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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: US FDA; This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate
EC Number:
915-335-6
Molecular formula:
C11H20O2 C10H20O
IUPAC Name:
Reaction mass of 2,6-dimethyloct-7-en-2-ol and 2,6-dimethyloct-7-en-2-yl formate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Cr1:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at arrival: 62 days (approximate)
- Weight at study initiation: 218 to 244 g
- Fasting period before study: None
- Housing: Individually in stainless steel, wire-bottomed cages (except during cohabitation)
- Diet: Ad libitum. Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO)
- Water: Ad libitum. Local water processed by reverse osmosis. Chlorine added as bacteriostat
- Acclimation period: Yes, but duration not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-26.1 (targeted)
- Humidity (%): 30 to 70 (targeted)
- Air changes (per hr): 10 (minimum)
- Photoperiod (hrs dark / hrs light): 12/12

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. Prepared formulations were stored at room temperature, protected from light.

VEHICLE
- Concentration in vehicle: The dosage preparations are shown in Table 1.
- Amount of vehicle: Constant dosage volume of 10 mL/kg
Details on analytical verification of doses or concentrations:
Samples were analyzed according to the method described in Charles River Laboratories Preclinical Services. Results of concentration and homogeneity analyses were within +/- 15% of calculated concentrations and
Details on mating procedure:
After a suitable acclimation, 130 virgin female rats were placed into cohabitation with 130 breeder male rats, one male rat per female rat. The cohabitation period was a maximum of five days. Gestation day 0 was considered the first day when spermatozoa were detected in a vaginal smear or a copulatory plug was observed. Pregnant rats were assigned to individual housing.
Duration of treatment / exposure:
Rats were treated on gestation days 7 through 17. Dosages were adjusted daily based on the individual body weights recorded prior to dosage administration.
Frequency of treatment:
Once daily
Duration of test:
Until gestation day 21
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
No. of animals per sex per dose:
25 female/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of a range-finding study with the same test article at a dosage level of 1000 mg/kg/day. This was selected as the highest dose in the current study.
- Rationale for route of administration: Oral (gavage) was selected for use as comparison with the dietary route; the exact dosage can be accurately determined.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (rats were observed for viability at least twice daily each day of the study and weekly for clinical observations and general appearance during the acclimation period and on DG 0)
- Cage side observations included: Clinical observations, abortions, premature deliveries and deaths before and approximately 3 hours after dosing, and once daily during the post-dosage period.

DETAILED CLINICAL OBSERVATIONS: No data

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Thoracic, abdominal and pelvic viscera. Gross lessions were retained in neutral-buffered 10% formalin for possible future evaluation. Unless specifically cited, all other tissues were discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- 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. Number and distribution of live and dead fetuses were recorded.
Fetal examinations:
- External examinations: Yes (all per litter)
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
- Head examinations: No data
- Anogenital distance of all live rodent pups: Not specified
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 obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Historical control data:
Historical control data was available from this laboratory for the period from January 2005 to January 2007.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minor incidences of sparse hair coat or localized alopecia, excess salivation, urine-stained abdominal fur, ungroomed coat, rales, ptosis, chromodacryorrhea (colored tears), and soft or liquid feces. These observations were all considered unrelated to the treatment because the incidences were observed in vehicle control rats, were not dosage dependent, or were transient and did not persist.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Body weight gains for the entire dosage period were reduced by 5% when compared with controls, an observation that was not statistically significant but was considered evidence of a threshold for maternal toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Absolute (g/d) and relative (g/kg/d) feed consumption values were significantly reduced for the entire dosage period.



Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions were noted at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Body weights for combined male and female fetuses were reduced approximately 3%. The reduction in female fetuses was statistically significant (P ≤ .05).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Significant increases in fetal variations attributable to the test article were observed only at the 1000 mg/kg bw/day dosage level. These changes consisted of reversible minor variations, including a threshold but statistically significant increase in supernumerary ribs, along with associated significant increases and decreases in the respective numbers of thoracic and lumbar vertebrae, and a small but statistically significant retardation in ossification of the metatarsal bones in the hindpaws, evident as a reduction in the mean number of ossified metatarsal bones.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
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:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: MATERNAL BODY WEIGHT GAINS - SUMMARY






















































































































































DOSE GROUP



 



I



II



III



IV



DOSAGE (mg/kg bwt/day)



 



0 (control)



250



500



1000



RATS TESTED



N



25



25



25



25



PREGNANT



N



22



24



24



25



MATERNAL BODY



 



 



 



 



 



WEIGHT CHANGE (G)



 



 



 



 



 



DAYS 0 - 7



MEAN±S.D.



+42.5 ± 7.2



+42.0 ± 8.2



+40.7 ± 10.6



+39.8 ± 7.5



DAYS 7 - 8



MEAN±S.D.



-2.6 ± 4.6



-0.4 ± 5.2



-1.2 ± 5.5



-4.1 ± 6.8



DAYS 8 - 9



MEAN±S.D.



+1.4 ± 4.3



+2.0 ± 5.6



+1.5 ± 4.0



-1.1 ± 6.2



DAYS 9 - 10



MEAN±S.D.



+3.2 ± 4.6



+4.0 ± 4.8



+6.1 ± 4.2



+5.4 ± 5.7



DAYS 7 - 10



MEAN±S.D.



+2.0 ± 6.5



+5.5 ± 7.0



+6.4 ± 8.6



+0.1 ± 9.4



DAYS 10 - 12



MEAN±S.D.



+12.0 ± 5.3



+14.8 ± 5.4



+12.9 ± 5.2



+11.8 ± 9.6



DAYS 12 - 15



MEAN±S.D.



+17.7 ± 8.5



+17.1 ± 9.5



+19.8 ± 5.8



+18.4 ± 7.3



DAYS 15 - 18



MEAN±S.D.



+41.2 ± 9.6



+39.4 ± 8.8



+41.2 ± 7.3



+39.0 ± 7.7



DAYS 7 - 18



MEAN±S.D.



+73.0 ± 17.0



+76.8 ± 15.2



+80.2 ± 14.5



+69.3 ± 15.1



DAYS 18 - 21



MEAN±S.D.



+59.4 ± 11.4



+54.2 ± 8.7



+61.0 ± 10.8



+58.1 ± 12.8



DAYS 7 - 21



MEAN±S.D.



+132.4 ± 22.6



+130.9 ± 16.7



+141.2 ± 20.5



+127.4 ± 20.5



DAYS 0 - 21



MEAN±S.D.



+174.9 ± 25.6



+173.0 ± 21.0



+181.9 ± 27.2



+167.2 ± 23.6



 


 


Table 3: MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY





































































































































































DOSAGE GROUP



I



II



III



IV



 



DOSAGE (MG/KG/DAY)a



 



0 (VEHICLE)



250



500



1000



RATS TESTED



N



25



25



25



25



PREGNANT



N



22



24



24



25



MATERNAL FEED



 



 



 



 



 



CONSUMPTION (G/KG/DAY)



 



 



 



 



DAYS 0 - 7



MEAN±S.D.



96.2 ± 6.8



94.2 ± 6.9



93.5 ± 10.6



92.5 ± 6.5



 



 



 



[ 23]b



[ 23]b



 



DAYS 7 - 10



MEAN±S.D.



58.6 ± 10.2



60.8 ± 9.7



60.4 ± 12.7



46.2 ± 11.3**



 



 



 



 



[ 23]b



 



DAYS 10 - 12



MEAN±S.D.



61.4 ± 9.6



65.4 ± 8.8



60.0 ± 7.9



54.2 ± 15.5*



DAYS 12 - 15



MEAN±S.D.



59.8 ± 7.2



63.5 ± 9.7



59.6 ± 7.1



54.9 ± 9.3



 



 



 



[ 22]b



[ 23]b



 



DAYS 15 - 18



MEAN±S.D.



63.8 ± 14.0



68.7 ± 7.6



64.2 ± 10.0



60.8 ± 8.9



 



 



[ 21]b



 



 



 



DAYS 7 - 18



MEAN±S.D.



61.0 ± 5.6



64.5 ± 6.0



61.4 ± 7.3



54.1 ± 6.1**



 



 



[ 21]b



 



 



 



DAYS 18 - 21



MEAN±S.D.



73.1 ± 8.1



71.8 ± 8.7



74.6 ± 7.1



76.2 ± 8.1



DAYS 7 - 21



MEAN±S.D.



63.7 ± 4.8



66.4 ± 5.4



64.7 ± 6.0



59.6 ± 5.3*



DAYS 0 - 21



MEAN±S.D.



69.5 ± 4.0



70.5 ± 4.6



69.0 ± 5.3



66.0 ± 3.8*



[ ] = NUMBER OF VALUES AVERAGED



  1. Dosage occurred on days 7 through 17 of gestation.

  2. Excludes values that could not be calculated or appeared incorrectly recorded, as well as those associated with spillage.


* Significantly different from the vehicle control group value (p≤0.05). ** Significantly different from the vehicle control group value (p≤0.01).


 


 


Table 4: Dosage-Dependent Statistically Significant Fetal Observations (Average Ossification Sites per Fetus per Litter)


 




























































































































































































Observation



Dosage Group (DGs 7 through 17)



 



 



mg/kg/day



0 (Vehicle)



250



500



1000



Historical Controla



 



 



 



 



 



 



Litters



22



24



24



25



982



Fetuses



155



164



183



184



7305



Rats tested, n



25



25



25



25



 



Rats pregnant, n (%)



22 (88)



24 (96)



24 (96)



25 (100)



 



 



 



 



 



 



 



Corpora lutea, mean ± SD



14.6 ± 2.4



14.9 ± 2.8



16.3 ± 2.4



15.8 ± 2.3



 



Implantations, mean ± SD



14.0 ± 2.1



13.8 ± 2.8



15.2 ± 1.7



14.8 ± 1.8



 



Resorptions, mean ± SD



0.6 ± 0.7



0.8 ± 0.9



0.6 ± 0.6



0.5 ± 1.2



 



Litter size, mean ± SD



13.4 ± 2.2



13.0 ± 2.4



14.6 ± 2.0



14.3 ± 2.4



 



Fetal body weight/litter, mean ± SD



5.50 ± 0.28



5.54 ± 0.30



5.55 ± 0.31



5.33 ± 0.33



 



Male fetuses



5.63 ± 0.28



5.73 ± 0.31



5.68 ± 0.28



5.45 ± 0.36



 



Female fetuses



5.38 ± 0.29



5.37 ± 0.28



5.42 ± 0.35



5.18 ± 0.32*



 



Live fetuses/group



295



311



350



358



 



Dead fetuses/group



0



0



0



0



 



 



 



 



 



 



 



Ribs (pairs)b,c



13.06 ± 0.11



13.06 ± 0.10



13.09 ± 0.13



13.19 ± 0.23d



13.06 (13.02-13.12)



Thoracic Vertebraeb,c



13.08 ± 0.15



13.08 ± 0.16



13.12 ± 0.17



13.24 ± 0.28*,d



13.08 (13.02-13.16)



Lumbar Vertebraeb,c



5.92 ± 0.15



5.91 ± 0.16



5.87 ± 0.17



5.75 ± 0.27**,d



5.92 (5.83-5.97)



Metatarsalsb,c



4.95 ± 0.12



4.87 ± 0.21



4.91 ± 0.12



4.78 ± 0.27**,d



4.83 (4.68-4.95)



 



 



 



 



 



 




  1. Studies (N = 44) conducted at the Testing Facility between January 2005 and January 2007 in rats of the same strain.

  2. Means and Standard Deviations

  3. Mean and range of means per study

  4. Bold = effect of test article because values were significant and/or exceeded historical ranges. DGs = Days of Gestation


* = Significantly different from the vehicle control group value (p≤0.05). ** = Significantly different from the vehicle control group value (p≤0.01).


Testing facility historical control data for mean fetal body weight ranges (82 studies): combined male/female, 4.94 to 5.93 g; male fetuses, 5.10 to 5.98 g; female fetuses, 4.80 to 5.78 g.


 

Applicant's summary and conclusion

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. At this dose level, maternal body weight gains were reduced (not significantly) and feed consumption was significantly reduced. The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.

The developmental NOAEL was also 1000 mg/kg bw/day. This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.
Executive summary:

The test item was administered in corn oil by gavage to groups of pregnant Sprague-Dawley rats at dosages of 0, 250, 500 or 1000 mg/kg bw/day on gestational days 7 to 17. Viability, clinical signs, body weights and feed consumption were observed or measured for all animals. Animals were sacrificed on gestational day 21 and Caesarean sectioning performed. Fetuses were weighed and examined for sex, gross changes, and soft tissue or skeletal alterations. No clinical signs attributable to the test item administration were observed. At the 1000 mg/kg bw/day dose level, mean maternal body weight gains were reduced 5% versus controls. Absolute and relative feed consumption were also reduced at this dose level. The minimal maternal effects noted were associated with minimal (approx. 3%) reductions in fetal body weights, reversible variations in ossification and increases in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Findings of delayed skeletal ossification are generally considered readily reversible (Carney and Kimmel, 2007 "Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs", Birth Defects Research 80:473 -496). The minimal developmental delays (reduced pup weights, delayed ossificaiton, changes in metatarsals) are all expected to be readily reversible based on studies that have investigated the progression and outcome of similar effects. On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bw/day. At this dose level, maternal body weight gains were reduced (not significantly) and feed consumption was significantly reduced.  The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.The developmental NOAEL was also 1000 mg/kg bw/day.  This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bw/day dose level was assigned as the NOEL for developmental effects.