Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Feb 2016 - 03 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Jan. 2001)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
(May 2008)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(August 1998)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Delta-Valerolactone
- Physical state: clear colorless liquid
- Analytical purity: 99.4 area-%
- Lot/batch No.: B2003-000STD77L0
- Expiration date of the lot/batch: 14 Oct 2016
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 to 14 weeks
- Weight at study initiation: 203 - 207 g
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum): ad lib. (SM R/M-Z from SSNIFF)
- Water (e.g. ad libitum): ad lib. (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12h / 12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (1.107) and vehicle (0.92). No correction was made for the purity/composition of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Concentration in vehicle: 100, 300, 1000 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (16 February 2016), according to a validated method (Project 512208). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also
determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy:Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
All animals were sacrificed on Day 21 post-coitum
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor and based the results of an OECD422 study, in which no systemic toxicity was observed up to 1000 mg/kg bw/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative assessment was introduced.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post-coitum
- Organs examined: All macroscopic abnormalities (external, thoracic, abdominal) were recorded, collected and fixed in 10% buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of implantations: Yes (In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites )
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: sex and weight of each fetus, weight of each placenta (of live fetuses only)
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter, i.e., the fetuses also used for visceral examination]
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
- The Steel-test many-to-one rank test)) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss(%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea x100
Post-implantation loss(%) = (number of implantation sites - number of live fetuses) / number of implantation sites x100
Viable fetuses affected/litter(%) = number of viable fetuses affected/litter / number of viable fetuses/litter x100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects: no

Macroscopic examination at necropsy revealed no treatment-related findings.
One female at 300 mg/kg bw/day showed a diaphragmatic hernia of the liver. This was considered as incidental and not treatment related.
A broken right upper incisor was noted for one female (no. 23) at 100 mg/kg bw/day, confirming the clinical sign observed during the in-life phase.

One female at 100 mg/kg bw/day and one female at 1000 mg/kg bw/day were non-pregnant. All pregnant females had litters with viable fetuses.
A relatively high mean value for pre-implantation loss (14.6% per litter) was noted in the control group compared to the treatment groups. As this value was within normal limits and concerned the control females, it is not considered toxicologically significant.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no treatment-related effects on litter size for any group

The male:female ratio was unaffected by treatment up to 1000 mg/kg bw/day

There were no effects on fetal body weights (per sex and combined for both sexes) noted by treatment up to 1000 mg/kg bw/day.
8.4.1. External malformations and variations
There were no treatment related effects on external morphology following treatment up to 1000 mg/kg
bw/day.

The only external malformation observed in this study was noted in a fetus of Group 3. The affected fetus (A063-10) had an omphalocele and since this finding occurred singly, it was considered a chance finding.
External variations were not seen in any group

There were no treatment related effects on visceral morphology following treatment up to 1000 mg/kg bw/day.

Two malformations were revealed at fetal visceral examination. Group 4 fetus A081-10 appeared to have an absent eye and a small eye at serial sectioning of the head. In control fetus A014-03 the aortic arch was on the right rather than on the left side. The single occurrence of an eye malformation at the highest dose level, a finding that was also previously noted among historical control fetuses, was not considered to be related to treatment.
The variation of appendix of the liver showed statistically significantly higher incidences in Groups 2 and 3. Mean litter incidences were 0.0%, 3.9%, 6.4% and 2.4% per litter for Groups, 1, 2, 3 and 4, respectively. However, because the group distribution of this variation occurs also spontaneously and does not denote a dose response, it was not considered to be attributed to treatment.
Other variations that were noted in this study were small supernumerary lobe(s) of the liver, partially undescended thymus horns and dilated and convoluted ureters. These variations occurred at low incidences, in the absence of a dose-related incidence trend and/or were noted in control fetuses only and therefore were not considered to be treatment related.

There were no treatment related effects on skeletal morphology following treatment up to 1000 mg/kg bw/day.
Only one skeletal malformation was observed (Group 2 fetus A042-02 had an interrupted cervical vertebral arch). The variations that were noted occurred in the absence of a dose-related incidence trend or occurred infrequently or were within the historical control range of the rat strain used. Therefore, all skeletal findings were not considered treatment related

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
IMaternal findings
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
Developmental findings
No developmental toxicity was observed in the 100, 300 and 1000 mg/kg bw/day groups.
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Delta-Valerolactone was established as being at least 1000 mg/kg bw/day.