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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Recent study according to OECD Guideline 415 under GLP, with full report and all individual data. Data are derived from the structural methyl ionone analog pseudoionone, for more information please refer to the read-across justification.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes
Remarks:
NOTOX BV
Limit test:
no
Species:
rat
Strain:
other: Wistar Crl: (WI) BR (outbred, SPF quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males 5 - 6 weeks old; females 11 - 12 weeks old
- Identification: tattoo on the tail
- Housing: suspended stainless-steel cages, 4 animals per sex per cage, with males and females being kept in separate rooms. Mated females and males were housed individually in labelled polycarbonate cages containing sawdust (SAWI bedding, Jelu-Werk, Rosenberg, Germany) as bedding material. During the final stage of the pregnancy period, from day 16 post coitum, and during lactation, paper (Enviro-dri, BMI, Helmond, The Netherlands) was supplied to the dams for incorporation into the nest. The paper was replaced when soiled.
- Diet (e.g. ad libitum): ad libitum, standard pelleted rat diet (Altromin, code VRF1, Lage, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Analyses for all batches of feed and quarter-yearly analyses of tap water are retained at NOTOX archives
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Pseudoionone was formulated daily

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1 from same dose group
- Length of cohabitation: until vaginal plug was found; max. 3 weeks
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing pairs were separated.
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were analytically confirmed to be stable for at least 4 hours at room temperature and to correspond to targeted concentrations.
Duration of treatment / exposure:
Exposure period: males: mean 106 (range 104-108) days;
females: mean 60 (range 36-65) days
Premating exposure period (males): 11 weeks
Premating exposure period (females): 2 weeks
Duration of test: 126 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
40, 120 and 360 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on a GLP 28-day subchronic toxicity study with the same dose levels that resulted in a NOEL of 50 mg/kg bw/d and a LOEL of 250 mg/kg bw/d with reversible effects (-> 7.5.1 Strobel1997.Repeated dose toxicity: oral rat 28d)
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter; Mated females were weighed on days 0, 7, 14 and 21 of gestation and during lactation on days 1, 4, 7, 14 and 21


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption of mated females was recorded on gestation days 0, 7, 14 and 21 and during lactation on days 1, 4, 7, 14 and 21


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, but only subjective appraisal was maintained

REPRODUCTIVITY
- numbers of animals mated, mating date, confirmation of pregnancy and day of delivery were recorded
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight (1, 4, 7, 14 and 21 of lactation), physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, males were killed after confirmation of the pregancy of the female they had been mated with or after successful delivery of the respective dam.
- Maternal animals: All surviving animals killed at day 21 post partum or shortly thereafter


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
-The tissues indicated were prepared for microscopic examination and weighed:
cervix plus uterus, epididymides (both together), kidney, liver, ovaries, pituitary (weighed after 24 h fixation), prostate (weighed after 24 h fixation), seminal vesicles together with coagulating gland and fluids, spleen and testes.
-All organ and tissue samples as listed below were processed, embedded, microtomed at 2-4 µm and stained with haematoxylin and eosin: kidneys and liver from 10 randomly selected animals per sex from all treatment groups. All slides were examined by a professional histopathologist, abnormalities were described and included in the histopathology report. The histopathologist was asked to add an interpretation of the findings
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at at day 21 post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Main offspring found dead or killed before day 14 of lactation were sexed and externally examined if practically possible. Main offspring found dead or killed on or after day 14 of lactation were sexed and subjected to external examination of the thoracic and abdominal tissues and organs


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- All abnormalities were recorded. If possible, defects or cause of death were evaluated.

Statistics:
For variables assumed to follow a normal distribution, the Dunnett test was applied; for other assumed distributions the Steel test was used. In those cases where variables could be dichotomised without loss of information, the exact Fisher test was applied. All tests were two-sided, significance was accepted at p < 0.05.
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
no
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
360 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Reproductive effects observed:
not specified

Protocol deviations
13 protocol deviations are listed in the report. All 13 were
evaluated and considered not to have affected the integrity
of the study or of the results.

Dose preparations
A first analysis of formulations prepared on 17-Jun-2002
showed values for accuracy within the range of 86-131% for
pseudoionone peak 1 and of 84-126% for peak 2, which was
considered insufficient. Additional analyses were performed
and the one of the formulations prepared on 24-Jun-2002
showed 98-102% for peak 1 and 97-102% for peak 2. The
insufficient results were considered to originate from
pipetting errors of the volatile solvent (n-hexane) during
sample pretreatment for chemical analysis. Preparations of
the formulations, however, were performed according to the
accurate method and it was concluded that the animals
received the complete and correct exposure to the test
substance.
Analyses for homogeneity of the low- and high-dose
formulations prepared on 17-Jun-2002, 24-Jul-2002 and
29-Aug-2002 all showed values within the range of 94-109%
for peak 1 and of 84-115% for peak 2, which were considered
acceptable for this type of formulations. 
A stability analysis of the low- and high-dose formulations
from 17-Jun-2002 showed decreases over 7 days of 15% (peak
1) and and 11% (peak 2) for groups 40 mg/kg bd/d and of 15%
(peak 1) and 13% (peak 2) for group 360 mg/kg bw/d, which
was considered sufficient in view of the fact that
formulations were prepared daily.


Mortalities
There were 3 unscheduled deaths out of a total of 192 main
parental animals; all 3 animals were females. Two were
killed in extremis, one each in the 120 and the 360 mg/kg
bw/d groups after 38 respectively 43 days of treatment. The
other animal, also a female from the 360 mg/kg bw/d group,
died spontaneously on day 38. All three were found to have
severe delivery difficulties, with 17 foetuses in the birth
canal, 16 dead pups and three foetal resorptions, and 19
foetuses in the birth canal, respectively. These deaths were
considered incidental and very possibly caused by the big
litter sizes. Therefore, these deaths were considered not to
be related to the treatment with the test substance.


Clinical signs
Salivation was observed in all males and females of the
highest dose group. Incidental findings consisted of
alopecia, lethargy, clonic spasms, rales, salivation, scabs,
nodule at the tail, red staining of the right eye, broken
teeth, hunched posture, piloerection, pale appearance,
emaciation, dull eyes and dark eyes. No relationship was
established with treatment for these observations or they
were considered to be within the normal biological variation
for rats of this age and strain. Animal no. 40 of group 2
(40 mg/kg bw/d) showed several signs of stress (compulsive
biting, saltator spasms, tremor and muscle twitching) just
before or after dosing during four days of treatment.


Body weight
Body weights and body weight gain rates were unaffected by
treatment up to 360 mg/kg bw/d.


Food consumption
Statistically significant increases in relative food
consumption were observed in some of the 120 and 360 mg/kg
bw/d males. No explanation for this increase can be given,
however, this finding was not considered an adverse effect,
it was considered incidental in nature and not to be
toxicologically relevant.


Macroscopic examination
No treatment-related macroscopic findings were identified
but a number of findings that were considered incidental in
nature. These findings included pelvic dilation of the left,
right or both kidneys, testes reduced in size, flaccid
testes, enlarged testes, accentuated lobular pattern of the
liver, pale discolouration of the liver, alopecia at several
parts of the body, dark red discolouration of the
mediastinal cranial lymph nodes, isolated yellowish hard
nodule at the tail of the left epididymis, dark red hard
nodule at the left and right tips of the epididymides,
epididymides reduced in size, enlarged liver, reddish soft
nodule at the papillary process of the liver, soft nodule at
the papillary process of the liver, stomach and spleen grown
together with a soft nodule at the papillary process of the
liver, dark red discolouration of the left mandibular lymph
node. These findings are occasionally seen among rats used
in this type of study and, in the absence of correlated
microscopic histopathological findings, were not considered
of toxicological significance. Fluid in the uterus (in one
female of the control group, in three females of the 40
mg/kg bw/d group, in one female of the 120 group and in one
female of the 360 group) is related to a stage in the
oestrous cycle and is a normal finding.
In the 120 mg/kg bw/d group, one female that was killed in
extremis showed 17 foetuses in the birth canal. Of the 360
group, one female that was killed in extremis showed 3
foetal resorptions and 9 placentas in the left uterus horn
and the thoracic cavity containing milky-cloudy fluid; one
female from the 360 group that died spontaneously showed 19
foetuses in the birth canal and beginning autolysis.


Organ weights
Males and females of the 360 mg/kg bw/d group showed
statistically significant increased absolute and relative
liver and kidneys weight. Males of the 120 group showed
significantly increased liver weight:
----------------------------------------------------------
Dose group,      Bodyweight-related organ weights (%bw),
mg/kg bw/d       group mean values
                 -----------------------------------------
                 liver                kidney
                 -------------------- --------------------
                 males      females   males      females
----------------------------------------------------------
   0    mean      3.33       4.28      0.68       0.69
        SD        0.23       0.31      0.05       0.04
        n        24         24        24         24
----------------------------------------------------------
  40    mean      3.39       4.16      0.68       0.69
        SD        0.19       0.45      0.06       0.05
        n        24         22        24         22
----------------------------------------------------------
 120    mean      3.61**     4.45      0.71       0.70
        SD        0.20       0.25      0.07       0.05
        n        24         23        24         23
----------------------------------------------------------
 360    mean      4.13**     4.75**    0.77**     0.73*
        SD        0.29       0.29      0.07       0.06
        n        24         22        24         22
----------------------------------------------------------
*/**: Dunnett's test on pooled variance, significant at 5%
(*) or 1% (**) level.
----------------------------------------------------------
In the absence of histopathological changes, both effects
were considered not to be toxicologically relevant but
rather manifestations of physiological adaptation to
additional metabolic and excretionary loads. 
Males of the 40 group showed statistically significantly
reduced seminal vesicles weight. In the absence of a
dose-response relationship, this finding was considered to
be caused by chance and not to be related to treatment.
Microscopic examination
There were no treatment-related findings. No
histopathological changes were found to correlate with the
observed increase in liver and kidney weights.


Reproduction
Reproduction parameters were unaffected by treatment up to
360 mg/kg bw/d. In the 40 group, one female did not mate and
one female was non-pregnant. In the 120 group, one female
showed delivery difficulties, and in the 360 group, two
females showed delivery difficulties. Mating performance,
duration of gestation and fertility parameters including
number of pups at birth were similar for the control and
treated groups.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
360 mg/kg bw/day
Species:
rat
Quality of whole database:
Data are derived from the structural methyl ionone analog pseudoionone, for more information please refer to the read-across justification.
Additional information

Due to the lack of studies concerning the toxicity to reproductive toxicity of methyl-ionone, studies with the structural analogue substances pseudo-ionone (CAS 141-10-6) and mixed ionone isomers (CAS 8013-90-9) have to be taken into account for assessment. For more information on the category approach for read-across, please refer to the read-across justification.

The developmental toxicity was analyzed in an One-generation reproduction toxicity study with pseudo-ionone performed under GLP according to OECD guideline 415 (Beekhuizen, 2003). 96 male and 96 female Wistar rats were randomly assigned to four treatment groups which were administered by gavage with 0, 40, 120 or 360 mg/kg bw/d pseudoionone in maize/corn oil. Exposure length was 11 weeks prior to mating up to termination for males, giving the mean of 106 days, with a range from 104 to 108 days. The females were exposed for 2 weeks prior to mating up to termination; the mean duration of treatment was 60 days, with a range of 36 to 65 days. The offspring was not treated. Females were paired one-to-one with males from the same group and allowed to give birth normally. Pups were killed either at adjusting litters on day 4 post partum or at the end of the study at day 21 post partum. At the end of the study, all survivors were killed, the males after confirmation of the pregnancy or successful delivery of the female they were mated with, the females at day 21 post partum or shortly thereafter. After killing or natural death all parental animals were subjected to external examination and to macroscopic examination during dissection, with special attention to the reproductive organs. Also, pups were sexed and externally examined. Three unscheduled deaths occurred, which were considered inci­dent­al and therefore considered not to be treatment-related. Males and females of the 360 mg/kg bw/d group showed statistically significant increased absolute and relative liver and kidneys weight, which was also seen in the males of the 120 mg/kg bw/d group. In the absence of histopathological changes, both effects were considered not to be toxicol­og­ically relevant but rather manifestations of physiological adaptation to additional metabolic and excretionary loads.

Regarding reproductive parameters it was found that one female did not mate and one female was not pregnant in the 40 mg/kg bw/d group. In the 120 mg/kg bw/d group, one female showed delivery difficulties, and in the 360 mg/kg bw/d group, two females showed delivery difficulties, all of which were not considered related to the treatment but rather to the high number of foetuses. All other parameters, specifically mating performance, duration of gestation and fertility parameters including number of pups at birth were similar for the control and the treat­ment groups. In summary, the reproductive respectively fertility parameters were not affected up to 360 mg/kg bw/d. Thus, the general toxicological parental NOAEL was 120 mg/kg bw/d (NOEL 40 mg/kg bw/d) while the reproductive NOEL was 360 mg/kg bw/d based on the liver- and kidney-weight changes in males and salivation by all high dose animals.

In addition to the described One-generation reproduction toxicity study, results of a two-generation study could also be taken for assessment where no adverse effects on reproduction were found, when 8-10 mg/kg bw/day of the structural analogue mixed ionone isomers (CAS 8013-90-9) were administered to the parental rats for 8 months (Sporn, 1963). Thereby, the female rats were studied through 3 reproduction cycles for number of pregnancies, weight and number of offspring, live pups, weight of pups at birth and after 7 and 21 days, and the viability of the pups after birth. The F1 generation (offspring) were allowed to reach maturity and were then treated with 15 mg/kg of ionone prior to being subject to reproductive toxicity testing. As a result, no effects on reproductive parameters and organs were observed in this study.

Because of the structural similarities it can be assumed, that also for methyl-ionone no effects on fertility have to be expected (see read-across justification).


Short description of key information:
Fertility:
- NOAEL = 360 mg/kg bw/day (OECD 415; CAS 141-10-6)

Justification for selection of Effect on fertility via oral route:
GLP OECD guideline study with full report.

Effects on developmental toxicity

Description of key information

Teratogenicity:
- NOAEL maternal: 100 mg/kg bw/day (OECD 414; CAS 79-77-6)
- NOAEL teratogenic: 360 mg/kg bw/day (OECD 415; CAS 141-10-6)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guideline 415 under GLP, with full report and all individual data. Data are derived from the structural methyl ionone analog alpha-iso-methylionone, for more information please refer to the read-across justification.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 415 (One-generation reproductive toxicity)
Principles of method if other than guideline:
AccordinhOECD Guideline 415, One-generation reproductive toxicity
GLP compliance:
yes
Remarks:
NOTOX BV
Limit test:
no
Species:
rat
Strain:
other: Wistar Crl: (WI) BR (outbred, SPF quality)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: males 5 - 6 weeks old; females 11 - 12 weeks old
- Identification: tattoo on the tail
- Housing: suspended stainless-steel cages, 4 animals per sex per cage, with males and females being kept in separate rooms. Mated females and males were housed individually in labelled polycarbonate cages containing sawdust (SAWI bedding, Jelu-Werk, Rosenberg, Germany) as bedding material. During the final stage of the pregnancy period, from day 16 post coitum, and during lactation, paper (Enviro-dri, BMI, Helmond, The Netherlands) was supplied to the dams for incorporation into the nest. The paper was replaced when soiled.
- Diet (e.g. ad libitum): ad libitum, standard pelleted rat diet (Altromin, code VRF1, Lage, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Analyses for all batches of feed and quarter-yearly analyses of tap water are retained at NOTOX archives
Route of administration:
oral: gavage
Vehicle:
maize oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Pseudoionone was formulated daily
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were analytically confirmed to be stable for at least 4 hours at room temperature and to correspond to targeted concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1 from same dose group
- Length of cohabitation: until vaginal plug was found; max. 3 weeks
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing pairs were separated.
- After successful mating each pregnant female was caged (how): individually
Duration of treatment / exposure:
Exposure period: males: mean 106 (range 104-108) days
females: mean 60 (range 36-65) days
Premating exposure period (males): 11 weeks
Premating exposure period (females): 2 weeks
Duration of test: 126 days
Frequency of treatment:
once daily
Duration of test:
Exposure period: males: mean 106 (range 104-108) days;
females: mean 60 (range 36-65) days
Premating exposure period (males): 11 weeks
Premating exposure period (females): 2 weeks
Duration of test: 126 days
Remarks:
Doses / Concentrations:
40, 120 and 360 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on a GLP 28-day subchronic toxicity study with the same dose levels that resulted in a NOEL of 50 mg/kg bw/d and a LOEL of 250 mg/kg bw/d with reversible effects (-> 7.5.1 Strobel1997.Repeated dose toxicity: oral rat 28d)
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter; Mated females were weighed on days 0, 7, 14 and 21 of gestation and during lactation on days 1, 4, 7, 14 and 21


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption of mated females was recorded on gestation days 0, 7, 14 and 21 and during lactation on days 1, 4, 7, 14 and 21


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes, but only subjective appraisal was maintained

REPRODUCTIVITY
- numbers of animals mated, mating date, confirmation of pregnancy and day of delivery were recorded
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
Fetal examinations:
- External examinations: Yes: all per litter
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

For general results, please refer to Beekhuizen2003.Toxicity to reproduction rat 1 generation


Reproduction
Reproduction parameters were unaffected by treatment up to
360 mg/kg bw/d. In the 40 group, one female did not mate and
one female was non-pregnant. In the 120 group, one female
showed delivery difficulties, and in the 360 group, two
females showed delivery difficulties. Mating performance,
duration of gestation, fertility parameters and number of
pups at birth were similar for the control and treated
groups.
----------------------------------------------------------
Dose group,   Litter size, live births, mean bodyweights,
mg/kg bw/d         n            n              g
----------------------------------------------------------
   0    mean      14.7         14.5            6.8
        SD         2.5          2.72           0.62
        n         24           24             24 
----------------------------------------------------------
  40    mean      15.0         14.1            6.6
        SD         2.5          3.98           0.44
        n         22           22             21
----------------------------------------------------------
 120    mean      15.7         15.1            6.5*
        SD         1.3          2.72           0.45
        n         23           23             23
----------------------------------------------------------
 360    mean      15.4         14.5            6.5
        SD         4.5          5.38           0.59
        n         23           23             22
----------------------------------------------------------
*: p < 0.05, Dunnett's test on pooled variance.
----------------------------------------------------------

Pups
Development of the pups was unaffected by treatment up to
360 mg/kg bw/d. Numbers of pups at birth were similar
between controls and all treatment groups. No teratogenic
malformations are reported. However, postnatal deaths were
significantly increased at 360 mg/kg bw/d during days 0-4
post partum, due to which the viability index was decreased
in this group (91.0 compared to 96.6 in controls).

Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
Study currently ongoing
Adequacy of study:
supporting study
Study period:
2019 - 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 Jun 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 00073177LO
- Expiration date of the lot/batch: 09 Jun 2019
- Physical state/appearance: Liquid/ yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Guaranteed by the Sponsor
- Stability of the test substance in the solvent/vehicle: Verified prior the start of the study (at room temperature over a period of 7d)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The specific amount of test substance were weighed, topped up with 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer.
Before and during administration, the preparations were kept homogeneous with a magnetic stirr.
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services,
Germany GmbH/ Charles River Laboratories, France
- Housing: Single housing
- Fasting period before study: Not applicable
- Diet (e.g. ad libitum): ad libitum; pelleted Kliba maintenance diet rabbit and guinea pig “GLP” (Granovit AG, Kaiseraugst/Switzerland)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: At least 5 d before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 h - 18:00 h); 12 hours darkness (18:00 h - 06:00 h)

Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer. Before and during administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guaranteed that the test substance concentrations in the vehicle remained stable.

VEHICLE
- Concentration in vehicle: 0, 0.5, 1.5, 5.0 g/100mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration control analysis at the beginning of administration. The samples of the mid and end of the study are analyzed only if any imprecision occurs during the analysis of the samples from the start of the study.
The stability of the test substance in the vehicle at room temperature over a period of 7 days had been verified prior to the start of the study.
Details on mating procedure:
- Impregnation procedure: Artificial insemination (GD 0)
- 0.2 mL of a synthetic hormone (Receptal, Intervet Deutschland GmbH, Unterschleißheim, Germany), which releases LH and FSH from the anterior pituitary lobe, was injected intramuscularly into the female rabbits about 1 h before insemination
- The samples of the ejaculate used for the artificial insemination were derived from male New Zealand White rabbits of the same strain as the females. The male donors were maintained under the same conditions (air conditions, feed and water) as the females used in the study. The day of insemination was referred to as GD 0.
Duration of treatment / exposure:
GD 6-28
Frequency of treatment:
daily
Duration of test:
GD 0-29
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment:
(Crl:KBL(NZW)) rabbits were chosen because of their general acceptance and suitability for embryotoxicity testing (including teratogenicity), availability of historical control data and the reliability of the commercial supplier
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity
- A check for moribund and dead animals twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 29)
- During administration period (GD 6-28): all animals were checked daily for abnormal clinical signs before administration as well as within 5 hours after the administration. Abnormalities and changes are documented for each animal

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Gross-pathological examination







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
- Other: Site of implantations in the uterus; Gross-pathological examination
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 (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus)
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:

- DUNNETT’s test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight,
weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and
live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the does) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Test group 0 (control): reduced defecation 6/25, no defecation 2/25
- Test group 1 (50 mg/kg bw/d): reduced defecation 5/25, no defecation 2/25, blood in bedding 1/25, aborted/sacrificed 1/25
- Test group 2 (150 mg/kg bw/d): reduced defecation 8/25, no defecation 2/25, aborted/sacrificed 1/25
- Test group 3 (500 mg/kg bw/d): reduced defecation 16/25, no defecation 6/25, vaginal hemmorhage 1/25, aborted/sacrificed 1/25
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Mortality 0 1 1 1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
BW (0) 3721 g 3720 g (100%) 3662 g (98%) 3694 g (99%)
BW (6) 3896 g 3879 g (100%) 3853 g (99%) 3875 g (99%)
BW (28) 4148 g 4060 g (98%) 4129 g (100%) 3932 g (95%)**
BW (29) 4174 g 4084 g (98%) 4144 g (99%) 3935 g (94%)**

BWC (0 - 6) 174.8 g 190.3 g 159.5 g 181.5 g
BWC (6 - 28) 251.9 g 274.5 g 180.4 g 56.9 g**
BWC (0 - 29) 452.8 g 482.2 g 373.5 g 239.6 g**
Food efficiency:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
FС (0 - 6) 171.7 g 166.5 g (97%) 166.1 g (97%) 171.9 g (100%)
FС (6 - 28) 122.7 g 122.3 g (100%) 108.6 g (89%) 92.0 g (75%)
FС (0 - 29) 132.5 g 130.9 g (99%) 119.9 g (91%) 108.0 g (82%)
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:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Uterus weight 477.7 g 483.7 g (101%) 502.4 g (105%) 427.0 g (89%)
Description (incidence and severity):
- Test group 0 (control): intestines: watery feces 1/25, intestines: bloated 1/25
- Test group 1 (50 mg/kg bw/d): intestines: watery feces 1/25
- Test group 2 (150 mg/kg bw/d): intestines: watery feces 2/25, large intestines: watery feces 1/25, stomach: erosin(s) 1/25
- Test group 3 (500 mg/kg bw/d): intestines: watery feces 3/25, large intestines: watery feces 2/25
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Carcass 3696.0 g 3660.5 g (99%) 3581.5 g (97%) 3507.7 g (95%)*
Net weight change from gestation day 6 -199.7 g -193.9 g -286.0 g -366.9 g*
Number of abortions:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Resorption 0 1 1 1
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Preimplantation 5.9% 5.4% 6.4% 6.6%
Postimplantation loss 11.3% 5.2% 6.5% 11.1%
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Does with all resorptions 1 0 0 1
Early or late resorptions:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
early 0.6 0.3 0.4 0.4
late 0.3 0.2 0.3 0.4
total mean 0.8 0.6 0.8 0.8
Dead fetuses:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Dead fetuses 0 0 0 0
Changes in pregnancy duration:
no effects observed
Dose descriptor:
NOAEL
Remarks on result:
other: ongoing study
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
females (mean) 4.8 4.7 5.2 4.6
males (mean) 4.9 4.4 4.8 4.5
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
Fetal weights of all viable fetuses 35.7 g 36.9 g 34.9 g 33.3 g
External malformations:
no effects observed
Description (incidence and severity):
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
fetal incidence 1 (short tail) 0 0 1 (umbilical hernia)
litter incidence 1 0 0 1
Description (incidence and severity):
examinations ongoing, results will be reported in a dossier update
Other effects:
no effects observed
Description (incidence and severity):
fetal external variations
Test group 0 (control) Test group 1 (50 mg/kg bw/d) Test group 2 (150 mg/kg bw/d) Test group 3 (500 mg/kg bw/d)
fetal incidence 1 (paw hyperflexion) 0 0 0
litter incidence 1 0 0 0
Dose descriptor:
NOAEL
Remarks on result:
other: currently ongoing study
Developmental effects observed:
not specified
Treatment related:
not specified

The high-dose of the test item (500 mg/kg bw/d) caused maternal toxicity, such as considerably reduced food consumption as well as correspondingly reduced gross and net body weights/body weight gain. Cesarean section and external or visceral examination of offspring revealed no relevant findings which could be associated with a potential developmental toxicity of the test item. Examination of skeletons and cartilage are still ongoing and will be reported in a dossier update.

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
360 mg/kg bw/day
Quality of whole database:
Data are derived from the structural methyl ionone analog pseudoionone, for more information please refer to the read-across justification.
Additional information

Developmental toxicity was evaluated in a study performed according to OECD Guideline 414 (BASF SE, 40R0397/09R158). The test substance Methylionon 70 was administered as a solution of 0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)

to 25 "time-mated" (mated by breeder) female New Zealand White rabbits/group by stomach tube at doses of 50, 150 and 500 mg/kg bw on day 6 through day 28 post coitum (p.c.). On day 29 p.c., all females were sacrificed and assessed by gross pathology including the uterus and the placentae where corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal findings.

The study is yet not finalized. However, first preliminaty results are available and can be reported. The high-dose of the test item (500 mg/kg bw/d) caused maternal toxicity, such as considerably reduced food consumption as well as correspondingly reduced gross and net body weights/body weight gain. Cesarean section and external or visceral examination of offspring revealed no relevant findings which could be associated with a potential developmental toxicity of the test item. Examination ofskeletons and cartilage are still ongoing and will be reported in a dossier update.

Due to the lack of further studies concerning developmental toxicity of methyl-ionone, studies with the structural analogue substances alpha-iso-methylionone (CAS 127-51-5), pseudo-ionone (CAS 141-10-6) and beta-ionone (CAS 79-77-6) were taken into account for assessment. Data are derived from the structural methyl ionone analog alpha-iso-methylionone, for more information please refer to the read-across justification. Furthermore, a study according to OECD 414 is currently ongoing.

In a GLP-study according to FDA guideline (1994) alpha-iso-methylionone (AIM) was evaluated for developmental toxicity in Sprague-Dawley rats (Politano 2007). One hundred presumed-pregnant rats were randomly assigned to four dose groups (25/group). The rats were dosed orally (gavage) at 0, 3, 10, or 30 mg/kg/day AIM on days 7-17 of gestation (GDs 7 through 17). The dosage volume was 10 ml/kg and the vehicle was corn oil. Rats were observed for viability, clinical signs, abortions, premature delivery, deaths, body weights, and feed consumption. On GD 21, all rats were Caesarean-sectioned and a gross necropsy was performed on the thoracic, abdominal and pelvic viscera. Pregnancy occurred in 21-25 rats in each dose group. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender and gross external changes. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations and the remaining for skeletal alterations. No deaths occurred. No treatment-related clinical signs were observed. Feed consumption, body weight gains, gross tissue changes at necropsy, and Caesarean-section and litter parameters, as well as fetal developmental morphology, were unaffected by dosages of AIM as high as 30 mg/kg/day. Based on these data, maternal and developmental no-observable-adverse-effect-levels (NOAELs) of more than 30 mg/kg/day were established for AIM. It is concluded that AIM is not a risk to human development.

Developmental toxicity was also evaluated in a study with beta-ionone performed according to OECD Guideline 414 (BASF AG, 2004). The test substance was administered as a solution in olive oil to 25 time-mated female Wistar rats/group by stomach tube at doses of 25, 100 and 400 mg/kg bw on day 6 through day 19 post coitum (pc). On day 20 post coitum, all females were sacrificed and assessed by gross pathology including the uterus and the placentae where corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal findings. As a results, administration of 400 mg/kg bw elicited substance-induced effects on the dams including signs of maternal toxicity like reduced body weight gain (-29%). The dosage of 100 mg/kg bw/day resulted in some substance-related findings (i.e. temporary salivation, marginally increased liver weights), which are not considered to be adverse, but mirror some adaptive responses of the animals. At the low dose (25 mg/kg bw/day) no substance-induced effects on the dams occurred. Thus, the test substance had no influence on gestational parameters and did not induce adverse signs of developmental toxicity or teratogenic effects at all dose levels.

In addition, developmental toxicity was analyzed in a One-generation reproduction toxicity study with pseudo-ionone performed under GLP according to OECD guideline 415 (Beekhuizen, 2003). 96 male and 96 female Wistar rats were randomly assigned to four treatment groups which were administered by gavage with 0, 40, 120 or 360 mg/kg bw/d pseudoionone in maize/corn oil. Exposure length was 11 weeks prior to mating up to termination for males giving the mean of 106 days, with a range from 104 to 108 days. The females were exposed for 2 weeks prior to mating up to termination; the mean duration of treatment was 60 days, with a range of 36 to 65 days. The offspring was not treated. Females were paired one-to-one with males from the same group and allow give birth normally. Pups were killed either at adjusting litters on day 4 post partum or at the end of the study at day 21 post partum. They were sexed and externally examined. Although the mean bodyweights of the pups in the 120 and 360 mg/kg bw/d groups, were slightly but significantly reduced (90.6% respect­ively 95.6% of concurrent controls), these values were within the range of historical data, so that this finding was not toxicologically relevant. The number of dead and living pups at first litter check, of living pups on day 4 post partum, of breeding losses during days 5–21 post partum, of living pups on day 21 post partum and the weaning index were similar for control and all treated groups. However, postnatal deaths were significantly increased at 360 mg/kg bw/d during days 0–4 post partum, due to which the viability index was decreased in this group (91.0 compared to 96.6 control). Thus, the reproductive toxicity NOAEL was set at 120 mg/kg bw/d. The development of the pups itself was unaffected by treatment up to 360 mg/kg bw/d and no teratogenicmalformations are reported. Hence, the developmental NOEL was set at 360 mg/kg bw/d.

Because of the structural similarities it can be assumed, that also for methyl-ionone no effects on development have to be expected.

Justification for classification or non-classification

Due to the negative data obtained for analogue substances evaluating toxicity on fertility and development in studies performed according to OECD Guidelines, no classification is required.

Additional information