Methyl 3-oxo-2-pentylcyclopentaneacetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 June 2006 to 12 April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-test procedure according to national standards (FDA guideline).

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Reference 2

Reference Type:

publication

Title:

Evaluation of the developmental toxicity of Methyl Dihydrojasmonate (MDJ) in rats

Author:

Politano VT, Lewis EM, Hoberman AM, Christian MS, Diener RM, Api AM

Year:

2008

Bibliographic source:

International Journal of Toxicology, 27: 295-300, 2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: U.S. Dept of Health and Human services Food and Drug Administration. Guideline for Industry: Detection of toxicity to reproduction for medicinal products, ICH-S5A; September, 1994, Rockville (MD).

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

EC Number:

246-495-9

EC Name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

Cas Number:

24851-98-7

Molecular formula:

C13H22O3

IUPAC Name:

methyl 3-oxo-2-pentylcyclopentaneacetate

Details on test material:

- Substance type: pure active substance
- Name of test material (as cited in study report): Methyl Dihydrojasmonate (MDJ)
- Physical state: colorless liquid
- Analytical purity: 99% (GLC)
- isomers composition: ca. 10-12 % cis- and 88-90 % trans- isomers
- Purity test date: 28 March 2006
- Lot/batch No.: 2F37994
- Expiration date of the lot/batch: 27 February 2008
- Storage condition of test material: at room temperature protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD (SD) IGS BR VAF/Plus

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC - USA
- Age at arrival: ca. 65 days
- Weight at study initiation: 218 - 246 g
- Housing: stainless steel, wire-bottomed cages. Individual cages were used.
- Diet: ad libitum, Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO), individual feeders. Quality Routinely analyzed.
- Water: ad libitum, from an automatic watering access system. Quality Routinely Analyzed
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12-h dark / 12-h light

IN-LIFE DATES: From: 13 June 2006 To: 13 July 2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared weekly at the Testing Facility and were stored at room temperature. Prepared formulations were stirred continuously during dosage. Stability of suspension were checked (See "Detailes on analytical verification of doses").

VEHICLE
- Justification for use and choice of vehicle (if other than water): Commonly used solvent for low soluble substance (MDJ WS: 561 mg/l)
- Concentration in vehicle: Corn oil (Product n°C8267 from Sigma Aldrich Inc., St-Louis, MO-USA and Product n°901414 from MP Biomedicials Inc, Solon OH-USA)
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): 103K0107, 015K0115 & 065K0077 for Sigma and 7371H for MP Biomedicials
- Purity: pure corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulation samples were quantitated using high performance liquid chromatography (HPLC-UV) with six matrix-matched calibration standards. Stability was evaluated for the 4 mg/mL formulation concentration level. Initial stability samples were analyzed on June 26, 2006. Final stability samples were stored at 22±5°C and analyzed on August 15, 2006: resulting in a 50-day stability period. The final stability sample results were compared with the initial results and expressed as percent errors. All of the results were within the acceptable range of ± 10% error.
Concentration samples from the first and the last days of preparation were within the acceptable limits of ± 15% error. The variance between nominal and actual dosage was acceptable.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days maximum
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: copulatory plug and/or spermatozoa in vaginal smear referred to as day 0 of pregnancy (DG 0)

Duration of treatment / exposure:

13 days (DG 7-20)

Frequency of treatment:

Daily

Duration of test:

21 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40, 80, 120 mg/kg bw/day
Basis:
nominal conc.
based on the most recent body weight determination

No. of animals per sex per dose:

25 animal/sex/dose
(See Table 1: Animal Assignment & Dose Levels in "Any other information on material and methods incl. Tables")

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages of MDJ were selected on the basis of findings from a dosage-range study in which MDJ dosages of 125, 250, 500 and 1000 mg/kg/day were administered to pregnant rats once daily for 14 consecutive days (DGs 7 through 20). No mortality occurred at dosages as high as 1000 mg/kg/day. At 250 mg/kg/day and higher, clinical signs of slight excessive salivation were observed, and at 1000 mg/kg/day apparent dehydration, moderate to extreme excessive salivation, red or brown perioral substance, ungroomed coat, substance on the fur, urine-stained abdominal fur and sparse hair coat were observed. Reductions in body weight gain occurred at 125 mg/kg/day and higher dosages during the entire dosage period (18% to 59% less than the vehicle control values). However, the most pronounced effects occurred during the first three days of dosage period (DGs 7 to 10) which reflected losses in body weight that occurred after the first dosage was administered to the rats on DG 7. Corresponding reductions in absolute and relative feed consumption values also occurred in the 125, 250, 500 and 1000 mg/kg/day dosage groups for the entire dosage period (12%, 12%, 25% and 28% less than the vehicle control value, respectively), with the most pronounced effects again occurring during the first three days of treatment (DGs 7 to 10). Relative to the vehicle control group values, fetal body weights (total, male and female) were reduced in all MDJ dosage groups (8% to 33% less than the vehicle controls). With the exception of the reduction in fetal body weights, no other adverse effects on Caesarean-sectioning or litter parameters, fetal gross external alterations, or fetal sex were observed at dosages as
high as 1000 mg/kg/day.

- Rationale for animal assignment (if not random): computer-generated (weight-ordered) randomization procedure

Examinations

Maternal examinations:

Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and deaths before and between one and two hours after dosage, and once daily during the postdosage period.
Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21.




CAGE SIDE OBSERVATIONS & DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and
deaths before and between one and two hours after dosage, and once daily during the postdosage period.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. Uteri of apparently nonpregnant rats were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rats were retained in neutral buffered 10% formalin and discarded at the end of the study when authorized by the Study Director.

OTHER:

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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae were examined for size, color and shape

Fetal examinations:

A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead (there were no dead fetuses). Each fetus was weighed and examined for sex and gross external alterations
- 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

Statistics:

Clinical observations and other proportional data were analyzed, using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed, using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data.
Count data were evaluated, using the procedures described above for the Kruskal-Wallis Test.

Indices:

none

Historical control data:

Results were compared with facility testing historical data provided in the study report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
See "Remarks on results including tables" for more details and for Tables of results.

All rats survived until scheduled sacrifice on gestational day 21 (GD 21).

Adverse clinical signs possibly associated with the test article were minor and limited to a tendency toward an increase in sparse hair coat and ungroomed appearance in the 120 mg/kg/day dosage group. At necropsy on GD 21, adverse findings were relegated to tan areas in liver lobes and a pale spleen in two rats at 120 mg/kg/day. Although the incidence rate (2/25) was not statistically significant, as compared with the vehicle controls, it was considered a possible effect of MDJ because this finding occurred only in the high dosage group. The tissues were not examined histologically.

Significant reductions in mean maternal absolute feed consumption and body weight gains occurred only in the high-dose group (120mg/kg/day, see Table 1 in "Remarks on results incl. tables and figures"). The body weight gain data from three dams with small litters (4, 4, and 9 live fetuses in the 40, 80, and 120 mg/kg/day dosage groups, respectively) were excluded from the data because litter size was unrelated to MDJ but affects maternal body weight gain, particularly late in gestation (GD 18–21) when fetal growth is greatest. Adjusted body weight gains for the entire treatment period (calculated as GDs 7-21) in the 40, 80, and 120 mg/kg/day dosage group were 93%, 92%, and 85% of the vehicle control group value, respectively, and 91%, 92%, and 84% prior to exclusion. Mean maternal body weights were also reduced in the 120 mg/kg/day dosage group on GDs 7-21; these reductions were statistically significant (p≤0.05) on GDs 11-15, when compared to the vehicle control group values.

Absolute and relative maternal feed consumption values were significantly reduced in the 120 mg/kg/day dosage group for the entire dosage period (calculated as DGs 7 to 21), as compared with the vehicle control group value. Within the dosage period, these values were reduced or significantly reduced in the 120 mg/kg/day dosage group at all tabulated intervals, as compared with the vehicle control group values, with the most severe reductions present on DGs 7 through 15.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
See "Remarks on results including tables" for more details and for Tables of results.
As indicated in Table 2 (“Details on results incl. Tables and Figures”), the 120 mg/kg/day dosage group had reduced mean fetal body weights, as compared with the vehicle control group (combined fetal body weights averaged 5.12 g, as compared with 5.24 g for the vehicle control group). The reductions in fetal body weights were not considered to be of toxicological significance because the values did not significantly differ from the vehicle control group values and were within the historical ranges (4.96–5.63 g) of the Testing Facility.
Apparent increases in fetal body weights in the 40 and 80 mg/kg/day dosage groups were associated with inclusion of the fetal values from small litters, which tend to have larger fetuses. After exclusion of values from these litters, fetal averages for the three respective dosage groups were 101%, 102% and 98% of vehicle control values. No other Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day.
Fetal evaluations were based on 342, 308, 293, and 310 live GD 21 Caesarean-delivered fetuses in 24, 23, 22, and 23 litters in the 0, 40, 80, and 120 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 165, 148, 141 and 148 fetuses were examined for soft tissue alterations, and 177, 160, 152 and 162 fetuses were examined for skeletal alterations and fetal ossification site averages. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of MDJ as high as 120 mg/kg/day (See Table 3 of “Details on results incl. Tables and Figures”). There were no significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. All ossification site averages were comparable among the dosage groups. All values were within the historical ranges of the Testing Facility.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Mortality and Delivery

All rats survived until scheduled sacrifice on gestational day 21 (GD 21).

 

One rat in the 80 mg/kg/day dosage group, described below, prematurely delivered on the day of scheduled sacrifice (DG 21). This delivery was considered unrelated to treatment with Methyl Dihydrojasmonate (MDJ) because it was anondosage-dependent event and the incidence was within the historical experience of the Testing Facility.

 

Clinical Observations

Adverse clinical signs possibly associated with the test article were minor and limited to a tendency toward an increase in sparse hair coat andungroomedappearance in the 120 mg/kg/day dosage group.

All other clinical observations were considered unrelated to treatment with MDJ because: 1) the incidences were not dosage-dependent (urine-stained abdominal fur); 2) the observation occurred in only one or two rats in any dosage groups (slight excess salivation,chromorhinorrhea, soft or liquid feces, localized alopecia on the limbs, dehydration (based on skintugor) and/ormucoidfeces); and 3) the findings were considered common clinical observations for this species and strain of laboratory rat.

 

Necropsy Observations

At necropsy on GD 21, adverse findings were relegated to tan areas in liver lobes and a pale spleen in two rats at 120 mg/kg/day. Although the incidence rate (2/25) was not statistically significant, as compared with the vehicle controls, it was considered a possible effect of MDJ because this finding occurred only in the high dosage group. The tissues were not examined histologically.

The only other necropsy observation, dark red fluid present in the uterine horns, was considered unrelated to treatment with MDJ because: 1) the incidence was not dosage dependent; and 2) the observation was probably associated with impending parturition. The litter of this rat consisted of only five implantation sites (one earlyresorptionand four live fetuses). No other gross lesions were revealed by necropsy of the rats.

 

Table 1: Effect of oral administration of MDJ on maternal absolute feed consumption and maternal body weight gains

Dosage Group (mg/kg/day)^a	0 (control)	40	80	120
Rats tested	25	25	25	25
Rats pregnant	24	23	23	25
Rats included in the analyses	24	22^c	21^b,c	22^c
Maternal absolute feed consumption in g/day (mean±SD)
GD 0-7	22.4± 1.8	22.6± 2.0	23.0± 1.8	22.6± 1.7
GD 7-10	18.6± 2.8	17.7± 3.5	17.8±3.1	16.2± 2.7
GD 10-12	19.0± 2.7	18.8± 3.4	18.9± 4.2	15.8± 3.2**
GC 12-15	19.2± 2.2	19.4± 3.2	18.1± 3.6	16.8± 3.7*
GD 15-18	21.3± 2.8	19.9± 4.3	20.2± 3.7	19.2± 2.8
GD 18-21	19.6± 3.6	19.3± 4.9	19.5± 5.4	17.5± 4.2
GD 7-21	19.6± 2.2	19.0± 3.3	19.3± 4.1	17.2± 2.1
GD 0-21	20.5± 1.7	20.2± 2.7	20.7± 3.5	19.0± 1.8
Maternal body weight gains in g (mean± SD)
GD 0-7	43.2± 4.8	44.3± 8.9	45.7± 4.6	41.6± 7.0
GD 7-10	0.8± 4.4	0.2± 3.7	-1.3± 3.4	-2.0± 3.8
GD 8-9	4.7± 3.7	2.6± 4.3	3.5± 4.1	0.9± 3.3**
GD 9-10	4.0± 4.3	6.4± 5.8	4.6± 4.1	4.8± 4.4
GD 7-10	9.4± 6.6	9.2± 7.8	6.8± 5.9	3.7± 6.5**
GD 10-12	10.6± 6.9	12.1± 4.8	9.0± 5.8	7.9± 6.0
GC 12-15	17.4± 7.6	15.4± 8.0	16.2± 7.5	16.2± 7.5
GD 15-18	40.6± 8.5	38.9± 10.2	40.0± 9.6	41.4± 11.6
GD 18-21	47.0± 9.7	40.4± 10.2	43.4± 15.5	39.1± 18.1
GD 7-21	125.3± 18.9	116.0± 35.1	115.6± 21.7	107.0± 23.5
GD 0-21	168.8± 19.9	160.3± 39.5	161.2± 22.2	148.6± 24.4
^aDosing occurred on days 7-20 of gestation ^bExcludes dam 18174, which delivered a litter before scheduled sacrifice. ^cExcludes dams that had small litters. GD=gestational day, the presence of spermatozoa and/or acopulatoryplug in situ was designated as GD 0. ∗ Significantly different from the vehicle control group value (p≤0.05). ∗∗ Significantly different from the vehicle control group value (p≤0.01).

 

Maternal Body Weights and Body Weight Changes

Significant reductions in mean maternal absolute feed consumption and body weight gains occurred only in the high-dose group (120mg/kg/day). The body weight gain data from three dams with small litters (4, 4, and 9 live fetuses in the 40, 80, and 120 mg/kg/day dosage groups, respectively) were excluded from the data because litter size was unrelated to MDJ but affects maternal body weight gain, particularly late in gestation (GD 18–21) when fetal growth is greatest. Adjusted body weight gains for the entire treatment period (calculated as GDs 7-21) in the 40, 80, and 120 mg/kg/day dosage group were 93%, 92%, and 85% of the vehicle control group value, respectively, and 91%, 92%, and 84% prior to exclusion. Mean maternal body weights were also reduced in the 120 mg/kg/day dosage group on GDs 7-21; these reductions were statistically significant (p≤0.05) on GDs 11-15, when compared to the vehicle control group values.

 

Maternal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values

Absolute and relative maternal feed consumption values were significantly reduced in the 120 mg/kg/day dosage group for the entire dosage period (calculated as DGs 7 to 21), as compared with the vehicle control group value. Within the dosage period, these values were reduced or significantly reduced in the 120 mg/kg/day dosage group at all tabulated intervals, as compared with the vehicle control group values, with the most severe reductions present on DGs 7 through 15.

 

Caesarean-Sectioning and Litter Observations

As indicated in Table 2 below, the 120 mg/kg/day dosage group had reduced mean fetal bodyweights,as compared with the vehicle control group (combined fetal body weights averaged 5.12 g, as compared with 5.24 g for the vehicle control group). The reductions in fetal body weights were not considered to be of toxicological significance because the values did not significantly differ from the vehicle control group values and were within the historical ranges (4.96–5.63 g) of the Testing Facility.

Apparent increases in fetal body weights in the 40 and 80 mg/kg/day dosage groups were associated with inclusion of the fetal values from small litters, which tend to have larger fetuses. After exclusion of values from these litters, fetal averages for the three respective dosage groups were 101%, 102% and 98% of vehicle control values. No other Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day.

Fetal evaluations were based on 342, 308, 293, and 310 live GD 21 Caesarean-delivered fetuses in 24, 23, 22, and 23 litters in the 0, 40, 80, and 120 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations. Of these respective fetuses, 165, 148, 141 and 148 fetuses were examined for soft tissue alterations, and 177, 160, 152 and 162 fetuses were examined for skeletal alterations and fetal ossification site averages. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosages of MDJ as high as 120 mg/kg/day (See Table 3 below). There were no significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. All ossification site averages were comparable among the dosage groups. All values were within the historical ranges of the Testing Facility.

 

Table 2: Effect of maternal administration of MDJ onCeasarean-section and litter data

Dosage Group (mg/kg/day)^a	0 (control)	40	80	120
Rats tested	25	25	25	25
Ratspregnant (%)	24 (96)	23 (92)	23 (92)	23 (92)
RatsCeasareansectioned GD 21	24	23	22^b	23
Rats included in the analysis	24	22^c	21^b,c	22^c
Corporealutea(mean± SD)	15.9± 2.2	15.6± 1.9	14.8± 1.8	14.9± 1.7
Implantations (mean± SD)	14.7± 1.8	14.6± 2.1	14.1± 1.9	14.4± 1.8
Resorptions(mean± SD)	0.4± 0.8	0.8± 0.9	0.4± 0.6	0.7± 1.0
Litter size (mean± SD)	14.2± 1.9	13.8± 2.1	13.8± 1.8	13.7± 1.7
Fetal body weight/litter (mean± SD)	5.24± 1.9	5.29± 0.52	5.35± 0.40	5.12± 0.29
Live fetuses	342	304	289	301
Dead fetuses	0	0	0	0
^aDosing occurred on days 7-20 of gestation. ^bExcludes dam 18174, which delivered a litter before scheduled sacrifice. ^cExcludes dams that had small litters. GD=gestational day, the presence of spermatozoa and/or acopulatoryplug in situ was designated as GD 0.

 

Table 3: Fetal alterations observed (gross external, soft tissue, or skeletal variations or malformations) following oral administration of MDJ to pregnant rats

Dosage Group (mg/kg/day)^a	0 (control)	40	80	120
Litters evaluated	24	22^c	21^b.c	22^c
Fetuses evaluated	342	304^c	289^b,c	301^c
Litters with fetuses with any observed alterations (%)	6 (25.0)	3 (13.6)	3 (14.3)	5 (22.7)
Fetuses with any observed alterations (%)	7 (2.0)	3 (1.0)	4 (1.4)	5 (1.7)
% Fetuses /litter with any alteration (mean± SD)	2.0± 4.0	0.9± 2.4	1.3± 3.3	1.6± 3.1
^aDosing occurred on days 7–20 of gestation. ^bExcludes dam 18174, which delivered a litter before scheduled sacrifice. ^cExcludes dams that had small litters. GD=gestational day, the presence of spermatozoa and/or acopulatoryplug in situ was designated as GD 0.

 

Remark: complete data results are attached in "Attached backroud material"

Applicant's summary and conclusion

Conclusions:

See executive summary

Executive summary:

Introduction.This oral (gavage) developmental toxicity study of methyl dihydrojasmonate (MDJ) in rats was performed according to the following FDA Guideline for Industry:Detection of toxicity to reproduction for medicinal products, ICH-S5A; September, 1994, Rockville (MD).

 

Methods. One hundred presumed pregnant Crl:CD(SD) rats were randomly assigned to four dosage groups (Groups I through IV), 25 rats per group. The test article, Methyl Dihydrojasmonate (MDJ), or the vehicle, Corn Oil, was administered orally via gavage once daily on days 7 through 20 of presumed gestation (DGs 7 through 20) at dosages of 0 (Vehicle), 40, 80 and 120 mg/kg/day to rats in Groups I through IV, respectively. The dosage volume (10mL/kg) was adjusted daily on the basis of the individual body weights recorded before intubation. The rats were administered the test article and/or the vehicle once daily at approximately the same time each day. All rats were examined for clinical observations of effects of the test article, abortions, premature deliveries and deaths before dosage, between one and two hours following dosage and once daily during thepostdosageperiod. Body weights were recorded on DG 0 and daily during the dosage andpostdosageperiods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21. All rats were sacrificed on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. All fetuses were weighed and examined for sex and gross external alterations. Approximately half of the fetuses in each litter were examined for soft tissue alterations (by dissection) or skeletal alterations (alizarin red S-staining).

 

Results. All prepared formulations were acceptable for use on this study.

All rats survived to scheduled sacrifice on DG 21. One dam in the 80 mg/kg/day dosage group prematurely delivered its litter on DG 21, a non dosage dependent event unrelated to MDJ.

 

The 120 mg/kg/day dosage of MDJ was associated with an increase in sparse hair coat and a significant increase in ungroomed coat that occurred at the end of the dosage period. Two rats in the 120 mg/kg/day dosage group each had tan areas in the liver lobes and a pale spleen. All other clinical and necropsy observations were considered unrelated to treatment with MDJ.

 

Toxicologically important reductions in maternal body weight gain occurred in the 120 mg/kg/day dosage group.

 

Both the 80 and 120 mg/kg/day dosage groups lost weight after the initial dosage was given (DGs 7 to 8), clear reductions in weight gain continued throughout the dosage period only in the 120 mg/kg/day dosage group. The 120 mg/kg/day dosage group had significantly reduced (p≤0.01) body weight gain on DGs 8 to 9 and DGs 7 to 10 and reduced weight gain on DGs 10 to 12. Average maternal body weights were reduced in the 120 mg/kg/day dosage group on DGs 7 through 21; these reductions were statistically significant on DGs 11 through 15, as comparison to the vehicle control group values.

 

Absolute and relative maternal feed consumption values were significantly reduced in the 120 mg/kg/day dosage group for the entire dosage period (calculated as DGs 7 to 21), as compared with the vehicle control group value. Within the dosage period, these values were reduced or significantly reduced in the 120 mg/kg/day dosage group at all tabulated intervals, as compared with the vehicle control group values, with the most severe reductions present on DGs 7 through 15.

 

No Caesarean-sectioning or litter parameters were affected by dosages of MDJ as high as 120 mg/kg/day.

Although the 120 mg/kg/day dosage group tended to have reduced fetal body weights (combined fetal body weights averaged 5.12 g, as compared with 5.24 g for the vehicle control group), the reductions in fetal body weights were not considered to be of toxicological importance because the values did not significantly differ from the vehicle control group values and were within the historical ranges of the Testing Facility.

 

No gross external, soft tissue or skeletal fetal alterations (malformations or variations) or differences in ossification site averages were attributable to dosages of MDJ as high as 120 mg/kg/day. All values were within the historical ranges of the Testing Facility.

 

Conclusions. On the basis of these data,the maternal no-observable-effect-level (NOAEL) of Methyl Dihydrojasmonate(MDJ) is 80 mg/kg/day.The 120 mg/kg/day dosage of MDJ caused maternal weight loss after the initial dosage and reduced maternal body weight gains and body weights during the dosage period as well as reduced absolute and relative maternal feed consumption values for the entire dosage period.

 

The developmental NOAEL is greater than 120 mg/kg/day. The only observation associated with the 120 mg/kg/day dosage of MDJ was a minimal reduction in fetal body weight that was not considered to be toxicologically important because the value was not statistically significant, as compared with the vehicle control group value, and was within the historical range of the Testing Facility. Based on these data,MDJ should not be identified as a developmental toxicant.