1,2,3-tris(2,3-epoxypropoxy)propane

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)

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague-Dawley rats was 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 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 326.6~396.2 g 208.7~270.0 g

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on exposure:

35 days for males, and 41~67 days for females, including before mating, gestation period, and lactation period

Details on mating procedure:

3.4.6.1. Paring method
1:1 pairing was use. The paring period was 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:

yes

Details on analytical verification of doses or concentrations:

2.3.2. Analysis of dosing formulation
The analysis of the content and homogeneity of the test substance preparation were conducted at
CentralBio Co., Ltd. during the first and final preparation. The analysis results were checked
according to the accuracy and precision acceptance criteria.

Nominal Concentration (mg/L) First preparation
Calculated concentration (mg/L) CV(%) (Homogeneity Precision) Concentration (%) (Accuracy)

44,000 47,695±1,500 3.1 108.4
87,500 85,108±599 0.7 97.3
175,000 158,035±4,001 2.5 90.3


Nominal Concentration (mg/L) Final preparation
Calculated concentration (mg/L) CV(%) (Homogeneity Precision) Concentration (%) (Accuracy)

44,000 42,137±1,047 2.5 95.8
87,500 89,144±1,612 1.8 101.9
175,000 188,933±4,590 2.4 108.0

Frequency of treatment:

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:

Quarantine and acclimation
Upon animal acquisition, all animals were quarantined in the quarantine room 1-1 for 3 days and acclimated in the animal room 25 for 18 days under the environment of the 1st animal test area of CentralBio Co., Ltd(14, Saebeol-ro, Bupyeong-gu, Incheon, Republic of Korea). During this period, all animals were observed daily and only selected healthy animals were used in the study.

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.


Treatment
The duration of administration of the test substance was 35 days for males, and 41~67 days for females, including before mating, gestation period, and lactation period.

Dose / conc.:

88 mg/kg bw/day

Dose / conc.:

175 mg/kg bw/day

Dose / conc.:

350 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

3.4.1. Clinical signs
Clinical signs were observed at least once daily and mortalities were observed at twice daily. The first day of dosing were designated as Day 1 and observation of clinical signs were made until the day of necropsy.

3.4.2. Moribund or dead animal procedure
Moribund and dead animals were not observed.

3.4.3. Body weights
Body weights were measured on the day of animal receipt, on the day of group assignment and on the day of administration(before administration), and thereafter, at least once a week including the following time.
Pregnant females : Gestation day 0, 7, 14 and 20
Lactation day 0 or 1(post-partum within 24 hours), 4, 8, 13
Male : At least weekly and at necropsy
Pups : Post-partum 0 or 1 day((post-partum within 24 hours), 4, 8, 13

3.4.4. Food and water consumptions
Food and water consumption were measured on Day 1 and subsequently once weekly during the test period other than the mating period, including the following period. The remaining quantity of food and water on each following day were subtracted from the weight of food and water supplied to each cage to calculate mean daily consumption(g/cage/day).
Pregnant females : Gestation day 0, 7, 14 and 20
Lactation day 0 or 1(post-partum within 24 hours), 4, 8, 12
Male : At least weekly

Oestrous cyclicity (parental animals):

3.4.5. Monitoring of oestrous cycles
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.

Litter observations:

Observation of pups
Gender classification was performed within 24 hours of post-partum. There was an error in the gender classification of the pups, it was corrected in PND 4.
- Clinical sign(including stillbirths, runts, gross abnormalities etc.)
- Number of total pups and sex ratio(% male) on PND 0
- Number of live pups and sex ratio(% male) on PND 4
- Number of dead pups on PND 0 and PND 1~4(PND 5-13, if necessary)
- Anogenital distance (AGD) on PND 4
- Number of nipples/areolas for male offspring on PND 13.

Culling of pups
At PND 4, the number of pups per litter was adjusted by random selection to 8(4 females and 4 males). Some of the pups to be adjusted was have blood drawn to measure hormones concentration, and the blood was pooled regardless of sex. If the number of pups less than 8 per litter, culling was not be carried out. The following index was calculated by checking the number of pups of PND 0 and 4.
Gestation index (%) = (No. of litters with live pups/No. of females pregnant)×100
Live-born index (%) = (No. of pups born alive/No. of total pups born)×100
Viability index (%) = (No. of pups alive on PND 4/No. of pups alive on PND 0)×100

Postmortem examinations (parental animals):

Necropsy was performed on the 36th day after administration for males and on the 13rd 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.

After blood sampling, animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs were removed and examined. In females, the following index were calculated after counting the implantation site in the uterus and the number of corpora luteum in the ovary.

Pre-implantation loss (%) = [(No. of corpora luteum - No. of implantation sites) / No. of corpora luteum] × 100
Post-implantation loss (%) = [(No. of implantation sites - No. of live fetuses) / No. of implantation sites] × 100

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.

After blood sampling, animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs were removed and examined.

Statistics:

3.6. Statistical Analysis
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, and insufficient individual data(G3), only dose-dependent responses are determined without statistical analysis.

Reproductive indices:

3.4.7. 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

Offspring viability indices:

3.4.8.1. Calculation of gestation period
The gestation period was calculated using the date of observation of mating evidence and the date of parturition. The day of parturition was designated as lactation day(LD) 0 in the parental females and post-natal day (PND) 0 in the pups.


3.4.8.2. Observation of pups
Gender classification was performed within 24 hours of post-partum. There was an error in the gender classification of the pups, it was corrected in PND 4.
- Clinical sign(including stillbirths, runts, gross abnormalities etc.)
- Number of total pups and sex ratio(% male) on PND 0
- Number of live pups and sex ratio(% male) on PND 4
- Number of dead pups on PND 0 and PND 1~4(PND 5-13, if necessary)
- Anogenital distance (AGD) on PND 4
- Number of nipples/areolas for male offspring on PND 13.

3.4.8.3.Culling of pups
At PND 4, the number of pups per litter was adjusted by random selection to 8(4 females and 4 males). Some of the pups to be adjusted was have blood drawn to measure hormones on centration, and the blood was pooled regardless of sex. If the number of pups less than 8 per litter, culling was not be carried out. The following index was calculated by checking the number of pups of PND 0 and 4.

Gestation index (%) = (No. of litters with live pups/No. of females pregnant)×100
Live-born index (%) = (No. of pups born alive/No. of total pups born)×100
Viability index (%) = (No. of pups alive on PND 4/No. of pups alive on PND 0)×100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in all males at 350 mg/kg/day .
In pre-mating, salivation was observed in 7 females at 350 mg/kg/day.
Else, loss of fur was observed in 2 males(#32, 34) at 350 mg/kg/day.
In pre-mating, abdominal distention was observed in 1 female(#47) at 0 mg/kg/day.
The changes were not considered to be test substance-related change because there were low cases and/or they were observed as a judgment error when confirmation of a vaginal plug.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights in dam at 88 mg/kg/day on GD 20 was significantly decreased(p<0.05) and in dam at 175 mg/kg/day on GD 20 was decreased.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumptions in dam at 88 mg/kg/day on LD 8 was significantly decreased(p<0.05).
The change was not considered to be test substance-related change because there was temporary observed.
Else, there were no test substance-related changes.

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

effects observed, non-treatment-related

Description (incidence and severity):

Water consumptions in male at 175 mg/kg/day on Day 21 and 34 were significantly increased(p<0.05) and in male at 350 mg/kg/day on Day 28 was significantly increased(p<0.05).
Water consumption in female at 350 mg/kg/day on Day 14 was significantly increased(p<0.05).
The changes were not considered to be test substance-related because there was temporary observed or no dose-response relationship.
Else, there were no test substance-related changes.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Infiltrated of epididymis was observed in 1 male(#32) at 350 mg/kg/day.
Cyst of ovary was observed in each 1 female(#44, 71) at 0 and 350 mg/kg/day.
The changes were not considered to be test substance-related change because there was observed in vehicle control group or to be background changes.
Else, There were no test substance-related changes.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Male and female fertility index and fecundity index, and no. of females pregnant at all test substance were decreased.
No. of corpora lutea in dam at 88 and 175 mg/kg/day was decreased.
No. of implantations in dam at 88 mg/kg/day was significantly decreased(p<0.05), and No. of implantation in dam at 175 mg/kg/day was decreased.
Pre-implantation at in dam 88 mg/kg/day was significantly increased(p<0.05) and at 175 mg/kg/day was increased.
Post-implantation in dam at 175 mg/kg/day was increased.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In clinical signs and external examination, bent tail was observed in 1 and male at 0 mg/kg/day.
The changes were not considered to be test substance-related because there was observed at vehicle control and/or low cases.
Else, there were no test substance-related changes.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

3 dead pups(3 males) was observed in 0 mg/kg/day, 1 dead pup(1 male) were observed at 88 mg/kg/day.
The changes were not considered to be test substance-related because there was observed at vehicle control and/or low cases.
Else, there were no test substance-related changes.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights in male and female on PND 4 at 88 mg/kg/day were significantly increased (p<0.05). the change was not considered to be test substance-related change because there was temporary observed.
Else, there were no test substance-related changes.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital index(AGI) in female at 88 mg/kg/day was significantly increased(p<0.05).
The change was not considered to be test substance related-change because there was not relevant in T4 revel and necropsy findings.
Else, there were no test substance-related change.

Nipple retention in male pups:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

350 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The purpose of this study was to confirm the initial information of reproduction/developmental toxicity of the KF EPIOL-PE311 (GPGE) following a repeated oral administration to Sprague-Dawley rats.
The test substance, KF EPIOL-PE311 (GPGE) was administered at a dose level of 88, 175, and 350 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 41~67 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 body weights, food consumptions, water consumptions, estrus cycle, organ weights, necropsy findings, hormone concentration and histopathological examination.
In clinical sign, salivation observed in the both sexes at 350 mg/kg/day was considered to be test substance-related change because there was observed high dose and after administration.
However, the change was not considered to be toxicologically significant change because there was observed due to taste or phase of test substance.
In body weights, dam body weight decrease at GD20 observed at 88 and 175 mg/kg/day were considered to be test substance-related change because there were dose-response relationship.
However, the change was not considered to be toxicologically significant change because there were recovered during the lactation period or not relevant changes in the food and water consumption.
In reproductive performance, no. of males siring a litter, male fertility index and male fecundity index decrease observed at all test substance were considered to be test substance-related change because there were dose-response relationship. However, the change was not considered to be toxicologically significant change because there were not relevant changes in the body weight, food and water consumption, and the histopathological examination of testis and epididymis.
An increase in the pre-/post-implantation loss at 88 and 175 mg/kg/day were observed in relevant with decease in the no. of corpora lutea and no. of implantations at 88 and 175 mg/kg/day.
The changes were considered to be test substance-related change because there were dose-response relationship. Also, the changes were considered to be toxicologically significant change because there were relevant change in no. of females pregnant, female fertility index, pregnancy index, and no. of total pups born decrease.

[Offspring]
There were not able to test substance-related effects in clinical signs, external examination, body weights, anogenital index, number of nipples and areolas, necropsy findings, hormone concentration and histopathological examination.
In pups index, no. of total pups born, no. of alive on PND 0/PND 0 per dam and no. of pups alive on PND 4/PND 4 per dam decrease observed at 88 mg/kg/day were considered to be test substance-related change because there were relevant change in reproductive performance decrease of dam.
However, the change was not considered to be toxicologically significant change because there were not relevant changes in the other results of pups, and there were observed due to lack of pregnancy female.
Based on the above results, KF EPIOL-PE311 (GPGE) administered orally to Sprague-Dawley rats at 88, 175 and 350 mg/kg/day, the NOAEL of the KF EPIOL-PE311 (GPGE) was determined to be 350 mg/kg/day for males and not observed for dam and offspring.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available