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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:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Read across data

Data source

Reference
Reference Type:
other: Secondary Literature
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one
EC Number:
201-224-3
EC Name:
(E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one
Cas Number:
79-77-6
Molecular formula:
C13H20O
IUPAC Name:
trans-beta ionone
Test material form:
liquid
Details on test material:
- Name of test material: β-Ionone
- Common Name: (E)-Beta-ionone
- IUPAC name: (E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Substance type: Organic
- Physical state: Colorless to Yellowish Liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Germany)
- Age at study initiation: about 70-84 days
- Weight at study initiation: 148.7 - 183.6 g

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Olive Oil
- Test substance preparation: At the beginning of the administration period and thereafter at intervals which took into account the analytical results of the stability verification. For the preparation of the solutions an appropriate amount of the test substance was weighed depending on the dose group, in a graduated beaker, topped up with olive oil, and subsequently thoroughly mixed using a magnetic stirrer.
- Concentration in vehicle: 500, 2000 and 8000 mg/100 ml
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): 10R0449/02050
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Check of stability and concentration control was performed by HPLC. Since the test substance was a true solutions, investigations concerning homogeneity were not necessary.
Details on mating procedure:
The animals were mated by the breeder ("time-mated") and supplied on day 0 post coitum (= detection of vaginal plug / sperm). The animals arrived on the same day (i.e. day 0 p.c.) at the experimental laboratory. The following day was designed "day 1" post coitum (p.c.). Animals were assigned to the test groups by taken random selection.
Duration of treatment / exposure:
Day 6 through Day 19 post coitum (p.c.)
Frequency of treatment:
Once Daily
Duration of test:
Up to GD 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control Group
Dose / conc.:
25 mg/kg bw/day
Remarks:
Low Dose Group
Dose / conc.:
100 mg/kg bw/day
Remarks:
Mid Dose Group
Dose / conc.:
400 mg/kg bw/day
Remarks:
High Dose Group
No. of animals per sex per dose:
25 animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
No Data Available

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No Data Available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No Data Available

BODY WEIGHT: Yes
- Time schedule for examinations: No Data Available

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day # 19
- Organs examined: All visceral and reproductive organs.

OTHER: No Data Available
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: Dead fetuses, Calculation of conception rate and pre- and post implantation losseswere carried out.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Statistical analyses were performed according to following
schedule:
- DUNNETT-test (two-sided): Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- FISHER'S EXACT test (one-sided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- WILCOXON-test (one-sided): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
- KRUSKAL-WALLIS-test (two-sided): Liver weights
Indices:
Implantation Index, Resorption Index, Viability Index.
Historical control data:
The historical control data used for interpretation of findings refer to the same test facility, the same rat strain and supplier of the animals and cover a period of about 24 months (June 2001 - June 2003, 15 studies).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All high dose and the majority (22 out of 25) of the mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. After cessation of treatment on day 19 p.c., salivation did not occur any longer.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the high dose dams was statistically significantly reduced (9% below the concurrent control value) at initiation of treatment (days 6 - 8 p.c.). On the following days of the treatment period, however, food consumption of the high dose rats reached or even exceeded control values. The food consumption of the females of low and mid dose dams was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reductions in food consumption at 400 mg/kg bw were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
21 high dose dams showed dark-yellow discolored urine on gestation days 12 - 20 p.c. which is probably related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air and does not represent a toxicologically relevant finding.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No indications for disturbances of the general behavior.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Uterus weight: The mean gravid uterus weights of the animals of all test groups were not influenced by the administration of the test substance.

Liver weight: Absolute and relative mean liver weights were statistically significantly increased at the mid and high dose groups and were about 9 or 29% (absolute) and 8 or 29% (relative) above control values. These weight increases, which are considered to be substance-induced, are indicative of hepatic changes primarily caused by microsomal enzyme induction. Absolute and relative liver weights of the low dose dams, however, were similar to the control values and did not show any toxicologically significant changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There occurred no substance-related observations at necropsy in any of the dams of all test groups. Only very few spontaneous findings were recorded for single low and mid dose rats (one hydrometra in low dose female which consequently did not become pregnant, hemorrhagic thymus in one mid dose female). No association to the test compound was assumed for these findings due to their scattered occurrence without any relation to dosing.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
All high dose and the majority (22 out of 25) of the mid dose animals showed transient salivation immediately after treatment on one or several days of the treatment period; however, the observed salivation persisted in the respective females only for a few minutes after the actual gavaging had taken place. After cessation of treatment on day 19 p.c., salivation did not occur any longer. There were no substance-related or spontaneous mortalities in any of the groups. Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20). The mean food consumption of the high dose dams was statistically significantly reduced (9% below the concurrent control value) at initiation of treatment (days 6 - 8 p.c.). On the following days of the treatment period, however, food consumption of the high dose rats reached or even exceeded control values. The food consumption of the females of low and mid dose dams was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls. The transient reductions in food consumption at 400 mg/kg bw were accompanied by corresponding impairments in body weight gain of these dams at initiation of dosing. 21 high dose dams showed dark-yellow discolored urine on gestation days 12 - 20 p.c. which is probably related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air and does not represent a toxicologically relevant finding. The mean gravid uterus weights of the animals of all test groups were not influenced by the administration of the test substance. Absolute and relative mean liver weights were statistically significantly increased at the mid and high dose groups and were about 9 or 29% (absolute) and 8 or 29% (relative) above control values. These weight increases, which are considered to be substance-induced, are indicative of hepatic changes primarily caused by microsomal enzyme induction. Absolute and relative liver weights of the low dose dams, however, were similar to the control values and did not show any toxicologically significant changes. There occurred no substance-related observations at necropsy in any of the dams of all test groups. Only very few spontaneous findings were recorded for single low and mid dose rats (one hydrometra in low dose female which consequently did not become pregnant, hemorrhagic thymus in one mid dose female). No association to the test compound was assumed for these findings due to their scattered occurrence without any relation to dosing.

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
The conception rate reached 96% in the controls, 92% in the low and the high dose groups, and 100% in the mid dose. There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of corpora lutea and implantation sites.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the pre- and the postimplantation losses. The pre- and the postimplantation loss values in the 25 and 100 mg/kg groups, however, were above the upper ranges of the historical control values and the mean number of live fetuses/low dose dam was statistically significantly below the concurrent and the historical control value. These differences appeared without any dose-response relationship and thus are not considered to reflect any substance-induced effect.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between the test groups in the number of early or late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no substance-related and/or biologically relevant differences between viable and dead fetus.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The conception rate reached 96% in the controls, 92% in the low and the high dose groups, and 100% in the mid dose. There were no substance-related and/or biologically relevant differences between the test groups in the conception rate, in the mean number of corpora lutea and implantation sites, the pre- and the postimplantation losses, and the number of resorptions and viable fetuses. The pre- and the postimplantation loss values in the 25 and 100 mg/kg groups, however, were above the upper ranges of the historical control values and the mean number of live fetuses/low dose dam was statistically significantly below the concurrent and the historical control value. These differences appeared without any dose-response relationship and thus are not considered to reflect any substance-induced effect. They can well be explained by the fact that one low dose dam and two mid dose dams resorbed all of their implants and thus had
no viable fetuses (the same was also observed for one control dam). Moreover, one low dose, which had 7 implantation sites, resorbed 6 implants and had only one live fetus at terminal sacrifice. If these 5 rats are excluded from the calculation of the means, pre- and postimplantation loss values as well as the mean number of live fetuses/dam fit well into the historical control ranges with one unimportant exception (preimplantation loss value at 25 mg/kg bw/day).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
dermal irritation
early or late resorptions
food consumption and compound intake
gross pathology
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
urinalysis
Dose descriptor:
LOAEL
Effect level:
<= 400 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in all test groups was comparable with that of the control fetuses.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Most of the observed malformations were limited to one multiply malformed mid dose fetus. The external examination of this fetus revealed gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like tail.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During its skeletal evaluation additional malformations like severely malformed sternum and absent vertebrae were recorded. The other malformations that occurred were anasarca (in one control fetus), situs inversus (in one low dose fetus), and cleft sternum (in another control fetus).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue variations, exclusively in the form of dilated renal pelvis and ureters were observed to be sporadic, spontaneous and non treatment related.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The mean fetal body weights in all test groups were not influenced by the test substance administration and were very similar to or even identical with concurrent control values. The sex distribution of the fetuses in all test groups was comparable with that of the control fetuses. Most of the observed malformations were limited to one multiply malformed mid dose fetus. The external examination of this fetus revealed gastroschisis, anal atresia, malrotated left hindlimb, and a thread-like tail. During its skeletal evaluation additional malformations like severely malformed sternum and absent vertebrae were recorded. The other malformations that occurred were anasarca (in one control fetus), situs inversus (in one low dose fetus), and cleft sternum (in another control fetus). Soft tissue variations, exclusively in the form of dilated renal pelvis and ureters were observed to be sporadic, spontaneous and non treatment related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day.
Executive summary:

The chemical was given by oral gavage to 25 presumed pregnant rats per dose levelat 0 (vehicle), 25, 100 and 400 mg/kg bw/day from GD 6 to 19. Dose levels were selected based on the results of dose range-finding study. All animals survived to planned death (scheduled on GD 20). Clinical signs were limited to transient salivation at ≥100 mg/kg and discolored urine at 400 mg/kg. None of these effects were considered to be of toxicological significance. No significant effects on maternal body weight were observed. Maternal body weight gain at 400 mg/kg on GD 8 to 10 was significantly decreased (by about 29%) compared to the control group. This effect was accompanied by transient reductions in food intake and a slight decreased in corrected body weight gain (net gain from GD 6 to 20). Collectively, the observed effects on body weight gain and food intake at 400 mg/kg were taken as signs of maternal systemic toxicity. No significant effects were observed on mean gravid uterus weight. Absolute and relative liver weights were significantly increased at ≥100 mg/kg. Necropsy findings were unremarkable. The conception rates were 96, 92, 100 and 92% at 0, 25, 100 and 400 mg/kg, respectively. Pre- and post-implantation losses were significantly increased at 25 and 100 mg/kg compared to the control data. This effect was not attributed to the test chemical as no such effect was observed at 400 mg/kg. The mean number of live fetuses was significantly lower at 25 mg/kg (mean, 7.4) compared to the control data (mean, 8.7). This effect was not attributed to the test chemical as no such effect was observed at 100 or 400 mg/kg. No significant effects were observed in the mean numbers of corpora lutea, implantation sites, or resorptions. The number of abortions were 1, 1, 2 and 0 at 0, 25, 100 and 400 mg/kg, respectively. Mean placental weight was significantly increased at 400 mg/kg (mean, 0.45 g) compared to the control group (mean, 0.41 g) but was well within the historical control range (0.32 to 0.58 g). No significant effects on mean fetal body weight were observed among the groups. Sex ratio was unaffected by treatment. No treatment-related gross, visceral or skeletal malformations were observed. The combined number of malformations expressed as % of fetuses affected were 1.0, 0.6, 0.5 and 0.0% at 0, 25, 100 and 400 mg/kg, respectively. No external variations were observed. No treatment-related gross, visceral or skeletal variations were observed. The combined number of variations expressed as % of fetuses affected were 53, 54, 52 and 49% at 0, 25, 100 and 400 mg/kg, respectively. The combined number of variations expressed as % of litters affected was 100% at all dose levels. NOAEL for maternal systemic toxicity was concluded at 100 mg/kg bw/day. This effect level was based on observed effects on body weight gain and food intake at 400 mg/kg bw/day. NOAEL for developmental toxicity was concluded at 400 mg/kg bw/day.