Dodecanoic acid, 2,2-dimethyl-3-oxopropyl ester

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2015 and 23 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

468-880-2

EC Name:

-

Cas Number:

102985-93-3

Molecular formula:

C17H32O3

IUPAC Name:

2,2-dimethyl-3-oxopropyl dodecanoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.,1103 Budapest Cserkesz u. 90., Hungary
- Age at study initiation: females (10 weeks of age at start of the mating period), males (25-26 weeks of age at start of the mating period)
- Weight at study initiation: 167-224 g
- Housing: before mating: 1-3 females per cage, 1-2 males per cage; mating: 1 male and 1-3 females/cage; during gestation: 2-3 sperm positive females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days for females, 119 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations will be prepared in the laboratory daily to every three days.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore Sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle: 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study. Concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 103 % of nominal concentrations at both analytical occasions confirming proper dosing.
Formulation samples are diluted with hexane and then analysed by a GC-FID method in the presence of heptadecane internal standard. Five samples were taken from the formulations. Two replicate dilutions were prepared from each sample. One dilution was carried out from the control samples. The samples were diluted with hexane in two consecutive steps. A volume of 0.5 mL of the internal standard solution was added in the second step before the samples were filled up to 10 mL final volume.
Gas chromatograph: Clarus 580 GC, No: 580S11081708
Balance: AB54-S, Mettler Toledo, No.: 1122092721

GC conditions:
Column: Elite-5MS, 30 m x 0.32 mm x 0.25 μm, No: 926895
Temperature program: 1 min hold, 165°C to 200°C at 10°C/min, 1 min hold
Injector: 250°C
Detector: FID, 250°C
Carrier gas: nitrogen, 2 mL/min, constant flow
Split ratio: 20
Injection volume: 1 μL
Retention times: Heptadecane 2.5 min, test item: 4.3 min
Evaluation: Ratio of the peak areas is calculated and used for the computations

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:3
- Length of cohabitation: The females were paired to males in the mornings for two to four hours until the number of sperm positive females per group achieves at least twenty four.
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

7 days per week every day at similar time

Duration of test:

Treatment period: from gestational day 5 to 19

No. of animals per sex per dose:

24 sperm positive females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on findings obtained in a previous GLP Reproduction/Developmental Toxicity Screening Test with the test item in the rat (OECD 421, Toxi-Coop Zrt. 66.421.2959)

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (observations for signs of morbidity and mortality were made twice daily, at the beginning and end of the working period)
- Examinations: signs of morbidity, mortality, toxicity as well as behaviour and general conditions

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20. The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule: between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and ovary

OTHER:
- Examination of placental signs: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

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 live fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Historical control data:

The results were compared to the laboratory's historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. No treatment related reduction on food consumption or body weight was observed.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations. There were two fetuses with different types of malformations during external examination.
Cleft palate observed in one fetus at 1000 mg/kg bw/day and umbilical hernia in another single fetus at 100 mg/kg bw/day were judged to be without a relationship to the treatment with the test item considering the historical control database and the experience with this strain at the lab as well as in the international scientific literature. During visceral examination malformations were found only in the control group. The malformations found sporadically in single fetuses during skeletal examination were judged to be unrelated from the treatment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Fetal abnormalities

Key result

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 1000 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 24 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room for 7 days at the concentrations of 50 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 94 and 103 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

In total, on gestation day 20 there were 22 evaluated litters each in the control, 100 and 1000 mg/kg bw/day and 20 in the 300 mg/kg bw/day group.

None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed.

There was no treatment related reduction on food consumption and body weight indicated.

The test item did not influence the number of implantations, intrauterine mortality and sex distribution of the fetuses. With regard to fetal examinations, there were no test item related differences in the fetal- and placental weight, body weight retardation and other external, visceral and skeletal variations.

There were two fetuses with different types of malformations during external examination.

Cleft palate observed in one fetus at 1000 mg/kg bw/day and umbilical hernia in another single fetus at 100 mg/kg bw/day were judged to be without a relationship to the treatment with the test item considering the historical control database and the experience with this strain at the lab as well as in the international scientific literature.

During visceral examination malformations were found only in the control group.

The malformations found sporadically in single fetuses during skeletal examination were judged to be unrelated from the treatment.

 

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day