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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Data are derived from the structural methyl ionone analog beta-ionone, for more information please refer to the read-across justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Remarks:
WIL Research Europe B.V.
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:
4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Beta Ionon R
- Physical state: Clear yellowish liquid
- Analytical purity: 97.2 area-%
- Batch No.: 0000997L0
- Expiration date of the batch: 11 April 2015
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Strain: female albino rabbits, New Zealand White (NZW) strain (SPF-Quality)
- Age at study initiation: females were approximately 18-19 weeks at delivery / adult males
- Fasting period before study: at least 10 air changes/hour
- Housing: housed individually in labelled cages with perforated floors (Ebeco, Germany) and shelters (Ebeco, Germany)
- Diet: standard powder rabbit diet (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) prepared in-house, ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- The test substance was mixed with powder food. Water (approximately 12% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance; this diet was also supplied to all treated groups for at least 17 days before start of treatment.
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rabbit diet (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy)
- Storage temperature of food: When stored in the freezer at ≤-15°C, diets were acclimatized at room temperature for at least one hour before use. When kept at room temperature in the diet store room in the animal facility, diets containing test substance were used within 4 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After termination, the actual test substance intake was estimated based on the body weight and food consumption values.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
from days 6 to 29 post-coitum
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 17, 50, 200 and 1000 mg/kg bw/day
Basis:
nominal in diet
corresponding to 0, 500, 1500, 6000 and 30000 ppm
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Dose levels were based on a maternal toxicity study (non-GLP Project 503434; BASF Project 20R0449/02X035). No adverse effects were observed in mated female New Zealand White rabbits following dietary treatment at target dose levels of 150 and 500 mg/kg bw/day. Findings at both dose levels consisted of reduced food consumption, body weight loss and reduced faeces production. However, as these changes were transient, followed by recovery towards values within the normal range of biological variation, they were not considered to be adverse. No changes were noted for the remaining parameters (mortality, haematology, clinical biochemistry, macroscopic findings or organ weights).
Based on these results, the target dose levels of 0 (Group 1), 50 (Group 2), 200 (Group 3), and 1000 (Group 4) mg/kg bw/day were selected for the main study.
Group 4 showed severely reduced food intake after introduction of the test diet. As no recovery occurred up to Day 10 post-coitum, it was decided to stop treatment, remove the high dose group from the study without further examination and to add an extra group of 22 mated females treated at a low target dose level of approximately 17 mg/kg bw/day (Group 5).

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was graded and recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 0-3, 3-6, and daily from Days 6-29 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

MORTALITY: At least twice daily
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

Animals sacrificed before planned necropsy were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail and weighed. All live fetuses were euthanised by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity.

- Soft tissue examinations: Yes: [all per litter]
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.
Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin.

- Skeletal examinations: Yes: [all per litter]
All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.
The eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses. All specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

- Head examinations: Yes: [half per litter]
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). The heads from the remaining one-half of the fetuses in each litter were processed for skeletal examination.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics was applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) * 100

Post-implantation loss (%) = ((number of implantation sites - number of live fetuses) / (number of implantation sites) * 100

Viable fetuses affected/litter (%) = ((number of viable fetusus affected/litter) / (number of viable fetuses/litter)) * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
- no mortalities up to 200 mg/kg bw/day
- one female (no. 27) at 50 mg/kg bw/day had an abortion on Day 20 post-coitum (considered not to be toxicologically relevant)

Clinical signs
- reduced faeces production was noted for several rabbits of all groups, including control animals (no dose-related increase in the number of affected animals but a trend towards a longer period of reduced faeces production at 200 mg/kg bw/day as compared to the lower dose)
- incidental findings such as alopecia, scales, red discolouration of the skin, focal erythema, scars, scabs, wounds, red staining of the vagina, faeces containing mucus, pale discoloration of the faeces, diarrhea, and enlarged faeces balls (within the range of background findings, no toxicological relevance)

Body weights
- 200 mg/kg bw/day: reduced body weights, lower body weight gain (-87%) and/or body weight loss, decreased body weight gains corrected for gravid uterus weights (statistically significantly)
- 17 and 50 mg/kg bw/day: no substance related effects

Food consumption
- 200 mg/kg bw/day: lower food consumption (absolute and relative (up to -89%) to body weight on Days 6-29 post-coitum, except for the period from Days 10-14 post-coitum with temporally recovery)
- 17 and 50 mg/kg bw/day: no substance related effects

Water consumption
- no treatment related effects

Macroscopic examination
- no treatment related effects
- incidental findings among control and treated animals such as alopecia, accentuated lobular pattern and/or pale discoloration of the liver, enlarged uterus, and a smaller gallbladder (within the range of background findings, no toxicological relevance)

Maternal pregnancy data
- number of litters with viable fetuses available on Day 29 post-coitum: 21, 21, 20 and 21 (0, 17, 50, 200 mg/kg bw/day, respectively)
- in all groups one female each was not pregnant (not treatment related, as treatment started from implantation onwards)
- one female at 50 mg/kg bw/day had an abortion on Day 20 post-coitum (isolated observation at the mid dose level, it was considered to be a chance finding rather than related to treatment
- no treatment related effects on gravid uterus weights

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter size
- no treatment related effects (mean litter sizes 9.7, 9.0, 7.7 and 9.5 fetuses per group for the control, 17, 50 and 200 mg/kg group, respectively)

Fetal viability
- no treatment related effects
- The statistically significantly higher pre-implantation loss (% per litter) noted at 50 and 200 mg/kg bw/day was not considered treatment related, as treatment did not start before completion of implantation (i.e. Day 6 post-coitum).

Sex ratio
No toxicologically relevant findings were noted for sex ratio for treatment up to 200 mg/kg bw/day.

Fetal body weight
- 200 mg/kg bw/day: slightly lower fetal body weights in males and females (reaching statistical significance for males only). This change was considered to be secondary to the reduced food intake and markedly decreased body weight gain of the dams.

The numbers of fetuses (litters) available for external, visceral and skeletal morphological evaluation were 203 (21), 188 (21), 153 (20) and 199 (21) in the groups at 0, 17, 50 and 200 mg/kg bw/day, respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups.

Fetal morphological examinations
- Malformations were observed in 4 (4), 9 (8), 9 (8) and 5 (4) fetuses (litters) in the groups at 0, 17, 50 and 200 mg/kg bw/day. Fetal examination revealed one late resorption at 17 mg/kg bw/day with the external malformation carpal flexure.

External malformations and variations
- There were no treatment related effects on fetal external morphology up to 200 mg/kg bw/day.
- External malformations were observed in 2 (2), 0 (0), 1 (1) and 1 (1) fetuses (litters) in the groups treated at 0, 17, 50, and 200 mg/kg bw/day, respectively but not considered to be related to treatment.
One fetus each in the control, 50 and 200 mg/kg bw/day groups had an omphalocele. At this low incidence and in the absence of any doserelated distribution, this finding was not considered to be caused by treatment with the test substance.
Two control fetuses in two different litters showed carpal flexure without any apparent skeletal origin. One of these control fetuses had also the omphalocele. Since only control fetuses were affected, a treatment-related cause could be excluded.
- No effects on fetal external morphology up to 200 mg/kg bw/day.
- No external developmental variations were observed in any of the fetuses.

Visceral malformations and variations
- Visceral malformations were observed in 1 (1), 6 (5), 6 (6) and 2 (2) fetuses (litters) in the groups treated at 0, 17, 50, and 200 mg/kg bw/day, respectively.
There were several fetuses with cardiovascular malformations of which a tetralogy of Fallot complex was noted most frequently. This abnormality, comprising of pulmonary stenosis, ventricular septum defect, dextraposed aorta overriding the ventricular septum, and thickened right ventricular wall, occurred in 4 fetuses from different litters in the 50 mg/kg bw/day and in one fetus at 200 mg/kg bw/day. Since there was no dose-dependency, a compound-related effect is not evident.
Other (combinations of) cardiac and/or vascular malformations in this study occurred in one fetus at 50 mg/kg bw/day (narrow pulmonary trunk combined with dilated aortic arch and atrial septum defect) and two fetuses at 17 mg/kg bw/day (narrow aortic arch combined with atrial septum defect, and large heart, respectively) or in one control fetus (interrupted aortic arch and ventricular septum defect).
Further incidental visceral malformations noted for fetuses in the control and/or treated group(s) included absent lung lobe or thyroid, abnormal lobulation of the liver, malpositioned testis or kidney, and intestine diverticulum. As these malformations were also noted in the historical control database, occurred in control fetuses only and/or at a low incidence without a treatment-related distribution, they were not considered treatment related.
- Visceral variations noted for fetuses in the control and/or treated group(s) were malpositioned left carotid, supernumerary artery, retrocaval ureter, partially undescended thymus horn, cysts on ovary, testis or liver, liver appendix, small gallbladder, absent renal papilla(e), and constricted spleen. These variations were not considered to be treatment related, because they occurred infrequently, in the absence of a dose related trend, at frequencies within the historical control range and/or were seen in control fetuses only.

Skeletal malformations and variations
- There were no skeletal malformations or variations that were considered to be toxicologically relevant.
- Malformations: non treatment related skeletal malformations were observed in 3 (3), 4 (4), 3 (3) and 2 (2) fetuses (litters) in the groups treated at 0, 17, 50, and 200 mg/kg bw/day, respectively (isolated findings like split skull bones, caudal vertebral anomaly, vertebral anomaly with or without associated rib anomaly, rib anomaly, costal cartilage anomaly, fused or severely malaligned sternebrae).
- Variations: The mean incidences of unossified metacarpals and/or metatarsals (12.4% per litter) and unossified sternebra nos. 5 and/or 6 (25.7 % per litter) were slightly higher in the fetuses at 200 mg/kg bw/day as compared to the concurrent control values (4.2% and 18.9% per litter, respectively).
Further skeletal variations seen in control and/or treated groups included rudimentary or full 13th rib(s) and 7th cervical rib(s), nodulated rib, caudal shift of the pelvic girdle, slightly to moderately malaligned sternebra(e), supernumerary sites (additional piece of bone at the skull or vertebra), sternum with supernumerary ossification site, no ossification or reduced ossification of different bones (vertebral centra, skull bone line, skull, tarsal(s), hyoid body and/or arches, sternebra(e) nos. 1, 2, 3 and/or 4, pubis), bent hyoid arch(es) and caudal vertebral anomaly.
None of these skeletal variations was considered to be treatment related as they occurred at a very low incidence, at frequencies that were similar to concurrent controls, within or just outside the range of available historical control data, and/or were seen in fetuses of the control group only.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Severely reduced food consumption and significant body weight loss (up to 9% at the individual level) were noted for animals of the high dose Group 4 treated at a target dose level of 1000 mg/kg bw/day after start of treatment on Day 6 post-coitum. As no recovery occurred up to Day 10 post-coitum, it was decided to stop treatment, remove the high dose group from the study without further examination and to add an extra group of 22 mated females treated at a low dose level of approximately 17 mg/kg bw/day (Group 5).

Mean test substance intake values:

Group 2: 50 mg/kg bw/day (nominal concentration 50 mg/kg bw/day)

Group 3: 160 mg/kg bw/day (nominal concentration 200 mg/kg bw/day)

Group 5: 16 mg/kg bw/day (nominal concentration 17 mg/kg bw/day)

Applicant's summary and conclusion