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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997
Reference Type:
publication
Title:
Developmental Toxicity Studies of Four Fragrances in Rats.
Author:
Christian, M.S., Parker, R.M., Hoberman, A.M., Diener, R.M.|and Api, A.M.
Year:
1999
Bibliographic source:
Toxicology Letters, 111: 169-174.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran
EC Number:
214-946-9
EC Name:
1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran
Cas Number:
1222-05-5
Molecular formula:
C18H26O
IUPAC Name:
4,6,6,7,8,8-hexamethyl-1H,3H,4H,6H,7H,8H-indeno[5,6-c]pyran
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage, Michigan
- Age at study initiation: approximately 66 days at arrival
- Weight at study initiation: 208 to 256 gr
- Fasting period before study: no
- Housing: individually, except during the cohabitation period. During cohabitation each pair of male and female rats was housed in the male rats's cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 26 (+/- 2 %)
- Humidity (%): 40% to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Amount of vehicle (if gavage): dosage volume was 5 mL/kg bw
- Lot/batch no. (if required): 827AG
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A procedure has been developed for analysis of test material in corn oil formulations at high and low range. The method uses HPLC and UV. Formulations used for dosing were analyzed for concentration verification and stability determination.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 7 -17 of gestation
Frequency of treatment:
once daily
Duration of test:
approximately 4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a dosage-range study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
for viability: twice during acclimation and on Day of Gestation (DG) 0.
clinical observations: after dosage (DGs 7 through 17)

BODY WEIGHT: Yes
- Time schedule for examinations:
twice during acclimation
On DG 0
Daily during dosage and postdosage periods

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus

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
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution
Continuous data (e.g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent
resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site 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>0.05)]. 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≤0.05)], 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 ind vidual 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 for the Kruskal-Wallis Test
Historical control data:
Historical control data were included in the report. These include reproductive indices, maternal necropsy observations, fetal external alterations, fetal soft tissue alterations, fetal skeletal alterations, fetal ossification sites. Period: June 1994 - June 1996

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The 500 mg/kg bw/day dosage group had four to nine (p -<0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dams on 500 and 150 mg/kg bw/day showed statistically significant dosage-dependent reductions in maternal body weight gains for the entire dosage period (days 7 to 18 gestation), to 78% and 91% of body weight gain in controls, respectively. These reductions in weight gain reflected significant weight loss (-10.9 g compared to +10.5 g in the control group) on days 7 to 10 at 500 mg/kg bw per day, while on days 10 to 12 the weight gain was increased (+17.9 g compared to +11.8 g in the control group)At 150 mg/kg bw/day, a significantly reduced weight gain (+5.5 g) was also found on days 7 to 10. For the remainder of the study, body weight gains at 150 and 500 mg/kg bw per day were comparable to the control group values . Body weights were significantly reduced compared to controls on GD20 by 2.5, 3,3 and 4.6% for 50, 150 and 500 mg/kg bw/day, respectively.
Gross pathological findings:
effects observed, non-treatment-related

Maternal developmental toxicity

Number of abortions:
no effects observed
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:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative feed consumption values for the entire dosage period were reduced in the 150 and 500 mg/kg/day dosage groups, reflecting significant reductions in absolute feed consumption values on DGs 10 to 15 in the 150 mg/kg/day dosage group and in absolute and relative feed consumption values in the 500 mg/kg/day dosage group. After completion of the dosage period, the 500 mg/kg/day dosage group had a significantly increased relative feed consumption value on DGs 18 to 20, a rebound phenomenon commonly observed in these types of studies. These effects of the test article resulted in significant reductions in absolute and/or relative feed consumption values in the 150 and 500 mg/kg/day dosage groups for the entire interval after initiation of dosage (DGs 7 to 20) and in the 500 mg/kg/day dosage group for the entire gestation period (DGs O to 20). Absolute and relative feed consumption values were unaffected by the 50 mg/kg/day dosage of the test article.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The 500 mg/kg bw group had significantly reduced fetal bodyweights compared to control group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Significant increases in foetal incidences of skeleton (vertebral/rib) malformations were found (2.0 compared to 0 in the control group). In addition, significant increases in foetal incidences of incomplete ossification and/or no ossification of sternal centra and a significantly decreased number of ossification sites in the metatarsals were seen at 500 mg/kg bw/day (5.4 compared to 0.5 in the control group). For litters, these incidences were also increased, 14.3 compared to 0 in the control group for the skeleton malformations, and 23.8 compared to 4.0 in the control group for the ossification problems.
Details on embryotoxic / teratogenic effects:
No other Caesarean-sectioning and litter parameters were affected by administration of HHCB to the dams at doses as high as 500 mg/kg bw/day. The litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
other: decreased ossification of sternal centra and metatarsals

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
yes

Any other information on results incl. tables

The effects were decreased mean bodyweights in mid and high-dose dams and axial skeleton (vertebral/rib malformations in high dose offspring.  


 




































































































































































































































































































































































































































Table 1 Maternal body weights            
DOSAGE GROUP IIIIIIIV
DOSAGE (MG/KG/DAY)a 0 (vehicle)50150500
RATS TESTEDN25252525
PREGNANTN25242421
MATERNAL BODY WEIGHT (G)                
DAY 0MEAN±S.D.241,5±8,4232,9±9,60**232,2±9,5**233,6±9,1**
DAY 7MEAN±S.D.275,2±9,5266,2±10,40 269,2±12,90 270,2±14,1 
DAY 8MEAN±S.D.276,3±10,9263,8±10,9**266±13,3**259,8±14,1**
DAY 9MEAN±S.D.280,1±10,2269,2±11,20**269,2±12,4**254,7±17,4**
DAY 10MEAN±S.D.285,7±10,1275,1±12,10 214,8±13,20 259,3±18,5 
DAY 11MEAN±S.D.291,6±10,5281,8±12,90*282,1±14,30*269,4±18,3**
DAY 12MEAN±S.D.297,8±11,3286,9±12,90*286,5±15,40**277,2±16,1**
DAY 13MEAN±S.D.301,3±10,7291,6±12,30*290,8±14,90**202,7±14,2**
DAY 14MEAN±S.D.304,2±11,4300,8±14,90 295,4±15,20*288,9±14,7**
DAY 15MEAN±S.D.313,7±12,5307,3±12,70 303,8±17,10*295,7±14,8**
DAY 16MEAN±S.D.326,6±14,6316,5±15,40*313,6±18,70**306,8±16**
DAY 17MEAN±S.D.340,5±16,8330±16,30*326,5±19,80**321,3±16**
DAY 18MEAN±S.D.355±18,9347,7±16,70 341,6±21,30*333,2±17,5**
DAY 19MEAN±S.D.372,8±21,4364±17,40 357,4±23,70*349,5±17,3**
DAY 20MEAN±S.D.391,9±21,6362,2±19,20**379,1±27,20*373,7±21,2**
DAY - DAY OF GESTATION                
a. Dosage occurred on days 7 through 17 of gestation            
* Significantly different from the vehicle control group value (p ≤ 0,05)            
**  Significantly different from the vehicle control group value (p ≤ 0,01)            

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test (OECD TG 414, GLP), the maternal NOAEL was determined to be >=50 mg/kg bw/day based on body weight effects. The developmental NOAEL is >= 150 mg/kg bw/day based on fetal body weight changes.
Executive summary:

In a developmental toxicity study (OECD TG 414, GLP) the test substance in corn oil was administered by gavage to groups of 25 female Sprague-Dawley rats on days 7 through 17 of presumed gestation at dosages of 0, 50, 150 and 500 mg/kg bw/day. The dams were observed for signs of toxicity and body weights and feed intake were recorded. On day 20 of gestation, the dams were sacrificed, and gross necropsy was performed. The number of corpora lutea in the ovaries was recorded and the uteri were examined for pregnancy, number and distribution of implantations, live and dead foetuses and early and late resorptions and the placenta were examined. All foetuses were weighed and examined for sex and gross external abnormalities. One half of the foetuses in each litter were examined for soft tissue alterations. The remaining foetuses were examined for skeletal alterations.


Results: Clinical signs: No mortality was noted. The 500 mg/kg bw/day dosage group had four to nine (p ≤ 0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals). Body weights: Maternal body weights were unaffected by the 50 mg/kg bw/day dosage group. The 150 and 500 mg/kg bw dosage groups had statistically significant dose-dependent reductions in maternal body weight gains for the entire dosage period (day 7 to 18). For the entire interval after initiation of treatment, (day 7 to 20) maternal body weights were reduced and significantly reduced in the 150 and 500 mg.kg bw/day dosage groups, respectively. Food consumption: Absolute and relative feed consumption values were unaffected by the 50 mg/kg bw dosage group. Absolute and relative feed consumption values for the entire dosage period were reduced in the the 150 and 500 mg/kg bw dosage group after initiation of dosage and in the highest group for the entire gestation period. Fertility: Pregnancy occurred in 21 to 25 female rats in each dosage group. There were 25, 24,24 and 21 pregnant dams Caesarean-sectioned on DG 20 in the 0 (vehicle). 50, 150 and 500 mg/kg bw day dosage group, respectively. No abortions or premature deliveries occurred during the study. Developmental: Foetuses in the 500 mg/kg bw/day dosage group showed significantly reduced body weights, considered test item related. Significant increases in the litter and foetal incidences of skeleton (vertebral/rib) malformations were found in the 500 mg/kg bw and considered to be associated with administration of this dosage of the test article to the dams. Malformations occurring in foetuses of dams administered dosages less than 500 mg/kg bw day were considered unrelated to the test article because the severity of the expression of the malformation was not consistently dose dependent. There were no dosage-dependent, or statistically significant differences in the litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.


Conclusion: Based on a reduction in adverse clinical observations (500 mg/kg bw/day group only), maternal body weight gains and feed consumption values for the dosing period (days 7 to 18 of gestation), the systemic NOAEL was determined to be 50 mg/kg bw/day.


Based on a reduction in foetal body weight, increased incidences of foetal-skeletal (vertebral/rib) malformations, and decreased ossification of sternal centra and metatarsals seen at 500 mg/kg bw/day, the developmental NOAEL was determined to be 150 mg/kg bw/day.


The test substance was not selectively toxic to development because adverse effects on development occurred only at dosages that produced toxic effects (adverse clinical observations, decreased body weight and feed consumption values) in the dams.  

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