Undecan-4-olide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 26, 2002 to June 14, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

OECD 414 guideline study in compliance with GLP. γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 2, 2002 to July 31, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Remarks:

OECD 407 guideline study in compliance with GLP. γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Laboratories, Inc., Raleigh, North Caroline
- Age at study initiation: 34 days old at receipt, approximately seven weeks old at study initiation
- Weight at study initiation: males: 214-265 g; females: 157-200 g
- Housing: individually, in clean, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): ad libitum except during the period of fasting prior to blood collection, PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (analysis certified)
- Water (e.g. ad libitum): ad libitum, reverse-osmosis-treated (on-site) municipal water (analysis certified)
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From April 2, 2002 To: May 31, 2002 (recovery necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The 1000 mg/kg bw/d test article formulation was a weight/volume (test article/vehicle) mixture. For this group, the appropriate amount of the test article was weighted into a tared weighting vessel. Approximately 60 % of the vehicle was added to a calibrated storage container, and a deep vortex was created with a magnetic stir bar. The test article was slowly transferred quantitatively into the rapidly stirring vehicle and stirred until dissolved (approximately one minute). The remaining amount of vehicle was added to bring the volume to the calibration mark. The formulation was stirred until uniform and throughout use, using a magnetic stirrer. The mixture was used as a stock formulation to prepare the 30, 100 and 300mg/kg bw/d group dosing formulations. For these groups, the appropriate volume of the stock formulation was added to achieve the total volume for each group. The dosing formulations were stored refrigerated, protected from light. The test article formulations were prepared approximately weekly, then separated into aliquots for daily dispensation. All test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dose administration, samples (10 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the all dosing formulations, including the control group. In addition, duplicate samples (10 mL each) for stability determinations were collected from the top and bottom strata of these same dosing suspensions; one set was stored refrigerated for 10 days and one set was stored at room temperature for 10 days. Both sets were protected from light. Samples (10 mL each) for concentration analysis were collected from the first, second and third weekly preparations from each dosing formulation (including the control group.
All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, Inc. The test article formulations were found to be homogenous (the RSD for the overall mean concentration was ≤ 10 % at a concentration that is within the acceptable limits), contain the amount of test article specified in the protocol (analyzed concentrations were within ± 10 % of the target dose concentrations) and stable for at least 10 days when stored refrigerated or at room temperature (the mean concentrations were no less that 90 % of the time zero concentrations).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals/sex/dose, 5 additional recovery animals in control and high-dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not detailed in the study report
- Rationale for animal assignment (if not random): computerized randomization procedure
- Rationale for selecting satellite groups: control and high dose groups
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

Not applicable

Observations and examinations performed and frequency:

MORTALITY and MORIBUNDITY: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, prior to dose administration and approximately one to two hours following dose administration. Once daily during the recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning the week prior to test article administration and prior to scheduled necropsies

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly, beginning two weeks prior to test article administration. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded prior to each scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): NOT A FEEDING STUDY, however individual food consumption was recorded weekly, beginning two weeks prior to test article administration. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): NOT A DRINKING WATER STUDY

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of dose administration and near the end of the treatment period
- Dose groups that were examined: all animals
- All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope, preceded by papillary dilation with 0.5 % mydriacyl.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study week 4 and 6).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all surviving animals, and all recovery groups animals
- Parameters checked in table 7.5.1/2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study week 4 and 6).
- Animals fasted: Yes, overnight
- How many animals: all surviving animals and all recovery groups animals
- Parameters checked in table 7.5.1/2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the scheduled necropsies (study week 4 and 6).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table 7.5.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observation battery (FOB)
- Dose groups and time schedule for examinations: Five animals/sex/group prior to the initiation of dose administration and during study week 3, and for five animals in the control and 1000 mg/kg bw/d groups during study week 5 (recovery period).
Sound-attenuated room equipped with a white noise generator set top operate at 70 ± 10 db with one exception; home cage observations were performed in the animal room.
- Parameters examined: see Table 7.5.1/3

Locomotor activity
- Dose groups and time schedule for examinations: Five animals/sex/group prior to the initiation of dose administration and during study week 3, and for five animals in the control and 1000 mg/kg bw/d groups during study week 5 (recovery period).
- Measured automatically using the San Diego Instrument, Inc. Photobeam Activity System (San Diego Instruments, Inc. San Diego, California). This personal computer-controlled system utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage t quantify each animal’s motor activity. The testing of treatment groups was done accordingly to replicate sequence. Each animal was tested separately. Data were collected in five-minute epochs (print intervals) and the test session duration was 60 minutes.
Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of one photobeam) and ambulatory motor activity (interruption of two or more consecutive photobeams).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (See table 7.5.1/4). Animals were anesthetized by isoflurane and exsanguinated.
HISTOPATHOLOGY: Yes (See table 7.5.1/4)

Other examinations:

ULTRASTRUCTURAL EVALUATION: liver specimens collected in McDowell-Trump fixative for electron microscopy were embedded at North Carolina State University. Samples for the primary necropsy were subjected to ultrastructural examination at EPL, Durham, North Caroline.

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animal was two or less. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ± 1 in the last significant figure.
Body weight, body weight change, food consumption, continuous FOB, Locomotor Activity Data, clinical pathology and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistical significance (p < 0.05), Dunnett’s test was used to compare the test article-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test. Clinical pathology values for white blood cell types that occur at a low incidence (i.e. monocytes, eosinophils and basophils) were not subjected to statistical analysis.

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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

lower cholesterol levels in the 1000 mg/kg bw/d group, recovery occured

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver weight at 1000 mg/kg bw, recovery occured

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale liver in 2/5 M and 4/5 F dosed with 1000 mg/kg bw/d, correlated with increased lipid droplets

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled necropsy.
Test article-related clinical findings consisted of clear, yellow or red material around the mouth and/or the urogenital area or forelimbs observed at the 1000 mg/kg bw/d group at low incidence at the time of dosing (females) and one hour following dosing (males and females).
In addition, hypereactivity was noted infrequently in 30, 100 and 300 mg/kg bw/d group females and in single occurrences in the 1000 mg/kg bw/d group males and females. However, since the occurrences were infrequent and no dose and/or temporal relationship was apparent, these findings were not considered to be test article-related. Other clinical findings were not observed in a dose-related manner, occurred in singles animals and/or are common findings in laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
No test article-related trends in body weights or body weight gains were noted at any dose level. The only statistically significant (p < 0.05 or p < 0.01) differences from the control group values were increases in the 100 mg/kg bw/d group females (study weeks 0-1 through 0-3) and the 300 mg/kg bw/d females (study week 0-3). No dose-relationship was evident.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test article related-effects on food consumption were observed at any dose level.

OPHTHALMOSCOPIC EXAMINATION
There were no test article-related ophthalmic findings.

HAEMATOLOGY
There were no test article-related changes in hematology. The only statistically significant (p < 0.05) difference from the control group was a slightly lower mean MCHC value in the 1000 mg/kg bw/d group females at the week 6 recovery evaluation. This difference from the control group was not attributed to the test article since a similar decrease was not observed in these females at the study week 4 evaluation or in the 1000 mg/kg bw/d group males at either evaluation.

CLINICAL CHEMISTRY
Test article-related lower cholesterol levels were noted in the 1000 mg/kg bw/d group males and females at the study week 4 evaluation. Some of the differences were statistically significant (p < 0.05 or p < 0.01).
Mean cholesterol levels in the 1000 mg/kg bw/d group males and females were lower (statistically significant at p < 0.05 or p < 0.01) than those in the control group. At the recovery evaluation (study week 6), the mean cholesterol levels in the 1000 mg/kg bw/d group males and females were similar to those in the control. Another possible treatment-related effect in the 1000 mg/kg bw/d group females was slightly lower mean sodium levels at study week 4. The difference from the control group was statistically significant (p < 0.05). However, the biological significance of the lower sodium level is unclear since the magnitude of the change from the control value was small.
Other statistically significant changes in mean serum chemistry values at study week 4 were also observed, but the lack of dose-related trends or similar findings for comparable dosed rats of the opposite sex suggests that these findings are more likely random occurrences that treatment-related effects. These changes included higher mean albumin, total protein, urea nitrogen and calcium levels in the 1000 mg/kg bw/d group females, higher mean asparatate aminotransferase levels in the 1000 mg/kg bw/d group males and lower mean chloride levels in the 100 and 300 mg/kg bw/d group males.

URINALYSIS
There were no test article-related changes in urinalysis parameters. The only statistically significant (p < 0.05) difference from the control group was a slight increase in specific gravity in the 1000 mg/kg bw/d group females at the week 6 recovery evaluation. This difference was not observed in these females at the study week 4 evaluation or in the 1000 mg/kg bw/d group males at either evaluation.

NEUROBEHAVIOUR
- FOB:
No remarkable differences on home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations were apparent between the control and test article-related groups during study weeks 3 and 5.
No test article-related physiological observations were noted. Statistically significant (p < 0.05 or p < 0.01) increases were observed in body weight for the 100 mg/kg bw/d group females during pretest and catalepsy for the 30 mg/kg bw/d group males at study week 3. Since these findings occurred during the pretest period or were not observed in a dose-related manner, they were not attributed to the test article. In addition, a statistically significant (p < 0.05) decrease in body temperature was observed in the 1000 mg/kg bw/d group females at the week 5 recovery. Since this finding occurred during the recovery period, the differences were not attributed to the test article.
- Locomotor activity:
There were no test article-related effects on motor activity.

ORGAN WEIGHTS
Test article-related increased in liver weights (absolute and relative to final body/brain weights) were observed in the 1000 mg/kg bw/d group females at the study week 4 primary necropsy. A slight increase in liver weight relative to final body weight in the 1000 mg/kg bw/d group males at study week 4 was considered equivocal. At the study week 6 recovery necropsy, liver weights in the 1000 mg/kg bw/d group males and females were comparable to the control.
Mean absolute and relative (to final body and to brain weight) liver weights in the 1000 mg/kg bw/d group females were increased 27-31 % compared to the control group at the primary necropsy (study week 4). The differences were statistically significant (p < 0.01). Mean relative (to final body weight) liver weight in the 1000 mg/kg bw/d group males was also increased by 13 % (p < 0.05) compared to the control group. Mean absolute and relative liver weight in the 1000 mg/kg bw/d group males and females were similar to those in the control group at the study week 6 recovery necropsy. No other test article-related changes in organ weights were noted at the study week 4 primary necropsy, and no test article-related effects on organ weights were observed at the study week 6 recovery necropsy.
Statistically significant increases (p < 0.01) were observed in mean absolute brain weights in the 100 and 300 mg/kg bw.d group females compared to the control. No dose-relationship was evident; therefore, these increases were considered incidental.

GROSS PATHOLOGY
Test-article findings were observed in the liver in the 1000 mg/kg bw/d group males and females.
At the primary necropsy, a pale liver was observed in two males and four females in the 1000 mg/kg bw/d group. These findings correlated with the increased lipid droplets observed by election microscopy. No other test article-related findings were observed at the primary necropsy, and no test article-related changes were observed at the recovery necropsy.

HISTOPATHOLOGY:
There were no histopathologic observations that were conclusively test article-related.
A very slight increased incidence of foamy cytoplasm of hepatocytes (termed “vacuolation, cytoplasm”) was observed in the livers of two females and one male in the 1000 mg/kg bw/d group relative to the control group.

ULTRASTRUCTURAL EVALUATION:
Representative liver sections were examined for three males and three females form the control and 1000 mg/kg bw/d groups collected at the primary necropsy.
The livers from the 1000 mg/kg bw/d group males had considerably higher numbers of lipid bodies in the hepatocytes. The numbers of lysosomes and dark glycogen granules were also slightly increased. The ultrastructural features of the livers from the 1000 mg/kg bw/d group females were characterized by test article-related lipid content to the extent seen in the 1000 mg/kg bw/d group males. The distribution and severity of increased glycogen granules were also comparable to that observed in these males.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects noted at 1000 mg/kg bw/d were very minimal and not indicative of toxicity

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects at 300 mg/kg bw/day

Critical effects observed:

not specified

none

Conclusions:

Under the test conditions, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL to be 1000 mg/kg bw/d. γ-Caprolactone is not classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and the GHS.

Executive summary:

In a sub-acute toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, γ-Caprolactone diluted in deionized water was administered by gavage to Crl:CD®(SD)IGS BR rats (5/sex/group) at 30, 100, 300 or 1000 mg/kg bw/day for 28 days A control group received vehicle alone at the same volume-dosage (10 mL/kg bw/day). Control and high dose group contained an additional 5 rats/sex/group for evaluation of recovery from the test article-related effects, following a 14-day recovery period.

For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Functional observation battery (FOB) and locomotor activity data were recorded for five animals/sex/group prior to the initiation of dose administration and during study week 3, and for the recovery animals during week 5. Clinical pathology evaluations were performed on all animals. Ophthalmic examinations were performed during weeks -2 and 3. Complete necropsies were conducted on all animals, and selected organs were weighted at the scheduled necropsies. Selected tissues were examined microscopically for all animals. Sections of the livers from six rats/sex/group were collected at the scheduled necropsies for possible ultrastructural evaluation. Samples collected at the primary necropsy were examined by electron microscopy.

 

All animals survived to the scheduled necropsies. Body weights, food consumption, hematology and urinalysis parameters were unaffected by test article administration. There were no test article-effects on FOB parameters or locomotor activity. No test article-related ophthalmic findings were noted. No test article-related changes were noted in the 30, 100 or 300 mg/kg bw/d groups.

Test article-related effects noted in the 1000 mg/kg bw/d group consisted of:

- Slightly increased incidence of wet and/or dried material (clear, red and/or yellow) around the mouth and/or the urogenital area of forelimbobserved at the 1000 mg/kg bw/d group at low incidence at the time of dosing (females) and one hour following dosing (males and females). These findings were not considered to be adverse.

- Lower mean cholesterol levels (males and females). This finding was not considered to be adverse.

- Increased mean liver weights (absolute and/or relative to final body and/or brain weights) were noted for both males and females at the primary (week 4) necropsy. This increase was not observed at the recovery period.

- Pale liver was noted in two males and four females at the primary necropsy and correlated with the increased lipid bodies seen by election microscopy.

- No conclusive test article-related light microscopic findings were noted. However, a very slight increase in the incidence of cytoplasmic vacuolation was observed in one male and two females in the 1000 mg/kg bw/d group. This observation may explain the increased liver weights and ultrastructural findings.

- Marked increase in lipid bodies and a slight increase in dark glycogen granules within the hepatocytes of those livers examined by electron microscopy. The increased lipid bodies may account for the increased liver weights and the gross observations of pale liver, particularly in females.

 

Based on the results of this study, the NOEL was 300 mg/kg bw/day and the NOAEL was 1000 mg/kg bw/d. The effects noted at the 1000 mg/kg bw/d were very minimal and not indicative of significant toxicity. There was no evidence of liver toxicity. The higher liver weights, the slightly higher incidence of hepatic cytoplasmic vacuolation observed by light microscopy in the females and the higher incidence of lipid bodies and dark glycogen granules observed by electron microscopy were most likely a physiological adaptation to the administration of the test article. Consequently, they were not considered as adverse. Furthermore, the changes were reversible as they were no longer observed following the 14-day recovery period.

 

Therefore, γ-Caprolactone is not classified for damage to organs through prolonged oral dose repeated exposure according to the Regulation (EC) No 1272/2008 (CLP) and the GHS.

This study is considered as acceptable and satisfies the requirement for repeated dose toxicty endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexan-4-olide

EC Number:

211-778-8

EC Name:

Hexan-4-olide

Cas Number:

695-06-7

IUPAC Name:

5-ethyldihydrofuran-2(3H)-one

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): gamma-CL
- Physical state: clear colourless liquid
- Storage condition of test material: room temperature protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD®(SD)IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Caroline
- Age at study initiation: approximately 12 weeks old whenn paired fro breeding
- Weight at study initiation: 237 to 239 g on Day 0 of gestation
- Housing: upon arrival and until paring, individually housed in clean, wire-mesh cages suspended above cage-board. The rats were paired for mating in the home cage of the males. Following positive identification of mating, the femles were returned to an individual suspended wire-mesh cage, nesting material was not required as the females were euthanized prior to the data of expected parturition.
- Diet (e.g. ad libitum): ad libitum, PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 (analysis certified)
- Water (e.g. ad libitum): ad libitum, reverse-osmosis-treated (on-site) municipal water (analysis certified)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): approximately 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 26, 2002 To: May 3, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deioniszed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The 1000 mg/kg bw/d test article formulation was a weight/volume (test article/vehicle) mixture. For this group, the appropriate amount of the test article was weighted into a tared weighting vessel. Approximately 60 % of the vehicle was added to a calibrated storage container, and a deep vortex was created with a magnetic stir bar. The test article was slowly transferred quantitatively into the rapidly stirring vehicle and stirred until dissolved (approximately one minute). The remaining amount of vehicle was added to bring the volume to the calibration mark. The formulation was stirred until uniform and throughout use, using a magnetic stirrer. The mixture was used as a stock formulation to prepare the 100 and 300mg/kg bw/d group dosing formulations. For these groups, the appropriate amount of the stock formulation was added to achieve the total volume for each group. The dosing formulations were stored refrigerated, protected from light. The test article formulations were prepared approximately weekly (as single formulations for each dose level), then separated into aliquots for daily dispensation. All test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity, re-suspension homogeneity and stability of the test formulations at a similar range of concentrations as used in this study were established in a companion study (Repeated dose toxicity: oral, 28d, Kirkpatrick).
Prior to the initiation of dose administration (April 2, 2002), samples (10 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the all dosing formulations, including the control group. In addition, duplicate samples (10 mL each) for stability determinations were collected from the top and bottom strata of these same dosing suspensions; one set was stored refrigerated for 10 days and one set was stored at room temperature for 10 days. Both sets were protected from light. Samples (10 mL each) for concentration analysis were collected from the first, second and third weekly preparations from each dosing formulation (including the control group.
All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, Inc. The test article formulations were found to be homogenous (the RSD for the overall mean concentration was ≤ 10 % at a concentration that is within the acceptable limits), contain the amount of test article specified in the protocol (analyzed concentrations were within ± 10 % of the target dose concentrations) and stable for at least 10 days when stored refrigerated or at room temperature (the mean concentrations were no less that 90 % of the time zero concentrations).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From GD6 through GD19

Frequency of treatment:

Once daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: selected based on the results of previous studies and were provided by the sponsor after consultation with the study director.
- Rationale for animal assignment (if not random):
- Other:

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 through 20 of gestation. Animals were also observed for signs of toxicity approximately one hour following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 6-20 (daily)
Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-20, 6-20 and 0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation day 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding bpdy weigh change intervals . On the occasions when food intake could not be measured for one of the days in a given interval (due to a weighting error, food spillage, obvious erroneous value, etc.), values were calculated using the appropriate number of days for that interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No, not a drinking water study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross observations of organs, maternal tissues were preserved

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

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examination: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Soft tissue examinations (head): Yes: half per litter

Statistics:

All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance levels of 5 %, comparing each test article-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ± 1 in the last significant figure.
Mean maternal body weights (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, number of corpora lutea, implantations sites and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistical significant (p < 0.05) intergroup variance, Dunnett’s test was used to compare the test article-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution) were subjected to the Kruskall-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p < 0.05) intergroup variance, the Mann-Whitney U-test was used to compare the test article-related groups to the control group. Mean litter proportions (percent per litter) or total fetal malformations and developmental variations (external, visceral, skeletal and combined) and of each particular external, visceral and skeletal malformation or variation were analyzed by the Kruskall-Wallis nonparametric ANOVA test followed by the Mann-Whitney U-test (if appropriate) as described above.

Indices:

- Postimplantation loss/litter= (No. dead fetuses, resorptions (Early/Late)/Group) / No. Gravid Females/Group.
- Summation per group (%) = Sum of postimplantation loss/litter (%) / No. litters/group.
- Postimplantation loss/litter (%) = (No. dead fetuses, resorptions (Early/Late)/litter) / No. implantation sites/litter * 100
- Viable fetuses affected/litter (%) = (No. viable fetused affected/litter) / No. viable fetuses/litter * 100

Historical control data:

WIL Developmental historical control data [Crl:CD(SD)IGS BR Rats] is incuded in Appendix D of the study report. External, visceral and skeletal malformations and visceral and skeletal deviations were reported for 27 studies (635 fetuses)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MATERNAL CLINICAL OBSERVATIONS AND SURVIVAL
All animals survived to the scheduled necropsy. No test article-related findings were observed. Clinical findings, including hair loss or various body surfaces, yellow staining on the ventral abdomen and/or red material around the mouth or nose, were present prior to the initiation of dose administration, occurred infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
No test article-related effects on mean body weights, body weight gains, net body weight gains or gravid uterine weights were observed.

MATERNAL FOOD CONSUMPTION
Food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test article administration. The only statistically significant (p < 0.05 or p < 0.01) differences from the control group were slight decreases in the 300 mg/kg bw/day group (g/kg/day only) during gestation days 17-19. The g/animal/day value in the 1000 mg/kg bw/day group was similar to that in the control group; therefore, the transient reduction in food consumed relative to body weight was not considered to be adverse. A reduction in the g/animal/day value similar to that observed in the 300 mg/kg /day group during gestation days 17-18 was not observed in the 1000 mg/kg bw/day group. Therefore, this transient decrease was not attributed to the test article.

MATERNAL NECROPSY DATA
There were no test article-related internal findings at the scheduled necropsy on GD20. One female in the 100 mg/kg bw/day group had cyst in the renal cortex and medulla (bilateral) and a rough surface of the liver. Another female in this same group had calculi in the urinary bladder, distended ureters and dilated renal pelves. In the 1000 mg/kg bw/day group, one female had dark red areas in the lungs. No other internal findings were observed.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Maternal abnormalities

Abnormalities:

not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: statistically significant reduced mean fetal body weight

Details on embryotoxic / teratogenic effects:
GD20 LAPAROHYSTERECTOMY DATA
Mean fetal body weights (male, female and combined) in the 1000 mg/kg bw/day group (3.5, 3.3 and 3.4 g, respectively) were reduced (statistically significant at p <0.05 or p < 0.01) compared to the control group value (3.7, 3.5 and 3.6 g, respectively). The values in the 1000 mg/kg bw/day group were equal to the minimum values in the WIL historical control data. The reduction in mean fetal body weight was attributed to the test article. Other intrauterine parameters, including mean viable litter size, fetal sex ratios and mean litter proportions of pre- and postimplantation loss, in this group were unaffected by test article administration.
Intrauterine growth and survival in the 100 and 300 mg/kg bw/day groups were unaffected by test article administration. The mean numbers of corpora lutea and implantation sites were similar in all groups.

FETAL MORPHOLOGICAL DATA
The number of fetuses (litters) available for morphological evaluation were 364(24), 376(24), 365(23) and 393(25) in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Malformations (visceral and skeletal) were observed in 3(3) fetuses (litters) in the 300 mg/kg bw/day group and not treatment relationship as described below.

EXTERNAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a meningocele (a portion of the meninges protruded through an opening of the cranium). No other external malformations were observed.
No external developmental variations were observed.

VISCERAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a malpositioned descending aorta (the aorta coursed laterally to the left adrenal gland, between the adrenal and the left kidney, and resumed the normal position prior to the branching of the iliac artery). No other soft tissue malformations were observed.
Soft tissue developmental variations in the test article-treated groups included distended ureters, major blood vessel variations (the right carotid and right subclavian arteries arose independently from the aortic arch with no brachiocephalic trunk or a retroesophageal right subclavian artery, which joined the aortic arch adjacent to the right carotid artery, with no brachiocephalic trunk), an accessory spleen and a small spleen. These findings were observed in single fetuses or did not occur in a dose-related manner. No relationship to treatment was evident.

SKELETAL MALFORMATIONS AND VARIATIONS
One fetus in the 300 mg/kg bw/day group had a vertebral centra anormality (the right half of lumbar centrum no. 2 was absent, and the right half of lumbar centrum no. 1 was malpositioned). No other skeletal malformations were observed.
Skeletal developmental variations occurred in all dose groups, including the control group, and consisted primarily of unossified sternebra(e) nos. 5 and/or 6, 14th rudimentary ribs, 7th cervical ribs, cervical centrum no. 1 ossified and unossified hyoid. A statistically significant (p < 0.05) increase in the mean litter proportion (% per litter) of 7th cervical ribs was observed in the 100 mg/kg bw/day group compared to the control group value. Similar increases were not observed in the 300 and 1000 mg/kg bw/day groups; therefore this increase was not attributed to test article. Other skeletal variants observed on the test article-treated groups occurred infrequently, were within the range of the WIL historical control data or did not occur in a dose-related manner. No relationship to treatment was evident.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL for maternal toxicity was 1000 mg /kg bw/d. Due to slightly reduced foetal mean body weight in the high dose group compared to control, the NOAEL for developmental toxicity was 1000 mg/kg bw/day.

Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, γ-Caprolactone diluted in deionized water was administered to 25 females Crl:CD®(SD)IGS BR rats/dose by gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. There were no test article-related maternal clinical observations or effects on mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights or food consumption at any dose level. There were no test article-related internal findings at the scheduled necropsy. The mean fetal sex ratio and mean numbers and/or litter proportions of viable fetuses and pre- and postimplantation losses in the 1000 mg/kg bw/day group were not affected by test article administration. Intrauterine growth and survival were unaffected in the 100 and 300 mg/kg bw/day groups. No test article-related fetal malformations or developmental variations were noted at any dose level in this study.

Test article-related effects noted in the 1000 mg/kg bw/day group consisted of statistically significant reduced mean fetal body weight (5.6 % lower than the control group value for combined fetal weight). This slight decrease was not considered of toxicological concern. No other indicators of developmental toxicity were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered to be the NOAEL for maternal toxicity and the dose level of 1000 mg/kg bw/day was considered to the NOAEL for developmental toxicity.

Under the test conditions, γ-Caprolactone is not classified according to Regulation (EC) No.1272/2008 (CLP) criteria.

This study is acceptable and satisfies the requirement for developmental toxicity endpoint.