2-Oxetanone, 3-C12-16-alkyl-4-C13-17-alkylidene derivs., (4E)-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

849705-80-2

Cas Number:

849705-80-2

IUPAC Name:

849705-80-2

Constituent 2

Reference substance name:

-

EC Number:

434-710-0

EC Name:

-

IUPAC Name:

434-710-0

Constituent 3

Reference substance name:

Reaction product containing 2-Oxetanone, 3-(C14-16 and C16-unsatd. branched and linear alkyl) 4-(C15-17 and C17-unsatd. branched and linear alkylidene) derivs.

IUPAC Name:

Reaction product containing 2-Oxetanone, 3-(C14-16 and C16-unsatd. branched and linear alkyl) 4-(C15-17 and C17-unsatd. branched and linear alkylidene) derivs.

Details on test material:

READ ACCROS
- Name of test material (as cited in study report): P-2290
- Physical state: liquid, light brown
- Analytical purity: 86.8%
- Lot/batch No.: Bu 0278
- Expiration date of the lot/batch: 10-Dec-2007
- Stability under test conditions: the test item was shown to be stable in the vehicle for at least 15 days placed in a refrigerator (2 - 8°C)
- Storage condition of test material: at room temperature (17 - 23°C)

Test animals

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH, Kisslegg, Germany
- Age at study initiation: at least 16 weeks
- Weight at study initiation: 2239 - 3006 g
- Housing: individually in stainless steel cages
- Diet: Pelleted standard Kliba-Nafag 3418 rabbit maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland) ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days (minimum)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Frequency of dose formulation at least every eight days
- Storage of dose formulations in a refrigerator (2 - 8°C)
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using an appropriate homogenizer a homogeneous suspension was prepared. During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneinty and stability (10 days) of dose formulations were taken from the first dose formulation. Additionally, samples for determination of concentration and homogeneity were taken from the last dose formulation. On each occasion three samples of approx. 2 g were taken before dosing from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. For determination of stability, samples were taken before dosing from the middle of the container and stored in a refrigerator (2 - 8°C) for 10 days. The samples were then frozen (- 25 to - 15°C) pending analysis. Analysis w as performed using a HPLC.

Details on mating procedure:

After acclimatization, females were placed in cages with sexually mature males (1:1) until copulation had been observed. After mating, females were removed and caged individually. The day of mating was designated day 0 post coitum.

Duration of treatment / exposure:

from day 6 post coitum(first treatment) through to day 27 post coitum (last treatment)

Frequency of treatment:

once daily

Duration of test:

until sacrifice on day 28 post coitum

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

20 mated females

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 28 post coitum


FOOD CONSUMPTION: Yes
- Food consumption was recorded for the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, and 24 - 28 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 28
- Organs examined: gross macroscopic examination of all internal organs

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: position of fetuses in the uterus

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- Head examinations: Yes, half per litter

Statistics:

Means and standard deviations of various data were calculated. lf the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons. The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
MORTALITIES AND/OR SIGNS OF REACTION TO TREATMENT: All females survived until scheduled sacrifice and no signs of discomfort or clinical signs of reaction to treatment with the test item w ere observed throughout the study. In group 2 (100 mg/kg/day), one female hurt its right eye at mating which healed by day 21 post coitum. In group 3 (300 mg/kg/day), one female was found without hair on the breast region between days 7 and 24 post coitum.

FOOD CONSUMPTION: With begin of treatment the mean food consumption was distinctly r duced in all groups. This reduction was considered to be caused by the vehicle corn oil. However, for the entire treatment period the calculation of the food-consumption showed, that the reduction of 56% in group 1 (vehicle control) was distinctly higher than that of 44% in group 2 (100 mg/kg/day), of 42% in group 3 (300 mg/kg/day) and of 14% in
group 4 (1000 mg/kg/day; percentage relative to the values of the period prior to treatment of the respective group). The differences were more distinct if the calculations were performed relative to the values of group 1. In this case the differences showed an increased food consumption of 15% in group 2, of 20% in group 3 and of 94% in group4 vs that in group 1. These effects may be caused by a better digestibility of the dosing mixtures thanof corn oil alone.

BODY WEIGHTS: During the treatment period body weight gains of 2.5% in group 1, of 4.7% in group 2, of 4.7% in group 3, and of 6.5% in group 4 were noted. The calculations of the mean corrected body weight gain (corrected for the weight of the gravid uterus) resulted in a reduction of 7.5% in group 1, of 6.1% in group 2, of 5.9% in group 3 and of 4.1% in group 4. These results correlated with the food consumption data and confirmed the possibly better digestibility of the dosing mixtures.

REPRODUCTION DATA: The relevant reproductive parameters (post-implantation loss and number of fetuses per dam) were similar in all groups and gave no indication of a test item-related effect. One damn in group 3 and one damn in group 4 were excluded from the calcutation of the reproduction data because the uterus contained one resorption, respectively, at Caesarean section. Total resorption is a common finding in rabbits when they are pregnant with a very small number of implantations (1 or 2). Therefore this finding was considered to be incidental. In group 4, the pre-implantation loss was statistically significantly increased and caused a reduced number of implantations (also statistically significant in % of corpora lutea). Because implantation occurred around day 6 post coitum(first day of treatment) this pre-implantation loss was considered to be incidental.

NECROPSY FINDINGS: During macroscopic examination no findings that were considered to be test item related were noted. Macroscopic findings were confined to six females in group 1, two females in group 2, three females in group 3, and three females in group 4. The findings noted were common findings such as raised crateriform areas at the pylorus, in testine filled with fluid, intestinal closure in the duodenum, discoloured, dark-reddish lungs, enlarged bile bladder, watery cyst at the liver edge, connatal abnormalities ( isconnected left uterine horn), ovarian cysts, and uterus containing red fluid.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
BODY WEIGHTS: The mean fetal bodyweight of group 4 was statistically significantly increased (+ 11.8% vs group 1, calculated on individual basis). This finding correlated with the higher food consumption in dose group 4. Mean fetal bodyweights of the groups 2 and 3 were similar to that of the control group.

SEX RATIOS: There was no test item-related effect on the sex ratio of fetuses.

EXTERNAL AND FRESH VISCERAL EXAMINATION OF FETUSES: External and fresh visceral examination of fetuses did not reveal any test item-related effects. Types and incidences of common abnormal findings (such as clear gall bladder content, missing or smaller gall bladders, pale colouration of liver and spleen, discoloured thymi with reddish areas, elongated thymi, tracheal cartilages disorganised, additional arteries arising from aortic arch or innominate artery or accessory lung lobe absent, and renal veins branched or duplicated) in groups 2 - 4 were similar to those noted in the vehicle control. Unilateral or bilateral internal eye opacity was found in some fetuses of groups 1 and 3. These alterations were considered to be the consequence of the time differences between the time of killing and of examination. The occurrence of a fetus with multiple findings in each dose group but not the control was considered incidental.

EXAMINATION OF SECTIONED FETAL HEADS (INCLUDING BRAINS): No test item-related effects were noted during head sectioning after a Bouin's fixation. Slightly enlarged second and third ventricles were seen in fetuses of all groups without any dose relationship. Findings such as peri-meningeal space in the brain, and space between thalamus/midbrain and cerebral hemispheres were assumed fixation artefacts.

SKELETAL EXAMINATION OF FETUSES - ABNORMAL FINDINGS: During skeletal examination of the fetuses, abnormal findings were noted in 11 of 118 fetuses (in 9 of 19 litters) of group 1, 11 of 117 fetuses (in 7 of 18 litters) of group 2, 14 of 115 fetuses (in 9 of 18 litters) of group 3 and 14 of 98 fetuses (in 6 of 17 litters) of group 4. The findings noted mainly comprised common findings such as forelimb flexures, fused and incompletely ossified sternebrae, displaced pelvic girdles, sagittal sutures with suture bones, fused parietals with interparietals, parietals with fontanel bones at margin, anterior fontanels enlarged, frontals and parietals with unossified areas, and shortened ribs. Neither type nor frequency of these abnormal findings was considered to be test item related.

SKELETAL EXAMINATION OF FETUSES - STAGE OF DEVELOPMENT AND COMMON VARIANTS: On a litter basis, the fetuses of groups 3 and 4, respectively, revealed less incompletely ossified medial phalanges on digit 5 on both forelimbs (statistically significant). On a fetus basis, the fetuses of groups 2, 3 and 4 revealed less incompletely ossified medial phalanges on digit 5 on both forelimbs. Additionally, the metacarpalia 1 of the right forelimb was less unossified in group 2. Additional ossification was also noted in the humeri of groups 3 and 4, and the tibiae of group 3 (statistically significant). Still, these occasional statistically significant differences showed no dose dependency and were within the range of historical control data for Himalayan rabbits, and therefore not considered to be test item-related.

CARTILAGE EXAMINATION OF FETUSES - ABNORMAL FINDINGS: During cartilage examination of the fetuses, abnormal findings were noted in 1 of 118 fetuses (in 1 of 19 litters) of group 1, 0 of 117 fetuses (in 0 of 18 litters) of group 2, 1 of 115 fetuses (in 1 of 18 litters) of group 3, and 1 of 98 fetuses (in 1 of 17 litters) of group 4. The findings noted mainly comprised common findings and were considered incidental.

CARTILAGE EXAMINATION OF FETUSES - COMMON VARIANTS: On a litter basis, no finding was noted statistically significant vs control. On a fetus basis, both costal cartilages 10 were considered long in groups 3 and 4, respectively. The left costal cartilage 7 did not reach the sternum in group 2 (statistically significant). Still, these occasional statistically significant differences revealed no dose dependency and were within the range of historical control data, and therefore not considered test item related.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal and fetal organisms was considered to be 1000 mg/kg bodyweight/day. The test substance did not reveal any teratogenic potential up to and including a dosage of 1000 mg/kg bodyweight/day.

Executive summary:

The study was performed according to OECD TG 414 in compliance with GLP.

In order to detect effects on embryonic and fetal development in mated Himalayan female rabbits, the test substance was administered orally, by gavage, once daily from day 6 through to day 27 post coitum at dose levels of 100, 300 and 1000 mg/kg bodyweight/day. Each group consisted of 20 mated female rabbits. A standard dose volume of 2 mL/kg bodyweight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). 

The test item was well tolerated in the animals of all groups. No animal died during the course of study. The mean food consumption during the treatment period was dose-dependent higher in the dose groups (15% at 100 mg/kg, 19.6% at 300 mg/kg and 93.5% at 1000 mg/kg) vs. controls. The body weight development correlated with the food consumption and showed increased body weight gains of 4.7% at 100 and 300 mg/kg and 6.5% at 1000 mg/kg vs. 2.5% in controls. The calculations of the corrected body weight gain (corrected for the weight of the gravid uterus) resulted in lower body weight losses in the dose groups than in the control group (6.1% at 100 mg/kg, 5.9% at 300 mg/kg, 4.1% at 1000 mg/kg vs. 7.5% in controls). During the macroscopic examination, none of the observed findings in any dam was considered test item-related. The relevant reproductive parameters (incidence of post-implantation loss and number of foetuses per dam) were similar in all groups and gave no indication of a test item-related effect.

On an individual basis, the mean fetal body weights of the high dose group were increased when compared with the controls. This finding fits to the fact that the dams of group 4 also revealed a higher body weight when compared with the controls. The mean fetal body weights of groups 1, 2 and 3 were similar. There was no test item-related effect on the sex ratio of foetuses. External and fresh visceral examination of foetuses did not reveal any test item-related effects. The found abnormalities, including head sections after Bouin's fixation, were considered incidental and within the range of biological variation. No test item-related effects were noted during skeletal examination of foetuses.

Abnormal findings were spread evenly in all groups without any dose dependency. Within the stage of development and common variants, the phalanges on digit 5, the humeri and the tibiae were more ossified in groups 3 and 4 but still within the range of historical control data. No test item-related findings were noted during cartilage examination of foetuses. One abnormality was observed in groups 1, 3, and 4, respectively. The common variants were within the range of biological variation (historical data).

 

Conclusion: The NOAEL for maternal and fetal organisms was considered to be 1000 mg/kg bodyweight/day. The test substance did not reveal any teratogenic potential up to and including a dosage of 1000 mg/kg bodyweight/day.