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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

2.4.2. Reason for test system selection
The Sprague-Dawley rats are widely used in reproductive 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 receipt, all animals were kept in quarantine and acclimatized for 21 days under the environment of the animal room of CentralBio Co., Ltd. During this period, all animals were observed daily and only selected healthy animals were used in the study.
2.4.4. Animal and cage identification
Animals were individually identified on the tail by permanent markers, red in the acclimation period, and blue in the test period. Offspring were numbered using blue markers (males) and red markers (females). 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. In the case of females, animals with irregular oestrus cycles for 14 days was excluded. The evenness for the average and standard deviation of body weights per group will be checked during the group assignment.
2.4.6. Remnant animals
The remnant animals were humanely killed using CO2 gas.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 44 50
Number at first dose 40 40
Age(week) at receipt 7 7
Age(week) at first dose 10 10
Body weight range at dose 327.6~397.6 g 204.3~243.1 g

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on exposure:

3.2.3. Dose volume
The dose volume is set as 5 mL/kg, and the dose volumes for each animal was calculated based on the most recently measured body weight.
3.2.4. Method
The test substance was orally administered once by gavage using a feeding needle(sonde).
3.3. Euthanasia
Animals that were in moribund conditions or judged to have severe pain were not observed.

Details on mating procedure:

3.4.6.1. Paring method
1:1 pairing was use. The paring period will be up to 14 days and animals confirmed to evidence of mating were separated immediately. Additional mating were conducted by remating of females with proven males of the same group in case pairing is unsuccessful during the 7 day mating period.
3.4.6.2. Confirmation of mating
Each morning the females were examined for the presence of sperm or a vaginal plug. Gestation day(GD) 0 will be defined as the observation day of mating evidence.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The duration of administration of the test substance was 35 days for males, and 41~54 days for females, including before mating, gestation period, and lactation period

Frequency of treatment:

The test substance was administered once a day. For females, if there was specific events such as parturition, the administration time was adjusted according to the decision of the study director.

Details on study schedule:

Clinical sign observation and body weight measurement for pups were performed up to 13 days after birth, and AGD was measured on the 4th day after birth.

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 per male, 10 per female

Control animals:

yes, concurrent vehicle

Details on study design:

3.1.1. Study group
Group Dose (mg/kg/day) Dose volume (mL/kg/day) Sex Number of animals Animal number
G1 Vehicle 0 5 Male 10 1-10
Female 10 41-50
G2 Low dose 100 5 Male 10 11-20
Female 10 51-60
G3 Middle dose 300 5 Male 10 21-30
Female 10 61-70
G4 High dose 1,000 5 Male 10 31-40
Female 10 71-80

3.1.2. Selection of dose
As a result of a 14-Day Repeated-dose Oral Toxicity DRF study(Study No.: 21-0599DF, CentralBio Co., Ltd.) at doses of 250, 500 and 1,000 mg/kg/day, salivation was observed in both sexes at 1,000 mg/kg/day in clinical sign. In the hematological test, HGB was decreased in male at ≥500 mg/kg/day. In the clinical biochemistry test, TP was decreased in both sexes at 1,000 mg/kg/day, AST and CK were increased in both sexes at 1,000 mg/kg/day, and Na+ was decreased in female at at ≥250 mg/kg/day. Based on the above results, the high dose for this study was set at 1,000 mg/kg/day, and the middle and low doses were set at 300 and 100 mg/kg/day by applying about three-fold intervals.

Oestrous cyclicity (parental animals):

Monitoring the regularity and duration of the oestrous cycle by vaginal smear during the pretreat and pre-mating period. In addition, a vaginal smear was performed prior to necropsy to determine the oestrous cycle phase.

Sperm parameters (parental animals):

Each morning the females were examined for the presence of sperm or a vaginal plug. Gestation day(GD) 0 will be defined as the observation day of mating evidence.

Postmortem examinations (parental animals):

Necropsy was performed on the 36th day after administration for males and on the 13th day after parturition for females. On the day of necropsy, all surviving animals were anesthetized with 2-4% isoflurane, and when anesthesia is confirmed, blood was collected from the abdominal aorta. Females with no evidence of copulation was necropsied 24 to 26 days after the last mating day. Females with delayed parturition was necropsied on 24 to 26 days after the observation day of mating evidence.

Postmortem examinations (offspring):

Anesthetize with 3-5% isoflurane, and when anesthesia was confirmed, blood was collected from the heart. Blood was drawn from two or more pups on PND 4 and pooled regardless of sex. At PND 13, blood was drawn from least two pups per sex.

Statistics:

The data of body weight, food and water consumption, hematological test, and 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 Levene’s test for equality of variances. In accordance with the result of Levene’s test, Dunnett test(equal variance) or Dunnett T3(unequal variance) were conducted as a post-hoc test to confirm the significance. In post-hoc test, p<0.05 is considered as statistically significant. The oestrous cycle during the acclimation period was presented only in appendix without statistical analysis. For parameters with only one result per group, such as an index, only dose-dependent responses are determined without statistical analysis.

Reproductive indices:

Reproductive performance[SOP-RPT-004, 005]
Calculated the index below.
Male mating index (%) = (No. of males mated/No. of males paired)×100
Female mating index (%) = (No. of females mated/No. of females paired)×100
Male fertility index (%) = (No. of males siring a litter/No. of males paired)×100
Female fertility index (%) = (No. of females pregnant/No. of females paired)×100
Male fecundity index (%) = (No. of males siring a litter/No. of males mated)×100
Pregnancy index (%) = (No. of females pregnant/No. females mated)×100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in all males at 1,000 mg/kg/day .
Salivation was observed in all females at 1,000 mg/kg/day on the pre-mating, gestation period
and lactation period.
In pre-mating and gestation period, soiled perineal region was observed in all females at 1,000
mg/kg/day.
Else, in pre-mating, abdominal distention was observed in 3 female at 0 mg/kg/day and observed
in 1 female at 100 mg/kg/day. The change was not considered to be test substance-related
because it was observed as a judgment error when confirmation of a vaginal plug.

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):

There were no test substance-related changes.
Food consumptions in male at 300 mg/kg/day on Day 14 was significantly increased(p<0.05).
Food consumptions in male at 1,000 mg/kg/day on Day 7 was significantly increased(p<0.05).
The change was not considered to be test substance-related because there was temporary
observed.

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

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes.
Water consumptions in male at all test substance on Day 28 were significantly increased(p<0.05)
and in male at 1,000 mg/kg/day on Day 34 was significantly increased(p<0.05).
The changes were not considered to be test substance-related because there was observed due to
low levels at vehicle control group or no dose-response relationship.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes.
Else, degeneration of testis and infiltrate of epididymis were observed at 0 mg/kg/day in males.
Degeneration of testis and cell debris of epididymis were observed at 1,000 mg/kg/day in males.
The changes were not considered to be test substance related-change because there was observed
in vehicle control group or to be background changes.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes.

In clinical signs and external examination, small was observed in each 1 and 2 males at 0 and
100 mg/kg/day. Small was observed in each 3, 4, 1 and 2 females at 0, 100, 300 and 1,000
mg/kg/day. Acaudate was observed in 1 female at 300 mg/kg/day. The changes were not
considered to be test substance-related because there was no dose-response relationship.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

In mortality, 4 dead pups(2 males and 2 females) was observed in vehicle control group, 3 dead
pups(2 males and 1 female) were observed at 100 mg/kg/day, and 1 dead pup(1 male) were
observed at 300 mg/kg/day, and 2 dead pups(2 males) observed at 1,000 mg/kg/day.

Body weight and weight changes:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Key result

Reproductive effects observed:

no

Conclusions:

The purpose of this study was to confirm the initial information of reproduction/developmental toxicity of the Fatty acids, C8-18 and C18-unsatd., zinc salts, following a repeated oral administration to Sprague-Dawley rats.
The test substance, Fatty acids, C8-18 and C18-unsatd., zinc salts was administered at a dose level of 100, 300, and 1,000 mg/kg/day to 10 males and 10 females per group and compared to the vehicle control group administered with DMSO. The duration of administration of the test substance was 35 days for males, and 41~54 days for females, including before mating, gestation period, and lactation period. Clinical sign observation and body weight measurement for pups were performed up to 13 days after birth, and AGD was measured on the 4th day after birth.

[Parents]
There were no test substance-related effects in clinical signs, body weights, food consumptions, water consumptions, estrus cycle, reproductive performance organ weights necropsy findings, hormone concentration and histopathological examination.

[Offspring]
There were no test substance-related effects in clinical signs, external examination, body weights, Pups index, anogenital index, number of nipples and areolas, necropsy findings, hormone concentration and histopathological examination.
Based on the above results, Fatty acids, C8-18 and C18-unsatd., zinc salts administered orally to Sprague-Dawley rats at 100, 300 and 1,000 mg/kg/day, the NOAEL of the Fatty acids, C8-18 and C18- unsatd., zinc salts was determined to be 1,000 mg/kg/day for males, pregnant females and offspring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat