Fatty acids, C8-18 and C18-unsatd., zinc salts

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc dioctanoate

EC Number:

209-156-6

EC Name:

Zinc dioctanoate

Cas Number:

557-09-5

Molecular formula:

C16H30O4Zn

IUPAC Name:

zinc(II) octanoate

Constituent 2

Reference substance name:

Zinc decanoate

EC Number:

235-909-3

EC Name:

Zinc decanoate

Molecular formula:

C10H20O2.1/2Zn

IUPAC Name:

zinc(II) decanoate

Constituent 3

Reference substance name:

Zinc dilaurate

EC Number:

219-518-5

EC Name:

Zinc dilaurate

Cas Number:

2452-01-9

Molecular formula:

C12H24O2.1/2Zn

IUPAC Name:

zinc(II) dodecanoate

Constituent 4

Reference substance name:

Zinc dimyristate

EC Number:

240-369-7

EC Name:

Zinc dimyristate

Cas Number:

16260-27-8

Molecular formula:

C28H54O4Zn

IUPAC Name:

zinc(II) tetradecanoate

Constituent 5

Reference substance name:

Zinc dipalmitate

EC Number:

225-652-5

EC Name:

Zinc dipalmitate

Cas Number:

4991-47-3

Molecular formula:

C16H32O2.1/2Zn

IUPAC Name:

zinc(II) hexadecanoate

Constituent 6

Reference substance name:

Zinc distearate

EC Number:

209-151-9

EC Name:

Zinc distearate

Cas Number:

557-05-1

Molecular formula:

C18H36O2.1/2Zn

IUPAC Name:

zinc(II) octadecanoate

Constituent 7

Reference substance name:

Zinc dioleate

EC Number:

209-154-5

EC Name:

Zinc dioleate

Cas Number:

557-07-3

Molecular formula:

C18H34O2.1/2Zn

IUPAC Name:

zinc(II) 9-octadecenoate

Constituent 8

Reference substance name:

Zinc dilinoleate

EC Number:

235-870-2

EC Name:

Zinc dilinoleate

Cas Number:

13014-44-3

Molecular formula:

C36H62O4Zn

IUPAC Name:

zinc(II) 9,12-octadecdienoate

Test material form:

solid: bulk

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague-Dawley rats are widely used in toxicity studies and a large amount of historical data have been accumulated, facilitating the interpretation and evaluation of the test results.

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.4. Test System
2.4.1. Animal information
Species and strain : NSam: Sprague-Dawley Rat
Microbiological grade : Specific Pathogen Free(SPF)
Breeder : Samtako Bio Korea (105, Seorang-ro, Osan-si, Gyeonggi-do, Republic of Korea)
Supplier : Young Bio Co., Ltd. (388, Dunchon-daero, Jungwon-gu, Seongnam-si,
Gyeonggi-do, Republic of Korea)


Sex Male Female
Number At receipt 33 33
At first dose 30 30
Age(week) At receipt 5 5
At first dose 6 6
Body weight ranges at first dose 176.6 ~ 200.8 g 136.3 ~ 160.3 g


2.4.2. Reason for test system selection
The Sprague-Dawley rats are widely used in toxicity studies and a large amount of historical data have been accumulated, facilitating the interpretation and evaluation of the test results.
2.4.3. Quarantine and acclimation
Upon animal acquisition, all animals was quarantined at the quarantine room 1-1 for 3 days and acclimated at the animal room 25 for 4 days under the environment of the 1st animal test area of CentralBio Co., Ltd. During this period, all animals was observed daily, and only selected healthy animals will be used in the study.
2.4.4. Identification
Animals were individually identified on the tail by permanent markers, red in the acclimation period, and blue in the test period. Identification cards including information such as colors by dose groups, study number and animal number were attached to each cage. A log sheet was attached to the entrance of the animal room to identify the study.
2.4.5. Group assignment
Following the quarantine-acclimation period, selected healthy animals were randomly grouped based on body weights. The evenness for the average and standard deviation of body weights per group were checked during the group assignment.
2.4.6. Remnant animals
The remnant animal was humanely killed using CO2 gas.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

DMSO

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 other: mg/kg/day

Doses / concentrations 2

Dose / conc.:

250 other: mg/kg/day

Doses / concentrations 3

Dose / conc.:

500 other: mg/kg/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

3.4. Observations
3.4.1. Clinical signs
Clinical signs were observed at least once daily, and observation of moribund or dead animals were
conducted twice daily. The first day of dosing was designated as Day 1.
3.4.2. Moribund or dead animal procedure
Moribund or dead animal was not observed.
3.4.3. Detailed clinical signs
At least once prior to the first exposure, and once a week thereafter, detailed clinical observations
were made in all animals. These observations were made outside the home cage, preferably in a
standard arena.
Changes of skin, fur, eyes and mucous membranes
Occurrence of secretions and excretions
Autonomic activity; lacrimation, piloerection, pupil size and unusual respiratory pattern
Change in gait, posture, response to handling
Presence of clonic or tonic movements
Stereotypes; excessive grooming and repetitive circling
Bizarre behavior; self-mutilation and walking backwards
3.4.4. Sensory reactivity
All animals were examined for following items in 4th week of dosing and 2nd week of recovery.
Visual
Auditory
Touch response
Pain response
Righting reflex

3.4.5. Functional observations
Grip strength and locomotor activity were measured in 4th week of dosing(main groups) and 2nd
week of recovery(recovery groups). Functional observations were omitted for groups that otherwise
reveal signs of toxicity to an extent that would significantly interfere with the functional test
performance.
3.4.5.1. Grip strength
The front leg was measured three times using a hand-held dynamometry(BIO-GS3, Panlab,
S.L.U., Spain) and averaged.
3.4.5.2. Motor activity
The distance moved was measured individually for each animal using open field test
system(SuperFlex Open Field, Omnitech Electronics, Inc.). Measurements are made for five
minutes and the total trave
3.4.6. Body weights
Animals were weighed on the day of animal receipt, on day of group assignment, on Day 1 (before
administration), subsequently once weekly, and on the day of necropsy. After fasting overnight,
body weight was measured at necropsy.
3.4.7. Food and water consumptions
Food and water consumption were measured on Day 1 and subsequently once weekly during the
observation period. The remaining quantity of food and water on each following day was subtracted
from the weight of food and water supplied to each cage to calculate mean daily consumption.
3.4.8. Ophthalmological examination
In 4th week of dosing(main groups) and 2nd week of recovery(recovery groups), Animals were
macroscopically observed for external appearance of the eyes. Mydriatic drops(Mydriacyl, Lot.
21A25AD) were used in both eyes to facilitate mydriasis. Then anterior parts of the eye, optic media
and fundus were observed with a fundus camera. No representative photographs were taken since
there were no abnormal findings.

Sacrifice and pathology:

3.5. Clinical pathology
3.5.1. Urinalysis
In 4th week of dosing and 2nd week of recovery, urine was collected for 16-hours using urine plate, and chemistry examination and general examination were conduct. Urine was collected for 24- hours to obtain the total urine volume for each animal.


Chemistry examination
pH -
Specific gravity(SG) -
Protein(PRO) mg/dL
Glucose(GLU) mg/dL
Ketone body(KET) mg/dL
Bilirubin(BIL) mg/dL
Urobilinogen(URO) E.U./dL
Nitrite(NIT) -
Blood(BLO) -
Leukocyte(LEU) -

Multistix 10 SG (Siemens, U.S.A.)
Urine analyzer (Clinitek status®, Siemens, Germany)


General examination
Volume mL Naked eye
Urinary sediments - Microscope
Color - Naked eye
Clarity - Naked eye


3.5.2. Blood sampling
Blood samples were collected from the posterior vena cava of all animals for scheduled necropsy under deep isoflurane anesthesia. Animals were fasted overnight (more than 16 hours) prior to sample collection.

3.5.3. Hematological test
Some blood samples were placed into CBC bottles containing EDTA as anticoagulant, and CBC(complete blood count), WBC 5 differential count and reticulocyte were analyzed using a Coulter counter(ADVIA 2120i, Siemens, Germany). Put the remained blood in a bottle containing 3.2 % sodium citrate and centrifuge(3000 rpm, 10 minutes) to separate plasma. Separate plasma was tested using a blood coagulation analyzer (CA660, Sysmex, USA).
The parameters are as follows:


Items Units
White blood cell count(WBC) 103 cells/μL
Differential leucocyte count 103 cells/μL, %
- Neutrophils(NEU)
- Lymphocytes(LYM)
- Monocytes(MONO)
- Eosinophils(EOS)
- Basophils(BASO)
Red blood cell count(RBC) 106 cells/μL
Hemoglobin(HGB) g/dL
Hematocrit(HCT) %
RBC indices
- Mean corpuscular volume(MCV) fL
- Mean corpuscular hemoglobin(MCH) pg
- Mean corpuscular hemoglobin concentration(MCHC) g/dL
Reticulocyte(RETI) 109 cells/L, %
Platelet(PLT) 103 cells/μL
Prothrombin time(PT) sec
Activated partial thromboplastin time(APTT) sec

3.5.4. Clinical biochemistry test
The remaining blood samples was placed into serum separating tube containing coagulation accelerator and a serum separation gel. The samples was coagulated at room temperature, and centrifuged to prepare the serum samples. The separated serum was analyzed using a biochemistry and electrolyte analyzer(Hitachi3500, HITACHI, Japan). The parameters are as follows:

Items Units
Total protein(TP) g/dL
Albumin(ALB) g/dL
Albumin/Globulin(A/G) ratio
Total bilirubin(T-BIL) mg/dL
Alkaline phosphatase(ALP) IU/L
Aspartate aminotransferase(AST) IU/L
Alanine aminotransferase(ALT) IU/L
Creatinine(CRE) mg/dL
Blood urea nitrogen(BUN) mg/dL
Total cholesterol(T-CHO) mg/dL
Triglycerides(TG) mg/dL
Glucose(GLU) mg/dL
Calcium(CA) mg/dL
Inorganic phosphorus(IP) mg/dL
Creatine phosphokinase(CK) IU/L
Sodium(Na+) mmol/L
Potassium(K+) mmol/L
Chloride(Cl-) mmol/L

3.5.5. Hormone concentration
Hormone concentration measurements were not performed because no adverse effects were observed in organ weight and histopathological examination results for the pituitary gland and thyroid gland.

Statistics:

The data of body weight, food and water consumption, hematological test, clinical biochemistry test, and organ weights were analyzed using SPSS statistical program (IBM, Ver 25.) to compare the homogeneity of variance. The One-way ANOVA(in assumption of normality, p>0.05) was performed followed by Leven’s test for equality of variances.
In accordance with the result of Levene’s test, the Dunnett test(equal variance, p>0.05) or Dunnett T3(unequal variance, p≤0.05) was conducted as a post-hoc test to confirm the significance. In posthoc test, p value< 0.05 was considered as statistically significant.
The results of general symptom observation, detailed observations, functional observations battery (sensory responses), urination tests(urine chemistry and urination tests), and histopathological tests provide observed cases without statistical analysis. All observation and measurement results were presented in appendix, and the table presents the mean and standard deviation or frequency of occurrence.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
Food consummation in male at 250 and 500 mg/kg/day on Day 1 was significantly decreased(p<0.05). The change was not considered to be test substance-related because there was no dose-response relationship and there was temporary observed.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, MCHC and PT in female at 500 mg/kg/day were significantly decreased(p<0.05).
PT in female at 125 mg/kg/day was significantly decreased(p<0.05). These changes were not considered to be test substance-related because there was no dose-response relationship.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
TP in male at 500 mg/kg/day was significantly decreased(p<0.05).
ALT in male at 125, 250 and 500 mg/kg/day was significantly decreased(p<0.05).
Else, CK in female at 250 mg/kg/day was significantly decreased(p<0.05). AST in female at 250 and 500 mg/kg/day was significantly decreased(p<0.05). These changes were not considered to be test substance-related because there was no dose-response relationship.
[Recovery group]
There were no test substance-related changes.
Else, A/G and ALB in female at 500 mg/kg/day were significantly decreased(p<0.05). T-CHO in male at 500 mg/kg/day was significantly increased(p<0.05). The changes were not considered to be test substance-related because there was not observed in the main groups.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

[Main group]
Distance moved in male at 125, 250 and 500 mg/kg/day was significantly increased(p<0.05).
[Recovery group]
There were no test substance-related changes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, relative weight of kidney in male at 500 mg/kg/day was significantly increased(p<0.05).
Related weight of thymus in male at 125 and 250 mg/kg/day was significantly increased(p<0.05).
These changes were not considered to be test substance-related because there was no dose-response relationship.
[Recovery group]
There were no test substance-related changes.
Else, relative weight of heart and pituitary gland in male at 500 mg/kg/day were significantly decreased(p<0.05). Absolute and relative weight of spleen in female at 500 mg/kg/day were significantly increased(p<0.05). These changes were not considered to be test substance-related because there was not observed in the main group.

Gross pathological findings:

not examined

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
There were no test substance-related changes.
Else, Right testis small and right epididymidis small were observed in one male at 500 mg/kg/day.
The change was not considered to be test substance-related because there was observed in only one animal.
[Recovery group]
There were no test substance-related changes.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

[Main group]
Test substance related-change was not observed.
Else, Changes observed in the kidney, pancreas, lung, prostate gland, testis, epididymis and thyroid in the male 500 mg/kg/day administration group were considered to be background changes. In addition, changes observed in the lung and harderian gland in the female 500 mg/kg/day administration group were considered to be background changes it was considered to be not test substance-related change.
[Recovery group]
As a result of histopathological examination for the main group, no findings observed as a test substance-related change were observed, so histopathological examination was not performed on the recovery group.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

histopathology: neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The purpose of this study was to confirm toxicity of Fatty acids, C8-18 and C18-unsatd., zinc salts in Sprague-Dawley rats after a 28-Day repeated-dose oral administration followed by a 14-Day recovery period and determine the no-observed-adverse-effect-level (NOAEL) identify the target organ.
The test substance was administered in a 28-Day repeated-dose oral administration at a dose level of 125, 250, and 500 mg/kg/day to 5 males and 5 females per group and compared with those of the vehicle control group administered with DMSO.
Also, each 5 animals/sex were assigned to the vehicle control and high dose test-substance group in order to evaluate recovery potential during 2 weeks of recovery period.
There were no test substance-related effects in clinical signs, detailed clinical signs, sensory reactivity, body weights, food consumptions, water consumptions, ophthalmological examination, urinalysis and urinary sediments, hematological, organ weights, necropsy findings and histopathological examination. In functional observations, increased distance moved in male at 125, 250 and 500 mg/kg/day was considered to be test sustance-related because there was a dose-response relationship. However, The change was not considered to be adverse effect because it was recovered during the two-week recovery period.
In clinical chemistry test, decreased TP in male at 500 mg/kg/day, decreased ALT in male at 125, 250 and 500 mg/kg/day were considered to be test sustance-related because there was a dose-response relationship. However, these changes were not considered to be adverse effect because it was recovered during the two-week recovery period.
Based on the above results, Fatty acids, C8-18 and C18-unsatd., zinc salts administered orally to Sprague-Dawley rats at 125, 250 and 500 mg/kg/day repeatedly for 4 weeks and a recovery period of 2 weeks, there was no adverse effect. Therefore, NOAEL of the Fatty acids, C8-18 and C18-unsatd., zinc salts was determined to be 500 mg/kg/day for both male and female, and no target organ was observed.