approved the study protocol and found it to be in accordance with provisions of the USDA Animal Welfare Act, the PHS Policy on Humane Care and Use of Laboratory Animals, and the US Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals. The purpose of this study was to determine the potential toxicity of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) when administered daily for fourteen days via oral gavage to male and female Sprague Dawley rats in order to present dose levels for the 90-day sub-chronic oral toxicity study.
GLP compliance:
no
Remarks:
non-GLP study; not required for dose range setting.
Limit test:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected because it is a standard species for use in toxicology studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
Species and Strain: Sprague Dawley Rate
Supplier: Envigo, Frederick, MD
Method of Identification: Ear tage/cage card
Number of Animals Received: Males – 22; Females – 22
Number Used on Study: Males – 20; Females – 20
Age at First Dose: Males – 9-10 weeks; Females – 9-10 weeks
Weight Range at First Dose: Males – 278.7-301.4 g; Females – 192.7-216.8 g
Disposition of Extra Animals: Transferred to training/stock colony

Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Husbandry
Feed: Certified Global Tekland Laboratory Diet 2018 (pellets) was provided ad libitum, unless otherwise noted.
Water: Filtered water was provided ad libitum via an automatic watering system
Bedding: Certified Sani Chips hardwood bedding
Housing: Animals were group housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were housed two or three to a cage.
Temperature Range: 20 to 26 °C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark, interrupted as necessary for study-related events
Air Changes: Minimum of 10 air changes per hour

The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analyzed for contaminants and specific microbes. The bedding was analyzed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, molds, and organophosphates prior to certification. No contaminants were known to be present in the feed, water, or bedding at levels that might have interfered with achieving the objectives of the study.
Route of administration:
oral: gavage
Details on route of administration:
The animals were dosed via oral gavage at a volume of 2 mL/kg for 14 days. Dose volumes were based on the animals’ most recent body weight. The first day of dosing was designated as SD 1 for each animal.
Vehicle:
peanut oil
Details on oral exposure:
The neat test and vehicle/control substances were considered 100% pure for formulation purposes.
The vehicle/control substance, peanut oil, was used as received; no formulations were necessary.
Group 4 (500 mg/mL) formulations were prepared daily by adding the appropriate amount of test substance into a pre-calibrated beaker. The required amount of vehicle was added and the solution was mixed until visually uniform. Formulations for Group 3 (150 mg/mL) were prepared daily by mixing together the required amount of Group 4 formulations with the required amount of vehicle. Formulations for Group 2 (50 mg/mL) were prepared daily by mixing together the required amount of Group 3 formulations with the required amount of vehicle. Formulations were maintained at room temperature until used for further formulating or dosing.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals (5 male/5 female) per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
The rat was selected because it is a standard species for use in toxicology studies.
This study was designed to use the fewest number of animals possible, consistent with the objective of the study, the scientific needs of the Sponsor, and contemporary scientific standards.
The oral route was selected because it is the anticipated route of exposure in humans.
The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor in rats through dermal exposure.
Animals were initially accepted into the randomization pool based upon prestudy body weights and physical examinations. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Males and females were randomized separately. Following randomization each study animal was assigned a unique number.
Positive control:
Not required
Observations and examinations performed and frequency:
Animal Observations/Measurements
Physical Examinations: Study Day 1 (prior to initiation of dosing); Weekly thereafter; Prior to necropsy; Unscheduled observations were recorded.
Cageside Observations: ≥ 2 times daily
Body Weights: Study Day1 (prior to initiation of dosing), 4 and 8; Day prior to necropsy (unfasted); Prior to necropsy (fasted).
Food Consumption (Quantitative): Study Day 1-4, 4-8 and 9-14 (Food was inadvertently discarded on the morning of SD 9).

Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.

Clinical Pathology
Blood samples and urine specimens were collected as shown below under "Serum Clinical Chemistry ". Animals were fasted overnight (with water available) prior to sample collection.
Blood Sampling/Urine Collection Information for Clinical Pathology
Chemistry
Collection Day: Prior to scheduled termination
Collection Method: Cardiac puncture under 70% CO2/30% O2 anesthesia.
Volume Collected: At least 800 μl
Tubes Used: serum separator

Hematology
Collection Day: Prior to scheduled termination
Collection Method: Cardiac puncture under 70% CO2/30% O2 anesthesia.
Volume Collected: At least 0.5 mL
Tubes Used: K2 EDTA

Coagulation
Collection Day: Prior to scheduled termination
Collection Method: Cardiac puncture under 70% CO2/30% O2 anesthesia.
Volume Collected: At least 1.8 mL
Tubes Used: sodium citrate

Urinalysis
Collection Day: Prior to scheduled termination
Collection Method: Metabolism cage
Volume Collected: Total volume recorded; up to 12 mL submitted
Tubes Used: Urintek

Samples/specimens were transported to the Clinical Pathology Laboratory for analysis. All remaining samples were discarded prior to finalization of the report.

Serum Clinical Chemistry
The Siemens Dimension XPand chemistry analyzer was used to measure the serum concentrations of the clinical chemistry parameters listed below.
Clinical Chemistry Tests
Albumin (ALB); Alkaline Phosphatase (ALPi); Alanine Aminotransferase (ALTi); Aspartate Aminotransferase (AST); Blood urea Nitrogen (BUN); Calcium (CA); Cholesterol (CHOL); Creatine Kinase (CK); Chloride (CL); Creatinine (CRE2): Gamma Glutamyltranspeptidase (GGT); Glucose (GLU); Potassium (K); Sodium (NA); Phosphorus (PHOS); Total Bilirubin (TBILI)
Total Protein (TPROT); Triglycerides (TRIG).

All reagents used during testing were obtained from Siemens. Commercially available controls were analyzed each day of testing.
Globulin (GLOB, g/dL) was calculated as the difference between total protein and albumin. The albumin to globulin ratio (A/G) was obtained by dividing the albumin value by the globulin value.
Values that were below the detection limit of the analyzer were reported as < the lowest limit of the measured parameter. For example, the lowest limit for triglyceride concentration determination was 15 mg/dL. A value that was observed to be less than this was reported as <15.

Hematology
The Siemens Advia 120 hematology analyzer was used to measure or calculate the hematology parameters listed below.
White Blood Cells (WBC); Red Blood Cells (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean Corpuscular Volume (MCV); Mean Corpuscular Hemoglobin (MCH); Mean Corpuscular Hemoglobin Concentration (MCHC); Mean Platelet Volume (MPV); Platelets (PLT); Red Cell Distribution Width (RDW); Absolute Neutrophils (ABNEUT); Absolute Lymphocytes (ABLYMP); Absolute Monocytes (ABMONO); Absolute Eosinophils (ABSEOS); Absolute Basophils (ABBAS); Absolute Reticulocytes (ABRETi).

All reagents used during testing were obtained from Siemens. Commercially available controls were analyzed each day of testing.
When the white blood cell differential or reticulocyte count could not be calculated by the Advia 120 due to mechanical or sample limitations, a manual count was performed. If platelet clumps were observed on the hematology smear, the platelet count and mean platelet volume were reported as clumped.
Blood smears for cellular morphology were prepared for each animal and stained using a Modified Wright-Giemsa stain. The slide was evaluated when the data results from the automated hematology evaluation for that particular animal were outside the system standards. Morphology findings (if any) were categorized as follows: 1+ = minimal; 2+ = mild; 3+ = moderate; 4+ = marked.

Coagulation
The Beckman Coulter Elite Pro coagulation analyzer was used to measure the coagulation tests listed below.
Prothrombin Time (PT); Activated Partial Thromboplastin Time (APTT); Fibrinogen (FIB).

All reagents used during testing were obtained from Beckman Coulter. Commercially available controls were analyzed each day of testing.

Urinalysis
Urine specimens were processed according to the procedures listed below. Reagent strips were run on a Siemens Clinitek 100 or Atlas analyzer.
Volume (TUVol); Color; Clarity; Specific Gravity (SG); Glucose (UGLU); Bilirubin (UBILI); Ketone (KET); Blood (BLO); pH (UpH); Protein (PRO); Uronilinogen (URO); Nitrites (NIT); Leukocytes (LEU). Microscopic Examination of Sediment: White Blood Cells (uWBC); Red Blood Cells (uRBC); Epithelial Cells (EPI); Bacteria (Bacter); Mucus; Yeast; Fecal; Sperm; Hyaline Cast (HYAL CA); Granular Cast (GRAN CA); Amorphous Crystals (Amorph); Triple Phosphate Crystals (TP CRYS); Calcium Oxalate Crystals (CAO CRYS); Other Observations (Other/Uri).

Commercially available controls were analyzed each day of testing. Qualitative measurements were made as follows: 1+ = 1 - 3, 2+ = 4 - 10, and 3+ = >10. Urine specimens with fecal contamination are designated as: 1+ = minimal, 2+ = moderate, and 3+ = heavy.
Sacrifice and pathology:
Termination and Necropsy
Termination
On SD 15, all animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

Necropsy
Animals were necropsied as soon as possible after the time of death. Animals were necropsied, bone marrow smears were prepared, required organs were weighed, and protocol-specified tissues were collected and preserved.
Gross necropsy included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Organ weights were collected as soon as possible after dissection and paired organs were weighed together. Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization
Statistics:
Quantitative data (body weights, body weight changes, food consumption, clinical pathology, and organ weight data) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques; sexes were analyzed separately. Prior to the ANOVA analysis, untransformed data were tested to determine if the data are normally distributed and have homogeneous variances among all groups. A Shapiro-Wilk test was used to test for normality, followed by the Levene’s test to test the hypothesis of homogeneity of variances. If either the normality or homogeneity test failed (as indicated by a Shapiro-Wilk or Levene’s test p-value ≤ 0.01), the data were transformed using log-transformed values and both the Shapiro-Wilk and Levene’s tests were repeated with the log-transformed values. If the results from either the Shapiro-Wilk or Levene’s test on the log-transformed data failed, the analysis of the data continued using rank-transformed data using Kruskal-Wallis ANOVA. If both the Shapiro-Wilk or Levene’s test were not statistically significant, the ANOVA was performed on the untransformed or log-transformed data, respectively. The Dunnett’s t-test was used to determine which groups (if any) differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. An arcsine square root transformation was used for some proportion/percentage data which do not meet the assumptions of parametric statistical tests in the attempt to normalize the data; this data was transformed prior to the ANOVA analysis. The term “significant” is used throughout the text of the report to indicate statistical significance at p ≤ 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on physical examinations. One incidental finding was an abrasion on the mouth in a Group 4 (1000 mg/kg) male on SD 5 and was therefore not considered test substance-related. No other observations were noted.
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on cageside observations; no abnormalities were noted in any animal throughout the study.
Mortality:
no mortality observed
Description (incidence):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on mortality. All animals survived until the scheduled termination.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on body weights or body weight changes. There were no statistically significant differences between the control and treated groups. All animals gained weight throughout the study and mean body weight gains were similar across groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on food consumption. Group 3 (300 mg/kg) males had significantly lower mean food consumption than the control from SD 1 to 4, but total food consumption for males from SD 1 to 8 and 9 to 14 was similar across groups. In females, significantly lower mean food consumption was measured for Groups 2 (100 mg/kg; SD 1 to 4, 4 to 8, 1 to 8 [total]) and 4 (SD 1 to 4, 1 to 8 [total]). Females in Group 3 (300 mg/kg) had significantly higher mean food consumption from SD 1 to 8 (total), 9 to 14, 9 to 14 (total). Total mean food consumption from SD 1 to 14 was lower than the control in Groups 2 and 4, but higher in Group 3. No statistical analysis was performed for this interval. Changes in food consumption did not correlate with body weights, were variable in the effect and there was no associated dose response, therefore the differences in food consumption were not considered test substance-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on hematology. Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on coagulation. In Group 2 males, fibrinogen was significantly increased and was within historical control range. Thus, it was considered toxicologically insignificant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had a limited effect on clinical chemistry parameters evaluated. Globulin (GLOB) levels and albumin/globulin (A/G) ratio were altered. GLOB was significantly higher for Group 3 (300 mg/kg) males and Group 4 (1000 mg/kg) males and females. The corresponding A/G ratio was significantly lower for Group 3 and 4 males and Groups 2 to 4 females. Although these parameters are statistically different compared to the control levels, they do not appear to be adverse. Several other parameters were significantly higher when compared to the control Group including TPROT (3M and 4M), AST (3M), and ALTi (3M). All of the changes in these parameters were within historical control range, due to a single outlier, did not have a corresponding dose response or only occurred in one sex, and therefore were consider toxicologically insignificant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no effect on urine parameters after treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect on organ weights after treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) for fourteen days.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) had no effect on macroscopic findings. There were no visible lesions found at necropsy except for two females, one in Group 1 (Animal 21331) and one in Group 2 (Animal 21345), which had a distended uterus. Since the finding was observed in a control animal, it was not considered test substance-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology, or organ weights.
Key result
Critical effects observed:
no

Animal Disposition and Physical Examinations

Group

Sex

Animal

Clinical Sign

Day Numbers Relative to Start Date

1

8

15

1

M

21326

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21327

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21328

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21329

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21330

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

2

M

21336

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21337

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21338

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21339

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21340

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

3

M

21346

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21347

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21348

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21349

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21350

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

4

M

21356

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21357

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21358

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21359

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21360

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Group

Sex

Animal

Clinical Sign

Day Numbers Relative to Start Date

1

8

15

1

F

21331

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21332

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21333

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21334

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21335

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

2

F

21341

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21342

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21343

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21344

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21345

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

3

F

21351

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21352

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21353

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21354

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21355

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

4

F

21361

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21362

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21363

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21364

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

21365

No Abnormalities Detected

Terminal Kill

X

-

X

-

X

X

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Body Weights (g)

Day Numbers Relative to Start Date

Group

Sex

Animal

1

4

8

14

15

Group

Sex

Animal

1

4

8

14

15

1

M

21326

21327

21328

21329

21330

297.3

286.6

280.9

295.6

294.0

308.4

291.7

283.7

303.4

302.1

326.1

315.6

309.6

331.02

330.2

343.5

332.6

326.4

348.5

342.2

322.2

317.6

309.6

333.6

328.9

3

M

21346

21347

21348

21349

21350

288.6

301.4

278.8

288.2

286.7

291.4

308.2

287.1

297.5

284.6

331.9

310.2

314.7

322.7

305.5

352.2

326.5

330.0

339.8

315.8

320.7

337.6

321.1

320.5

304.6

Mean

S.D.

N

290.88

6.91

5

297.86

9.98

5

322.50

9.47

5

338.64

8.94

5

322.38

9.42

5

Mean

S.D.

N

288.74

8.12

5

293.76

9.44

5

317.00

10.47

5

322.84

13.83

5

320.90

11.67

5

2

M

21336

21337

21338

21339

21340

289.8

279.3

298.3

292.7

278.7

298.7

287.3

231.6

302.6

284.6

327.4

307.7

330.3

329.1

308.2

346.5

325.1

348.1

347.6

318.3

322.6

311.3

337.1

332.4

301.4

4

M

21356

21357

21358

21359

21360

291.7

288.4

280.2

286.9

286.7

302.4

292.1

291.6

297.0

295.7

327.1

311.1

310.6

316.1

323.7

340.1

322.1

317.8

327.4

335.5

326.6

309.4

308.0

312.8

321.9

Mean

S.D.

N

287.79

8.56

5

296.76

10.80

5

319.94

11.17

5

337.12

14.59

5

322.96

15.67

5

Mean

S.D.

N

286.78

4.19

5

295.76

4.37

5

317.72

7.43

5

328.58

9.22

5

315.74

8.13

5

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Day Numbers Relative to Start Date

Group

Sex

Animal

1

4

8

14

15

Group

Sex

Animal

1

4

8

14

15

1

F

21331

21332

21333

21334

21335

206.2

204.1

208.9

216.8

192.7

209.3

202.4

205.9

220.0

196.3

222.2

215.5

223.4

226.1

209.3

228.2

219.8

227.4

228.0

212.1

217.2

209.8

221.4

220.5

203.1

3

F

21351

21352

21353

21354

21355

201.9

200.4

198.2

207.3

214.4

204.0

199.4

200.6

209.2

212.4

216.5

213.7

214.3

220.7

214.8

218.5

220.8

233.8

230.6

229.0

208.8

208.2

214.2

217.1

215.0

Mean

S.D.

N

205.74

8.7

5

206.79

8.82

5

219.30

6.82

5

223.10

7.08

5

214.40

7.79

5

Mean

S.D.

N

204.44

6.50

5

205.12

5.57

5

216.00

2.83

5

226.54

6.57

5

212.66

3.95

5

2

F

21341

21342

21343

21344

21345

204.6

203.1

206.6

202.3

196.2

208.0

208.9

207.5

207.4

200.3

215.3

223.7

215.8

216.6

211.7

220.4

225.5

218.4

219.2

213.1

209.5

214.4

206.8

210.7

201.0

4

M

21361

21362

21363

21364

21365

206.3

207.2

203.2

207.0

193.4

200.4

203.4

205.6

206.8

200.4

216.3

211.6

218.6

220.4

206.5

221.0

213.9

223.0

217.9

207.2

209.9

198.8

210.1

212.9

196.6

Mean

S.D.

N

202.56

3.91

5

206.42

3.47

5

216.62

4.38

5

219.32

4.44

5

208.48

4.99

5

Mean

S.D.

N

203.42

5.83

5

203.32

2.93

5

214.68

5.64

5

216.60

6.28

5

205.66

7.40

5

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Body Weight Changes (g)

Group

Sex

Animal

From:

To:

Day Numbers Relative to Start Date

1

4

4

8

8

14

Absolute Change

Percent Change

14

15

1

14

1

14

1

M

21326

21327

21328

21329

21330

 

11.1

5.1

2.8

7.8

8.1

17.7

23.9

25.9

27.6

28.1

17.4

17.0

16.8

17.5

12.0

46.2

46.0

45.5

52.9

48.2

15.54

16.05

16.20

17.90

16.39

-21.3

-15.0

-16.8

-14.9

-13.3

Mean

S.D.

N

 

6.98

3.16

5

24.64

4.21

5

16.14

2.33

5

47.76

3.05

5

16.42

0.89

5

-16.26

3.08

5

2

M

21336

21337

21338

21339

21340

 

8.9

8.0

12.3

9.9

5.9

25.7

20.4

19.7

26.5

23.6

22.1

17.4

17.8

18.5

10.1

56.7

45.8

49.8

54.9

39.6

19.57

16.40

16.69

18.76

14.21

-13.9

-13.3

-11.0

-15.2

-16.9

Mean

S.D.

N

 

9.00

2.36

5

23.18

3.06

5

17.18

4.37

5

49.36

6.94

5

17.12

2.11

-14.16

2.17

5

3

M

21346

21347

21348

21349

21350

 

2.8

6.8

8.3

9.3

-2.1

40.5

2.0

27.6

25.2

20.9

20.3

16.3

15.2

17.1

10.2

63.6

25.1

51.2

51.6

29.0

22.04

8.33

18.36

17.90

10.12

-31.5

11.1

-8.9

-19.3

-11.1

Mean

S.D.

N

 

5.02

4.69

5

23.24

13.94

5

15.84

3.67

5

44.10

16.40

5

15.35

5.85

5

-11.94

15.63

5

4

M

21356

21357

21358

21359

21360

 

10.7

3.7

11.4

10.1

9.0

24.7

19.0

19.0

19.1

28.0

13.0

11.0

7.2

11.3

11.8

48.4

33.7

37.6

40.5

48.8

16.59

11.69

13.42

14.12

17.02

-13.5

-12.7

-9.8

-14.6

-13.6

Mean

S.D.

N

 

8.98

3.08

5

21.96

4.17

5

10.86

2.18

5

41.80

6.66

5

14.57

2.23

5

-12.84

1.83

5

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Group

Sex

Animal

From:

To:

Day Numbers Relative to Start Date

1

4

4

8

8

14

Absolute Change

Percent Change

14

15

1

14

1

14

1

F

21331

21332

21333

21334

21335

 

3.1

-1.7

-3.0

3.2

3.6

12.9

13.1

17.5

6.1

13.0

6.0

4.3

4.0

1.9

2.8

22.0

15.7

18.5

11.2

19.2

10.67

7.699

8.86

5.17

10.07

-11.0

-10.0

-6.0

-7.5

-9.0

Mean

S.D.

N

 

1.04

3.13

5

12.52

4.08

5

3.80

1.56

5

17.36

4.11

5

8.49

2.18

5

-8.70

1.99

5

2

F

21341

21342

21343

21344

21345

 

3.4

5.8

0.9

5.1

4.1

7.3

14.8

8.3

9.2

11.4

5.1

1.8

2.6

2.6

1.4

15.8

22.4

11.8

16.9

16.9

7.72

11.03

5.71

8.35

8.61

-10.9

-11.1

-11.6

-8.5

-12.1

Mean

S.D.

N

 

3.86

1.89

5

10.20

2.98

5

2.70

1.44

5

16.76

3.79

5

8.29

1.91

5

-10.84

1.39

5

3

F

21351

21352

21353

21354

21355

 

2.1

-1.0

2.4

1.9

-2.0

12.5

14.3

13.7

11.5

2.4

2.0

7.1

19.5

9.9

14.2

16.6

20.4

35.6

23.3

14.6

8.22

10.18

17.96

11.24

6.81

-9.7

-12.6

-19.6

-13.5

-14.0

Mean

S.D.

N

 

0.68

2.03

5

10.88

4.86

5

10.54

6.69

5

22.10

8.26

5

10.88

4.31

5

-13.88

3.61

5

4

F

21361

21362

21363

21364

21365

 

-5.9

-3.8

2.4

-0.2

7.0

15.9

8.2

13.0

13.6

6.1

4.7

2.3

4.4

-2.5

0.7

14.7

6.7

19.8

10.9

13.8

7.13

3.23

9.74

5.27

7.14

-11.1

-15.1

-12.9

-5.0

-10.6

Mean

S.D.

N

 

-0.10

5.10

5

11.36

4.06

5

1.92

2.96

5

13.18

4.84

5

6.50

2.42

5

-10.94

3.76

5

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Gross Pathology Observations

Day Numbers Relative to Start Date

Animal No.

Mode of Death

Death

Observation(S)

Day

(Week)

Group: 1          Sex: Male

21326

21327

21328

21329

21330

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Group: 2          Sex: Male

21336

21337

21338

21339

21340

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Group: 3          Sex: Male

21346

21347

21348

21349

21350

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Group: 4          Sex: Male

21356

21357

21358

21359

21360

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Day Numbers Relative to Start Date

Animal No.

Mode of Death

Death

Observation(S)

Day

(Week)

Group: 1          Sex: Female

21331

 

21332

21333

21334

21335

TERMINAL KILL

 

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

 

15

15

15

15

(3)

 

(3)

(3)

(3)

(3)

Uterus; distention; fluid-filled; bilaterial; clear

Any remaining protocol required tissues, which have been examined, have no visible lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Group: 2          Sex: Female

21341

21342

21343

21344

21345

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Uterus; distention; fluid-filled; bilaterial; clear

Any remaining protocol required tissues, which have been examined, have no visible lesions

Group: 3          Sex: Female

21351

21352

21353

21354

21355

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Group: 4          Sex: Female

21361

21362

21363

21364

21365

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

TERMINAL KILL

15

15

15

15

15

(3)

(3)

(3)

(3)

(3)

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

No Visible Lesions

Nominal Dose: Group 1 – 0 mg/kg Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Conclusions:
Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) at doses up to 1000 mg/kg/day via oral gavage for 14 consecutive days was well tolerated by male and female Sprague Dawley rats. There was no effect of the test substance on all parameters evaluated including mortality, physical examinations, cageside observations, body weight, body weight changes, food consumption, clinical pathology, macroscopic findings, and organ weights.
Minor changes in food consumption and physical exams were noted after treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) for 14 days. These included changes in variable food consumption in females and an abrasion on the mouth of one Group 4 male. These changes were not considered to be test substance-related due to the lack of a dose related response and the incidental nature.
GLOB and A/G levels were found to be significantly different from control levels and did not appear to have toxicological significance due to no corresponding observations. Several other clinical chemistry (TPROT, AST, and ALTi), and coagulation (FIB) parameters were found to be statistically significantly increased for Group 3 and 4 (TPROT only) males. The findings were inconsistent, due to a single outlier or within historical control and were not considered to be test substance-related.
There were no apparent test substance-related effects at all doses of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) (up to 1000 mg/kg/day) evaluated. There were no effects on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology, or organ weights. At the dose levels tested, a no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day for male and female rats dosed with the test substance, decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510).
Executive summary:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510): A Fourteen-Day Repeat Dose Toxicity Study Following Oral Gavage Administration to Male and Female Sprague Dawley Rats.

 

The purpose of this study was to determine the potential toxicity of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) when administered daily for fourteen days via oral gavage to male and female Sprague Dawley rats.

 

Forty (20/sex) Sprague Dawley rats were randomly assigned to four groups (5 animals/sex/group). Animals were administered control substance (peanut oil) or decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) at 100, 300, or 1000 mg/kg/day once daily via oral gavage for 14 days. Animals were subjected to a full gross necropsy on Study Day (SD) 15.

 

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, and absolute and relative organ weights.

 

Treatment with decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) at doses up to 1000 mg/kg/day had no effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, or absolute and relative organ weights.

 

There were no apparent test substance-related effects at all doses of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) (up to 1000 mg/kg/day) evaluated. There were no effects on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology, or organ weights. At the dose levels tested, a no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day for male and female rats dosed with the test substance, decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
The following deviations from the protocol were noted:
Samples for concentration verification were shipped to Smithers Viscient for analysis during study week 2 (Mix 2) and not retained as specified in the protocol.
Samples were not prepared for concentration verification from Mixes 11 and 12 due to error.
Upon arrival of the terminal kill tissues to VPS for histological processing, the tissues for Animal 21985 (4f) could not be located. The paperwork for the shipment: FORM0901, Inventories, and Individual Animal Data records could not be located with the shipping box either.
Per protocol, gross lesions were to be trimmed for all groups for the main phase animals. Only identified targets were to be trimmed for the recovery phase animals. Gross lesions from recovery animals (1F-21920, 1F-21922 and 4F-21991) were inadvertently processed to slide. These gross lesions are not reported by the study pathologist and the paraffin blocks and H&E slides will be retained with the study materials.
Some tissues were not microscopically examined, or only one of a paired organ was examined, due to sectioning difficulties, technical errors, or other reasons. Non-examined tissues are identified with the individual histopathology data.
The above-mentioned deviations did not impact this study, nor did they affect the quality or integrity of the study or the interpretation of the results in this report.
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Test material form:
liquid
Details on test material:
Name: Decanoic acid, mixed esters with heptanoic acid, octanoic acid, and trimethylolpropane
Synonym: Hatcol 1510
Lot No.: 2015189219 (TR269-131)
CAS No./EC#: 68130-53-0/268-596-7
Purity: 100%
Expiration Date: 15 April 2018
Upon receipt at Smithers Viscient, the test substance (SMV No. 8807) was stored at room temperature in a dark, ventilated cabinet in the original container.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected because it is a standard species for use in toxicology studies and used as a rodent species per current OECD 408 testing guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
Species and Strain: Sprague Dawley Rat
Supplier: Envigo, Frederick, MD
Method of Identification: Microchip/Cage card
Number of Animals Received: Males: 55; Females: 55
Number Used on Study: Males: 50; Females: 50
Age at First Dose: Males: 9 – 10 weeks; Females: 9 – 10 weeks
Weight Range at First Dose: Males: 276.7 – 303.9 g; Females: 190.8 – 219.4 g
Disposition of Extra Animals: Transferred to training/stock colony or sentinel program

Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Husbandry
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum, unless otherwise noted.
Water: Filtered water was provided ad libitum via an automatic watering system.
Bedding: Certified Sani Chips hardwood bedding.
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were housed two or three to a cage.
Temperature Range: 20 to 26 °C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark, interrupted as necessary for study-related events.
Air Changes: Minimum of 10 air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected because it is the relevant route of exposure to humans and is the route used per current OECD 408 testing guidelines.
Vehicle:
peanut oil
Details on oral exposure:
The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control substance, peanut oil, was used as received; no formulations were necessary.
Group 2 (50 mg/mL), 3 (150 mg/mL), and 4 (500 mg/mL) formulations were prepared on a schedule within the established stability by adding the appropriate amount of test substance into a pre-calibrated beaker. The required amount of vehicle was added and the solution was mixed until visually uniform. Formulations were maintained at room temperature until used for dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (2-mL, in duplicate) for concentration verification were collected from each test substance dose formulation and the vehicle/control substance. Samples were stored under conditions set to maintain 5 ± 3°C. The full analytical report is appended below.
Stability of the dose formulations has been previously determined by the Sponsor’s designee.
One set of samples from SD 1, 28, 56, and 84 were shipped (on cool packs) to Smithers Viscient MRC for test substance concentration analysis.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/group for the main phase and 5 animal/sex/group in Groups 1 and 4 for the recovery phase
Control animals:
yes, concurrent vehicle
Details on study design:
The study design was based on OECD testing guidelines 408: Repeat dose 90-day oral toxicity study in rodents.
The rat was selected because it is a standard species for use in toxicology studies and used as a rodent species per current OECD 408 testing guidelines.
This study was designed to use the fewest number of animals possible, consistent with the objective of the study, the scientific needs of the Sponsor, contemporary scientific standards, and in consideration of applicable regulatory requirements cited previously in the study protocol.
The oral route was selected because it is the relevant route of exposure to humans and is the route used per current OECD 408 testing guidelines.
The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor at Smithers Avanza under study 2386-13844.
Animals were initially accepted into the randomization pool based upon prestudy body weights, physical examinations, and ophthalmic examinations. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Males and females were randomized separately. Following randomization each study animal was assigned a unique number.

The animals were dosed via oral gavage at a volume of 2.0 mL/kg for at least 90 days. Dosing volumes were based on the animals’ most recent body weights. The first day of dosing was designated as SD 1 for each animal.
Excess formulations were disposed of in accordance with company SOPs, appropriate regulatory requirements, and/or information contained in the Material Safety Data Sheets.
Positive control:
Not required for this study.

Examinations

Observations and examinations performed and frequency:
Animal Observations/Measurements
Physical Examinations: Study Day 1 (prior to initiation of dosing). Weekly thereafter. Prior to necropsy.
Cageside Observations: ≥ 2 times daily.
Body Weights: SD1 (prior to initiation of dosing). Weekly thereafter. Day prior to necropsy (unfasted). Prior to necropsy (fasted).
Food Consumption: Weekly quantitative.
Ophthalmologic Examinations: Prior to randomization. Prior to scheduled necropsy (within 2 days; all surviving animals).
Functional Observations Battery (FOB): Once on SD 90
Locomotor Activity: Once on SD 85

Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behaviour patterns.

Ophthalmologic observations were conducted using indirect ophthalmoscopy and slit-lamp biomicroscopy (as needed) following administration of 1% Tropicamide mydriatic solution.

For neurotoxicity observations (FOB, and locomotor activity), animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing. For locomotor activity, animals were placed into an activity chamber for 30 minutes. The Kinder Scientific Motor Monitor II recorded the total number of occurrences of basic movement, fine movement, and rearing during the 30-minute testing timeframe. Data was tabulated in 10-minute intervals.

Functional Observation Battery (FOB) Assessment-See table under “Any other information” for further details.

Clinical Pathology
Blood samples and urine specimens were collected for all animals. Animals were fasted overnight (with water available) prior to sample collection.
Blood Sampling/Urine Collection Information for Clinical Pathology-See table under “Any other information” for further details.

Serum Clinical Chemistry
Clinical Chemistry Tests-See table under “Any other information” for further details.

Hematology
Hematology Tests--See table under “Any other information” for further details.

Coagulation
Coagulation Tests-See table under “Any other information” for further details.

Urinalysis
Urinalysis Tests-See table under “Any other information” for further details.
Sacrifice and pathology:
Termination
On SD 92 for main phase animals and 120 for recovery animals, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

Necropsy
Animals were necropsied as soon as possible after the time of death. Animals were necropsied, bone marrow smears were prepared, required organs were weighed, and protocol-specified tissues were collected and preserved.
Gross necropsy included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Organ weights were collected as soon as possible after dissection and paired organs were weighed together. Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization.

Histopathology
Preserved tissues were transferred to VPS where the protocol-required tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist. The stomach, identified as a possible target tissue in Group 4 main phase animals, was processed to slides and microscopically examined in Groups 2 and 3 (main phase animals), and Groups 1 and 4 (recovery animals).
Animal 21985 (4F) tissues were collected on SD 92, however the tissues were not processed as they were went missing during transportation.
Other examinations:
No further examinations specified in the study report.
Statistics:
Quantitative data (body weights, body weight changes, food consumption, clinical pathology, and organ weight data) from the treated groups were compared statistically to the data of the control group using one-way Analysis of Variance (ANOVA) techniques; sexes were analyzed separately. Prior to the ANOVA analysis, untransformed data were tested to determine if the data are normally distributed and have homogeneous variances among all groups. A Shapiro-Wilk test was used to test for normality, followed by the Levene’s test to test the hypothesis of homogeneity of variances. If either the normality or homogeneity test failed (as indicated by a Shapiro-Wilk or Levene’s test p-value ≤ 0.01), the data were transformed using log-transformed values and both the Shapiro-Wilk and Levene’s tests were repeated with the log-transformed values. If the results from either the Shapiro-Wilk or Levene’s test on the log-transformed data failed, the analysis of the data continued using rank-transformed data using Kruskal-Wallis ANOVA. If both the Shapiro-Wilk or Levene’s test were not statistically significant, the ANOVA was performed on the untransformed or log-transformed data, respectively. The Dunnett’s t-test was used to determine which groups (if any) differed from the control group. Group comparisons were evaluated at the 0.05 (two-tailed) probability level. An arcsine square root transformation was used for some proportion/percentage data which do not meet the assumptions of parametric statistical tests in the attempt to normalize the data; this data was transformed prior to the ANOVA analysis. The term “significant” is used throughout the text of the report to indicate statistical significance at p ≤ 0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Treatment with Hatcol 1510 had no effect on physical examinations. Incidental findings included abrasion of the nose (Group 1 – one female), alopecia of the left and right forelimb (Group 1 – one male), squinting of the left eye (Group 2; 100 mg/kg – one male), and rough haircoat (Group 2 –one male). Animal 21986 (4f; 1000 mg/kg) had a small movable tissue mass of 0.3 to 1.0 cm on the right inguinal region on SD 92 (day of necropsy).
Treatment with Hatcol 1510 had no effect on cageside observations. The only abnormality observed was squinting of the left eye in Animal 21925 (2m; 100 mg/kg) from SD 69 to 87. This finding was not considered test substance-related as it was limited to one animal.
Mortality:
no mortality observed
Description (incidence):
Treatment with Hatcol 1510 had no effect on mortality. All animals survived until the scheduled termination.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with Hatcol 1510 had no effect on body weights or body weight changes. Mean body weights of Group 4 males (1000 mg/kg) were significantly lower than that of the control on SD 15, and were consistently lower than means of the other groups throughout the study. No differences in mean body weights were observed for females. Significant differences in mean body weight change compared to the control were noted from SD 1 to 8 for Group 4 males and females (decreased), SD 8 to 15 for Group 4 males (decreased), and SD 29 to 36 for Group 2 (100 mg/kg) females (increased).
During the recovery phase, mean body weight change of Group 4 males and females were significantly higher than the control from SD 92 to 99, and mean absolute change for Group 4 males was significantly higher than the control from SD 85 to 119. No significant differences in absolute change were noted from SD 1 to 91 for main phase animals or SD 1 to 119 for recovery animals.
The changes in body weight during the dosing phase were only observed in a single sex (males) in Group 4 and were not significantly different in total change or percent change in body weight at either the main study interval or recovery. Additionally, no corresponding reduction in food consumption were noted in any Group throughout the study and therefore were not considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with Hatcol 1510 had no effect on food consumption. Total mean food consumption for main phase animals (SD 1 to 91) was significantly higher in Group 2 (100 mg/kg) females, Group 3 (300 mg/kg) males, and Group 4 (1000 mg/kg) males compared to the control. Significantly lower mean food consumption was observed in Group 4 males from SD 85 to 92 and SD 92 to 99 and in Group 4 females from SD 106 to 113. For both Group 4 males and females, total mean food consumption was also significantly lower than that of the control during the recovery phase (SD 85 to 119) and throughout the study (SD 1 to 119). Lower food consumption did not correlate to body weight changes. The findings were not considered test-substance related as there was no correlated dose-response effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Treatment with Hatcol 1510 had no effect on ophthalmology; no ocular lesions were seen in any animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study Day 92
Test substance-related alterations in hematology parameters consisted of minimally increased mean red cell distribution width (RDW) in males given ≥300 mg/kg/day and females given 1000 mg/kg/day. The values were within the range for historical data controls and there were no correlating microscopic findings; therefore, these changes were considered non-adverse.
In one female (21986) given 1000 mg/kg, there were slight decreases in red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT), as well as increased RDW, percent reticulocyte (RET) and absolute reticulocyte count (ABRETi). Increased RET and ABRETi correlated microscopically with erythroid hyperplasia, which was observed in the bone marrow (sternum and femur). With the exception of increased RDW, the hematology changes in this female were considered unrelated to the test substance.
Study Day 120
There were no test substance-related alterations in hematology parameters at the recovery necropsy.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study Day 92
Test substance-related alterations in clinical chemistry parameters consisted of minimally decreased mean chloride (CL) in males given ≥100 mg/kg/day, and minimally increased mean potassium (K) in males given 1000 mg/kg/day. The values were within the range for historical data controls and were considered non-adverse.
Study Day 120
There were no test substance-related alterations in clinical chemistry parameters at the recovery necropsy.

Coagulation
Study Day 92
Test substance-related alterations in coagulation parameters consisted of minimally increased mean activated partial thromboplastin time (APTT) in males given 1000 mg/kg/day. Because the mean value was within the range for historical data controls and there were no associated clinical observations or correlation microscopic findings, this change was considered non-adverse.
Study Day 120
There were no test substance-related alterations in coagulation parameters at the recovery necropsy.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Study Day 92
There were no test substance-related alterations in urinalysis parameters at the terminal necropsy.
Study Day 120
There were no test substance-related alterations in urinalysis parameters at the recovery necropsy.
All remaining changes in mean clinical pathology test values at both timepoints, including those that were statistically significant in treated groups, were considered unrelated to Hatcol 1510 because they were negligible in magnitude, within normal biological variation, lacked a dose-response, were limited to a single sex and/or there were no correlating microscopic findings.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional Observational Battery and Grip Strength
Treatment with Hatcol 1510 had no effect on the functional observational battery. A score of zero was considered normal. Three animals had ptosis (squinting) of the left eye (Group 1 – one female; Group 2; 100 mg/kg – one male, one female), and each received a score of one for palpebral closure in handheld observations. All other animals were normal for observations. There were no differences in rearings, grooms, grip strength, or hindlimb splay noted.
Locomotor Activity
Treatment with Hatcol 1510 had no effect on locomotor activity. Mean number of basic movements, fine movements, and rearings decreased over the three ten-minute intervals, showing normal habituation in all groups. Additionally, females had lower overall motor activity compared to males, as expected.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The following organs were collected/weighed as follows: thyroid with parathyroids, testes with epididymides, and uterus with cervix. Organ weight data were presented as terminal body weights (TBW, g), absolute weights (Abs, g), organ-to-body weight ratios (/BW, %), and organ-to-brain weight ratios (/BR).
There were no test substance-related organ weight changes at either the terminal or recovery necropsies.
Any statistically significant changes in absolute and/or relative organ weights at the terminal and recovery necropsies were considered unrelated to Hatcol 1510 administration and due to individual variation, as there were no correlating microscopic findings and/or similar changes were not present in the opposite sex.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related macroscopic observations. All gross necropsy observations at the terminal and recovery necropsies were also considered incidental findings common in Sprague Dawley rats of this age.
In one female (21986) given 1000 mg/kg/day a firm, tan, red mass was observed in the mammary gland in the right inguinal region, correlating microscopically with mammary adenocarcinoma. This finding was considered incidental and unrelated to the test substance (see Microscopic Findings). In one female (21956) given 300 mg/kg/day, a semi-firm, tan mass was observed in the esophagus, correlating with an abscess. This finding was also considered unrelated to the test substance.
In one male (21977) given 1000 mg/kg/day, a raised brown area was observed in the medial lobe of the liver. Although microscopic examination of this tissue was not conducted (per protocol), this solitary observation in the liver of a high dose animal was considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal Necropsy (SD 92)
There were no test substance-related microscopic findings.
Minimal or mild glandular dilation was observed in the glandular stomach of control and Hatcol 1510-dosed groups, with the exception of control females, with a higher incidence or severity in high dose animals. Microscopic examination of stomachs in low and mid dose animals, however, revealed an absence of a clear dose response; therefore, this finding was considered a background lesion unrelated to Hatcol 1510.
Mammary adenocarcinoma, described grossly as a firm, tan, red mass, was observed in the mammary gland of one female (21986) given 1000 mg/kg/day. Neither hyperplastic nor neoplastic mammary lesions were observed in other animals at the terminal necropsy, and there were no mammary masses observed grossly in recovery animals. Adenocarcinoma is a common neoplasm in female rats (Giknis and Clifford, 2013; Son and Gopinath, 2004); therefore, the occurrence of one mammary tumor in the current study was considered incidental and unrelated to the test substance. Also, in female 21986, moderate erythroid hyperplasia was observed in the bone marrow of the sternum and femur, correlating with increased RET and ABRETi. This finding was also considered unrelated to the test substance.

Recovery Necropsy (SD 120)
There were no test substance-related microscopic findings in the stomach of recovery animals. Glandular dilation in the glandular stomach occurred at a similar incidence in control and high dose groups.
Remaining microscopic findings at the terminal and recovery necropsies were considered unrelated to Hatcol 1510 due to low incidence rates and/or because they are common findings in Sprague Dawley rats of this age.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Veterinarian Examinations/Treatments
One animal was evaluated/treated by the staff veterinarian. Animal 21928 (2m; 100 mg/kg) exhibited body weight loss, rough haircoat, and intermittent audible breathing when examined on SD 78. Treatment included subcutaneous fluids (0.9% sodium chloride) and diet gel recovery packs. Findings were considered incidental and not test substance-related.
Details on results:
Stability data demonstrates that the formulated test substance was stable under refrigerated storage (2 - 4°C) for a period of 20 days.

Analysis of the control samples confirmed absence of the test substance. Test substance dose formulations from Intervals 1 to 4 demonstrated an average recovery of 92.5% to 95.6% for concentration verification samples. Since all test substance dose formulations mean concentrations for concentration verification recovered within 70 to 120% of the target concentration, it can be determined that the appropriate dose formulation was achieved for each peanut oil sample.

Following 13 weeks of once daily oral gavage administration of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) at 0, 100, 300 or 1000 mg/kg, no effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, ophthalmic examination findings, functional observational battery, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, absolute and relative organ weights, or histopathology findings. There were no test article-related effects noted on any parameter examined.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no test article-related effects noted on any parameter examined.

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)

Any other information on results incl. tables

Summary of Animal Disposition and Physical Examinations

Animal Disposition

Day Numbers Relative to Start Date

Group:

1

2

3

4

Sex: Male

 

Terminal Kill

           Number of Animals

           Days from – to

 

10

92 – 92

 

10

92 – 92

 

10

92 – 92

 

10

92 – 92

Recovery Kill

           Number of Animals

           Days from – to

 

5

120 – 120

 

-

-

 

-

-

 

5

120 – 120

Sex: Female

 

Terminal Kill

           Number of Animals

           Days from – to

 

10

92 – 92

 

10

92 – 92

 

10

92 – 92

 

10

92 – 92

Recovery Kill

           Number of Animals

           Days from – to

 

5

120 – 120

 

-

-

 

-

-

 

5

120 - 120

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Animal Disposition and Physical Examinations

Physical Examinations

Day Numbers Relative to Start Date

Group:

1

2

3

4

Sex: Male

 

Alopecia

           Number of Observations

           Number of Animals

           Days from – to

 

3

1

106 – 120

 

-

-

-

 

-

-

-

 

-

-

-

 

Rough Haircoat

           Number of Observations

           Number of Animals

           Days form – to

 

-

-

-

 

1

1

78 – 78

 

-

-

-

 

-

-

-

 

Squinting

           Number of Observations

           Number of Animals

           Days form – to

 

-

-

-

 

4

1

71 – 92

 

-

-

-

 

-

-

-

Sex: Female

 

Abrasion

           Number of Observations

           Number of Animals

           Days from – to

 

2

1

85 – 92

 

-

-

-

 

-

-

-

 

-

-

-

Alopecia

           Number of Observations

           Number of Animals

           Days from – to

 

1

1

99 – 99

 

-

-

-

 

-

-

-

 

-

-

-

Small Movable Tissue Mass

           Number of Observations

           Number of Animals

           Days from – to

 

-

-

-

 

-

-

-

 

-

-

-

 

1

1

92 – 92

Squinting

           Number of Observations

           Number of Animals

           Days form – to

 

3

1

106 – 120

 

-

-

-

 

-

-

-

 

-

-

-

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Body Weights (g)

 

Day Numbers Relative to Start Date

Group

Sex

 

1

8

15

22

29

36

43

1

M

Mean

S.D.

N

289.35

7.42

15

316.31

11.41

15

337.31

14.03

15

352.63

17.69

15

366.35

21.04

15

377.87

23.43

15

393.43

24.36

15

2

M

Mean

S.D.

N

290.97

5.98

10

318.06

8.10

10

336.40

9.64

10

352.30

12.11

10

364.73

15.06

10

375.79

14.88

10

391.04

16.29

10

3

M

Mean

S.D.

N

289.64

5.15

10

317.44

6.07

10

337.86

9.80

10

354.92

12.97

10

367.91

13.38

10

380.97

15.54

10

397.25

14.78

10

4

M

Mean

S.D.

N

289.60

8.18

15

310.05

13.64

15

325.65*

14.76

15

341.97

16.08

15

354.53

17.99

15

366.57

18.59

15

380.36

19.42

15

1

F

Mean

S.D.

N

203.64

5.66

15

215.87

6.54

15

226.19

6.66

15

232.69

7.65

15

240.87

10.87

15

247.05

11.84

15

248.31

9.10

15

2

F

Mean

S.D.

N

203.67

5.17

10

214.43

4.80

10

225.60

9.21

10

231.09

8.78

10

235.71

9.23

10

247.56

9.13

10

225.05

13.47

10

3

F

Mean

S.D.

N

204.25

6.30

10

214.22

6.41

10

254.64

10.33

10

230.60

7.16

10

239.85

11.99

10

243.10

9.58

10

246.73

9.69

10

4

F

Mean

S.D.

N

204.83

6.04

15

212.47

9.14

15

224.33

12.05

15

225.56

10.35

15

234.80

14.04

15

246.16

11.88

15

246.05

11.84

15

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Body Weights (g)

 

Day Numbers Relative to Start Date

Group

Sex

 

50

57

64

71

78

85

91

92

1

M

Mean

S.D.

N

406.96

26.03

15

414.35

27.74

15

422.47

28.75

15

426.85

28.77

15

431.78

30.81

15

439.29

31.24

15

451.89

35.65

10

432.83

34.24

10

2

M

Mean

S.D.

N

401.85

17.53

10

411.18

17.73

10

418.73

18.88

10

425.83

16.94

10

424.37

24.31

10

433.99

21.19

10

440.35

22.55

10

423.52

20.54

10

3

M

Mean

S.D.

N

408.30

16.86

10

416.39

17.43

10

420.93

18.18

10

427.48

16.90

10

434.01

17.12

10

442.10

18.72

10

447.42

17.84

10

431.30

16.98

10

4

M

Mean

S.D.

N

392.74

20.09

15

397.34

21.32

15

407.13

22.95

15

412.92

22.99

15

419.16

23.97

15

424.12

23.13

15

438.58

18.56

10

419.15

17.80

10

1

F

Mean

S.D.

N

253.41

9.84

15

255.82

9.94

15

260.47

10.43

15

262.77

13.06

15

265.73

11.59

15

268.21

11.31

15

268.66

9.12

10

257.76

8.24

10

2

F

Mean

S.D.

N

252.13

8.76

10

255.23

8.79

10

257.59

10.06

10

258.06

11.09

10

263.50

10.83

10

265.35

11.73

10

266.20

10.83

10

255.78

9.81

10

3

F

Mean

S.D.

N

251.37

11.72

10

255.29

13.20

10

257.59

10.06

10

261.16

9.23

10

264.13

10.61

10

262.77

9.81

10

263.92

11.11

10

253.08

9.15

10

4

F

Mean

S.D.

N

248.19

13.77

15

249.75

11.125

15

251.47

13.60

15

255.30

13.57

15

259.84

12.99

15

260.57

14.24

15

261.50

13.90

10

249.71

13.83

10

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Body Weights (g)

 

Day Numbers Relative to Start Date

Group

Sex

 

92

99

106

113

119

120

1

M

Mean

S.D.

N

430.06

18.31

5

435.46

18.50

5

439.46

21.43

5

442.56

20.40

5

449.78

21.07

5

427.60

20.30

5

4

M

Mean

S.D.

N

411.04

28.15

5

425.28

26.95

5

430.44

26.61

5

439.14

28.04

5

447.78

31.01

5

427.12

28.79

5

1

F

Mean

S.D.

N

271.24

18.74

5

274.96

15.79

5

279.90

14.29

5

277.86

19.00

5

280.20

20.60

5

267.32

21.30

5

4

F

Mean

S.D.

N

261.36

12.22

5

273.64

7.05

5

275.30

13.15

5

275.70

13.17

5

276.10

10.46

5

264.74

12.47

5

Nominal Dose:    Group 1 – 0 mg/kg             Group 4 – 1000 mg/kg

 

Summary of Body Weight Changes (g)

 

Day Numbers Relative to Start Date

Group

Sex

From:

To:

1

8

8

15

15

22

22

29

29

36

36

43

43

50

1

M

Mean

S.D.

N

26.95

5.75

15

21.01

3.90

15

15.31

5.11

15

13.72

4.11

15

11.52

4.90

15

15.57

3.35

15

13.53

4.20

15

2

M

Mean

S.D.

N

27.09

4.45

10

18.34

4.21

10

15.90

4.58

10

12.43

4.64

10

11.06

3.80

10

15.25

4.63

10

10.81

3.31

10

3

M

Mean

S.D.

N

27.80

3.34

10

20.42

4.37

10

17.06

4.59

10

12.99

3.81

10

13.06

3.85

10

16.28

3.22

10

11.05

5.88

10

4

M

Mean

S.D.

N

20.45*

6.44

15

15.60*

4.40

15

16.31

3.79

15

12.57

3.36

15

12.04

2.30

15

13.79

3.16

15

12.38

3.86

15

1

F

Mean

S.D.

N

12.23

2.65

15

10.32

4.71

15

6.51

4.29

15

8.18

6.22

15

6.18

6.34

15

1.26

4.72

15

5.10

4.37

10

2

F

Mean

S.D.

N

10.76

3.48

10

11.17

6.94

10

5.49

6.59

10

4.62

4.66

10

11.85*

7.14

10

4.49

7.26

10

0.08

6.69

10

3

F

Mean

S.D.

N

9.97

7.35

10

10.42

5.58

10

5.96

9.49

10

9.25

7.61

10

3.25

6.17

10

3.63

4.19

10

4.64

4.14

10

4

F

Mean

S.D.

N

7.63*

5.66

15

11.87

7.34

15

1.23

6.67

15

9.24

7.29

15

11.6

8.29

15

-0.11

9.15

15

2.13

5.23

15

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Body Weight Changes (g)

 

Day Numbers Relative to Start Date

Group

Sex

From: To:

50

57

57

64

64

71

71

78

78

85

85

91

Absolute Change

Percent Change

91

92

1

91

1

91

1

M

Mean

S.D.

N

7.39

3.03

15

8.12

3.99

15

4.39

4.81

15

4.93

4.44

15

7.51

4.11

15

6.23

4.26

10

161.34

30.06

10

55.40

9.49

10

-19.06

2.88

10

2

M

Mean

S.D.

N

9.33

4.73

10

7.55

4.47

10

7.10

4.98

10

-1.46

17.04

10

9.62

6.19

10

6.36

4.01

10

149.38

19.88

10

51.32

6.55

10

-16.83

2.58

10

3

M

Mean

S.D.

N

8.09

3.73

10

4.54

5.09

10

6.55

3.82

10

6.53

4.11

10

8.09

3.91

10

5.32

4.23

10

157.78

15.62

10

54.46

5.28

10

-16.12

2.54

10

4

M

Mean

S.D.

N

4.60

3.85

15

9.79

3.78

15

5.79

3.06

15

6.24

3.57

15

4.96

3.26

15

7.52

3.44

10

147.65

15.38

10

50.75

5.13

10

-19.43

3.42

10

1

F

Mean

S.D.

N

2.41

6.32

15

4.65

5.09

15

2.29

4.00

15

2.97

5.17

15

2.47

4.09

15

-0.86

3.15

10

65.12

9.35

10

32.06

5.10

10

-10.90

1.35

10

2

F

Mean

S.D.

N

3.10

3.72

10

2.36

2.71

10

0.47

3.15

10

5.44

4.24

10

1.84

3.21

10

0.86

4.85

10

62.53

9.38

10

30.72

4.60

10

-10.42

2.33

10

3

F

Mean

S.D.

N

3.92

8.06

10

2.35

6.16

10

3.52

2.87

10

2.97

3.50

10

-1.36

4.73

10

1.15

3.96

10

59.67

11.98

10

29.30

6.19

10

-10.84

2.64

10

4

F

Mean

S.D.

N

1.56

5.88

15

1.73

6.33

15

3.83

5.28

15

4.54

3.76

15

0.73

4.93

15

1.49

2.60

10

56.18

11.39

10

27.37

5.49

10

-11.79

1.69

10

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Body Weight Changes (g)

 

Day Numbers Relative to Start Date

Group

Sex

From:

To:

85

92

92

99

99

106

106

113

113

119

Absolute Change

Percent Change

Absolute Change

Percent Change

119

120

85

119

85

119

1

119

1

119

1

M

Mean

S.D.

N

3.52

4.40

5

5.40

3.21

5

4.00

3.61

5

3.10

5.67

5

7.22

2.91

5

23.24

6.50

5

5.44

1.44

5

162.82

20.27

5

56.78

7.24

5

-22.19

1.99

5

4

M

Mean

S.D.

N

0.80

3.85

5

14.24*

1.54

5

5.16

3.36

5

8.70

3.23

5

8.64

3.72

5

37.54*

8.99

5

9.13

2.02

5

160.84

27.03

5

56.05

9.29

5

-20.66

3.51

5

1

F

Mean

S.D.

N

5.66

5.51

5

3.72

5.47

5

-4.06

3.14

5

6.96

8.09

5

2.34

6.57

5

14.62

6.76

5

5.46

2.40

5

76.36

17.91

5

37.43

8.41

5

-12.88

3.22

5

4

F

Mean

S.D.

N

-0.32

2.72

5

12.28*

5.62

5

1.66

6.31

5

0.40

1.25

5

0.40

3.81

5

14.42

3.90

5

5.56

1.66

5

72.24

9.81

5

35.46

4.89

5

-11.36

3.02

5

* - Significantly different from the control value, p ≤ 0.05

Nominal Dose:    Group 1 – 0 mg/kg             Group 4 – 1000 mg/kg

 

Summary of Functional Observational Battery Data

 

Day 90 Relative to Start Date

Activity Arousal:

Group

Sex

 

Handling Reactivity

Vocalisation

Posture

(Home Cage)

Posture

(Open Field)

Rearings

1

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

10.1

4.0

15

2

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

11.0

3.6

10

3

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

9.9

4.3

10

4

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

11.1

4.1

15

1

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

16.6

3.9

15

2

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

17.6

2.5

10

3

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

13.2

2.6

10

4

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

14.4

2.5

15

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Functional Observational Batter Data

 

Day 90 Relative to Start Date

Autonomic:

Group

Sex

 

Exopthalmus

Lacrimation

Palpebral Closure

Piloerection

Pupillary Status

(Hand Held)

Salivation

Pupillary Response

(Elicited)

Number of Fecal Boli

Number of Urine Pools

1

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.1

0.3

15

2

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.1

0.3

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.1

0.3

10

3

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

1

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.1

0.3

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.1

0.3

15

2

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.1

0.3

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.1

0.3

10

3

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.2

0.8

15

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Functional Observational Battery Data

 

Day 90 Relative to Start Date

Neuromuscular:

Group

Sex

 

Activity

(Home Cage)

Activity

(Open Field)

Fur Appearance

Unusual Behaviour

(Home Cage)

Unusual Behaviour

(Open Field)

1

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

2

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

3

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

1

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

2

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

3

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Functional Observational Battery Data

 

Day 90 Relative to Start Date

Neuromuscular:

Group

Sex

 

Gait

(Home Cage)

Gait

(Open Field)

Righting Response

Grooms

Forelimb Grip (kg)

Hindlimb Grip (kg)

Hindlimb Splay (mm)

1

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.3

0.9

15

1.469

0.176

15

0.649

0.148

15

73.0

9.2

15

2

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.2

0.6

10

1.508

0.168

10

0.588

0.120

10

77.8

14.7

10

3

M

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.4

1.0

10

1.435

0.123

10

0.593

0.142

10

69.3

6.5

10

4

M

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.3

0.6

15

1.469

0.149

15

0.598

0.094

15

76.5

13.4

15

1

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.2

0.4

15

1.422

0.116

15

0.562

0.092

15

50.7

10.2

15

2

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.4

0.7

10

1.345

0.078

10

0.494

0.070

10

59.5

11.9

10

3

F

Mean

S.D.

N

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.2

0.4

10

1.324

0.080

10

0.516

0.078

10

57.0

10.1

10

4

F

Mean

S.D.

N

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.1

0.4

15

1.309

0.080

15

0.511

0.072

15

53.0

8.0

15

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Functional Observational Battery Data

 

Day 90 Relative to Start Date

Physiological:

Group

Sex

 

Respiration

SD 85

Body Weight (g)

Sensory:

Approach Response

Auditory Response

Tail Pinch

Pinna Response

1

M

Mean

S.D.

N

0.0

0.0

15

439.29

31.24

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

2

M

Mean

S.D.

N

0.0

0.0

10

433.99

21.19

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

3

M

Mean

S.D.

N

0.0

0.0

10

442.10

18.72

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

M

Mean

S.D.

N

0.0

0.0

15

424.12

23.13

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

1

F

Mean

S.D.

N

0.0

0.0

15

268.21

11.31

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

2

F

Mean

S.D.

N

0.0

0.0

10

265.34

11.73

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

3

F

Mean

S.D.

N

0.0

0.0

10

262.77

9.81

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

0.0

0.0

10

4

F

Mean

S.D.

N

0.0

0.0

15

260.57

14.24

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

0.0

0.0

15

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

 

Summary of Locomotor Activity Data

 

Day 85 Relative to Start Date

Group

Sex

Interval

Basic Movements

Fine Movements

Rearing

Mean

SE

N

Mean

SE

N

Mean

SE

N

1

M

1

2

3

1961.47

958.87

725.60

91.06

77.91

72.17

15

15

15

858.60

543.33

422.60

48.49

46.43

37.48

15

15

15

5.48

5.22

6.27

132.47

56.20

36.67

15

15

15

2

M

1

2

3

2044.00

968.20

739.30

108.33

105.15

95.14

10

10

10

996.20

571.40

457.90

55.49

54.70

55.20

10

10

10

5.57

5.83

10.62

118.30

44.80

31.50

10

10

10

3

M

1

2

3

1913.50

1057.30

827.20

116.70

108.04

68.64

10

10

10

861.90

580.80

485.30

54.05

68.05

37.98

10

10

10

7.64

5.05

5.42

118.850

53.80

38.40

10

10

10

4

M

1

2

3

2091.80

1161.53

886.07

89.35

69.02

87.25

15

15

15

969.27

686.00

521.33

40.50

44.30

50.05

15

15

15

6.61

11.75

7.01

114.13

62.48

41.07

15

15

15

1

F

1

2

3

1849.60

1145.90

730.40

55.92

82.68

90.06

15

15

15

591.40

499.07

404.67

25.36

25.01

29.70

15

15

15

4.56

5.24

3.77

99.80

55.40

30.27

15

15

15

2

F

1

2

3

1970.70

1108.90

730.40

55.93

82.68

90.06

10

10

10

54.90

480.30

342.10

28.38

29.91

39.09

10

10

10

6.36

6.64

5.18

101.80

48.40

30.00

10

10

10

3

F

1

2

3

1857.00

1173.40

728.50

61.30

91.34

102.55

10

10

10

573.00

594.30

359.10

25.42

37.07

39.75

10

10

10

4.49

5.98

5.17

113.70

55.60

26.60

10

10

10

4

F

1

2

3

1881.73

985.20

706.87

48.47

47.95

88.27

15

15

15

353.60

449.67

349.13

27.25

22.97

39.23

15

15

15

6.13

4.87

3.87

90.73

41.00

21.47

15

15

15

SE = Standard Error

Nominal Dose:    Group 1 – 0 mg/kg             Group 2 – 100 mg/kg         Group 3 – 300 mg/kg         Group 4 – 1000 mg/kg

Applicant's summary and conclusion

Conclusions:
Following 13 weeks of once daily oral gavage administration of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) at 0, 100, 300 or 1000 mg/kg, no effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, ophthalmic examination findings, functional observational battery, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, absolute and relative organ weights, or histopathology findings. There were no test article-related effects noted on any parameter examined.
Based on these results, the no observed adverse effect level (NOAEL) of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) in Sprague Dawley rats following 90-day oral gavage dose is 1000 mg/kg/day, the highest dose level tested.
Executive summary:

Decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510): A 90-day Repeat Dose Toxicity Study following Oral Gavage Administration to Male and Female Sprague Dawley Rats With a 28-Day Recovery Period.

 

The purpose of this study was to determine the potential toxicity of decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane (Hatcol 1510) when administered daily for 90 days via oral gavage to male and female Sprague Dawley rats and to determine the persistence or reversibility of any toxic effects over a 28 day recovery period.

 

One hundred (50/sex) Sprague Dawley rats were randomly assigned to four groups (15 animals/sex in the control and high-dose groups; 10 animals/sex in the low and mid-dose groups). Animals were administered control substance (peanut oil) or Hatcol 1510 at 100, 300, or 1000 mg/kg once daily via oral gavage for at least 90 consecutive days. Animals were subjected to a full gross necropsy on Study Day (SD) 92 (terminal) or 120 (recovery).

 

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, ophthalmic examination findings, functional observational battery, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, absolute and relative organ weights, and histopathology findings.

 

Treatment with Hatcol 1510 at doses up to 1000 mg/kg had no effect on mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, ophthalmic examination findings, functional observational battery, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology findings, absolute and relative organ weights, or histopathology findings.

 

All animals survived to the scheduled necropsies. On SD 92 (terminal necropsy), test substance-related alterations in clinical pathology parameters were minor and consisted of decreased mean chloride (CL) levels in males given ≥100 mg/kg/day, increased mean potassium (K) in males given 1000 mg/kg/day, increased mean red cell distribution width (RDW) in males given ≥300 mg/kg/day and females given 1000 mg/kg/day, and minimally increased mean activated partial thrompboplastin time (APTT) in males given 1000 mg/kg/day. All values were within the range for historical data controls, had recovered by the recovery necropsy, and were considered non-adverse. There were no test substance-related organ weight changes, macroscopic observations or microscopic findings at either necropsy (SD 92 or SD 120).

 

Based on these results, the no observed adverse effect level (NOAEL) of Hatcol 1510 in Sprague Dawley rats following 90-day oral gavage dose is 1000 mg/kg/day, the highest dose level tested.