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EC number: 202-888-7 | CAS number: 100-79-8
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 01 FEB 2022 to 12 APR 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study performed according to the OECD TG 414 without any deviation.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- Reference substance 001
- Cas Number:
- 100-79-8
- Test material form:
- liquid
- Remarks:
- colorless
- Details on test material:
- - Density: 1.069
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Temperature set to maintain controlled room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability analysis performed in another study demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations between 1 and 200 mg/mL for 9 days. Dose formulation homogeneity was also demonstrated by GC/FID analysis.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not expected
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not relevant
TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None
Test animals
- 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 Laboratoires France -ESD, Chatillon sur Chalaronne, France
- Age at study initiation: 4 to 5 months
- Weight at study initiation: 2950 to 4265 g
- Fasting period before study: no
- Housing: single housed in noryl cages. For psychological / environmental enrichment, animals were provided with dumbbell and hay except when interrupted by study procedures/activities. Music was also put on for the same purpose.
Females were gently handled every day from arrival in order to allow habituation to manipulation.
- Diet: Pelleted complete diet, batch No. 102494 (Diet Reference No. 3409, KLIBA) and compacted hay pellets, batch No. 6801103 (SSNIFF), sterilized by irradiation, ad libitum. In addition, in case of drastically reduced food consumption during the acclimation period (≤ 50 g/animal/day), carrots were distributed daily. It was considered that there were no known contaminants in the feed that interfered with the objectives of the study.
- Water: Municipal tap water filtered with a 0.22 μm filter, ad libitum. It was considered that there were no known contaminants in the water that interfered, with the outcome of the study.
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 21
- Humidity (%): 30 to 70
- Air changes (per hr): 5 to 15 (filtered, non-recycled air)
- Photoperiod (hrs dark / hrs light): 8 / 16
IN-LIFE DATES: From: 02 MAR 2022 To: 12 APR 2022
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- other: Phosphate Buffer Saline solution (PBS) pH 7.4 (1X)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dose formulations were prepared according to stability data and divided into aliquots where required to allow them to be dispensed on each dosing occasion.
Control item was administered as a solution and test item as a suspension.
VEHICLE
- Justification for use and choice of vehicle: Stability analyses performed previously in Study No. 39833 AHS demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
- Lot/batch no.: 2276826 (expiry JUN 2023) and 2276826 (NOV 2023)
- Purity: not specified - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose formulation samples were analyzed by Gas Chromatography with FID detection (GC/FID) for the determination of AUGEO SL 191.
The dose formulations prepared in the first, second and last weeks of treatment were all found to be within the acceptance criteria (measured concentrations within ± 15% of the theoretical concentrations). No test item was found in the control dose formulation.
Dose formulation homogeneity was also demonstrated, as the Relative Standard Deviation (RSD) was found to be ≤ 10% for each tested group. - Details on mating procedure:
- - Impregnation procedure: purchased timed pregnant. Females were mated by the supplier and delivered on GD0 and GD1.
- Duration of treatment / exposure:
- From GD6 to GD28, inclusive
- Frequency of treatment:
- Once daily at approximately the same time of the day
- Duration of test:
- From GD 6 to GD 28 inclusive
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group No. 1 (control item)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group No. 2
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group No. 3
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Group No. 4
- No. of animals per sex per dose:
- 22
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
- 1. Results from a tolerability study of the test material in non-pregnant rabbits where two groups of three females were administered by oral gavage for two periods of 7 days (with at least a 7-day washout) and staggered start between dose levels and groups (Study No. 49529 TSL (Hauray et al., 2022)). In this study, there were no unscheduled death and no test item-related clinical signs. A minimal body weight loss was in females treated at 1000 mg/kg bw/day, after 7 days of treatment (- 0.86% on Day 22 vs. Day 15 value) associated with minimal lower food intake. There were no effects at 300 mg/kg/day.
- 2. Results from a dose-range finding study of the test material in the pregnant rabbits where three group of six females were administered by oral gavage from GD6 to 28 inclusive, at 100, 300 or 1000 mg/kg bw/day. An additional group of 6 females received the vehicle only [Phosphate Buffer Saline solution (PBS) pH 7.4 (1X)] and served as a control group (Study No. 49530 RSL; Spézia et al., 2022). In this study, there were no test item related clinical signs, no adverse effects on body weight/body weight change, no effects on food consumption and no test-item related findings at maternal necropsy or hysterectomy. At external fetal examination, there were no test-item related findings.
- Rationale for animal assignment: computerized randomization procedure based on body weight recorded on GD0 or GD1 and provided by the breeder, to ensure comparatively similar mean body weights of the groups.
Examinations
- Maternal examinations:
- MORTALITY: Yes
At least twice dailya (at the beginning and end of working day) beginning upon arrival through termination.
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily beginning upon arrival through termination.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In addition to the daily cageside observation, animals were observed between 1 and 3 hours post-dosing. A full clinical examination was performed on GD2 and on each weighing day during dosing phase and on the day of euthanasia.
BODY WEIGHT: Yes
- Time schedule for examinations: on GD2, 5, 6, 9, 12, 15, 18, 21, 24, 27 and 29.
FOOD CONSUMPTION: Measured daily from GD2.
WATER CONSUMPTION: Visual inspection of the water bottles.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #29 (intravenous injection of sodium pentobarbital)
- Organs examined: abdominal and thoracic cavities including the uterus. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all fetuses
- Soft tissue examinations: Yes: all live fetuses
- Skeletal examinations: Yes: all live fetuses
- Head examinations: Yes: half of the fetuses
- Anogenital distance of all live rodent pups: No - Statistics:
- Body weight, food consumption and reproductive data:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions). - Indices:
- See details in "Any other information on materials and methods incl. tables".
- Historical control data:
- Historical control data - 2021 on Reproductive toxicity provided by Charles River.
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Only a few findings [loud breathing, wound/scab(s) and/or soft feces] were observed on only a few occasions and/or with no-dose level relationship. In addition, they are common observations in animals dosed by gavage and maintained under the experimental conditions of this study.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No effects on mean body weight.
No statistically significant differences in mean body weight changes compared to controls.
At 1000 mg/kg bw/day, there was a minimal body weight loss (-9 g vs. +27 g in controls on GD 6-9) with a return toward control values from GD 9-12. A test item-related effect cannot be excluded, but taking into account the reversibility and the amplitude of the change, this finding was considered to be non-adverse.
At 300 and 100 mg/kg bw/day, there were no effects on mean body weight change. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, mean food consumption was low from the beginning of the treatment period (down to -31% vs. controls on GD 7-8 and GD 8-9 with p < 0.01 and 0.001, respectively) with a return towards control values from GD 22-23. Taking into account the amplitude of the differences and the duration of the effect, this finding was considered to be test-item related and adverse.
At 300 and 100 mg/kg bw/day, there were no effects on mean food consumption. - Food efficiency:
- not examined
- Description (incidence and severity):
- Not relevant.
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- Not relevant.
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- not specified
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on mean gravid uterus weight, mean carcass weight or mean net body weight change.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test-item related findings at the macroscopic examination.
No statistically significant differences in gross pathology findings compared to controls. Only a few findings (thymus/gall bladder discoloration, stomach with discolored area and/or periovarian serous cysts) were observed on only a few occasions and/or with no dose level relationship. In addition, they are common observations in the female New-Zealand white rabbit of this age and physiological condition. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- not examined
- Changes in number of pregnant:
- not examined
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- There were no effects on hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses).
Effect levels (maternal animals)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- food consumption and compound intake
Maternal abnormalities
- Key result
- Abnormalities:
- no effects observed
Results (fetuses)
- Fetal body weight changes:
- no effects observed
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- not specified
- Anogenital distance of all rodent fetuses:
- not examined
- Changes in postnatal survival:
- not examined
- Description (incidence and severity):
- not relevant
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- No statistically significant differences compared to the controls.
Variations:
At 1000 mg/kg bw/day, one litter had 2 fetuses (4508-06 and 4508-12) with protruding tongue (variation) and open eyes (malformation). In addition, fetus 4508-12 had a local edema and an enlarged abdomen (variations).
At 300 and 100 mg/kg bw/day, there were no variations at external examination of the fetuses.
Malformations:
At 1000 mg/kg/day, three fetuses from 2 litters had a malformation that was not observed in controls:
• fetuses 4508-06 and 4508-12 with open eyes (bilateral),
• fetus 4522-10 with omphalocele (intestine protruding through the abdominal wall and
covered by a thin transparent membrane).
At 300 and 100 mg/kg bw/day, there were no malformations at external examination of the fetuses. - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- No statistically significant differences compared to controls.
At 1000 mg/kg bw/day, absences of some cartilages were noted in fetus(es) with hemivertebra(e) (and hence absence of ribs).
From 100 mg/kg bw/day, the cartilage of hyoid was present, but not ossified.
Variations:
At 1000 mg/kg bw/day, when compared to the upper limit of the Historical Control Data, there were increased litter incidences of thoracic vertebra(e) with unossified arch(es) (4.8% vs. 0.0% in HCD) and unossified rib(s) (4.8% vs. 0.0 in HCD). These finding were considered to be test item-related.
From 100 mg/kg bw/day and when compared to controls, there was a dose-related increase in the incidence of unossified 5th sternebra. While the incidences remained close to upper limit of the HCD, a test-item relationship cannot be excluded.
All these findings were considered to represent delays in ossification (non-adverse test item effects).
All other fetal skeletal variations not presented in the above table were of isolated fetal and/or litter incidences, were not dose-related, and/or were of similar/lower incidence when compared to controls or Historical Control Data.
Malformations:
Fused sternebra and, supernumerary or absent pre-sacral vertebrae are not presented in the above table:
• fused sternebra(e) are no longer considered to be malformations, but normal stages in the development of a sternebra,
• supernumerary pre-sacral vertebra is described as supernumerary lumbar vertebra,
• absence of pre-sacral vertebra is described as absent lumbar vertebra.
At 1000 mg/kg bw/day, three fetuses from 2 different litters had at least one malformation at skeletal
examination (ossified bones):
• fetus 4502-04 with supernumerary lumbar vertebra (also described as supernumerary pre-sacral vertebra),
• fetus 4503-01 with fused thoracic vertebra(e),
• fetus 4503-02 with fused thoracic vertebra(e) and absent thoracic hemivertebra(e).
At 300 mg/kg/day, 2 litters had a fetus with a skeletal malformation (ossified bones):
• fetus 3517-05 with absent thoracic hemivertebra(e),
• fetus 3514-02 with absent lumbar vertebra (also described as the absence of pre-sacral vertebra).
At 100 mg/kg bw/day, 2 litters had a fetus with a skeletal malformation (ossified bones):
• fetus 2512-02 with branched rib(s),
• fetus 2507-07 with absent lumbar vertebra (also described as the absence of pre-sacral vertebra).
In controls, two fetuses from 2 different litters had a malformation at skeletal examination (ossified bones):
• fetus 1504-05 with split nasal bone,
• fetus 1514-04 with absent lumbar vertebra (also described as absence of pre-sacral vertebra). - Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- No statistically significant differences compared to controls.
Variations:
At 1000 mg/kg bw/day, one litter had 2 fetuses (4507-06 and 4507-09) with liquid content in the cranial cavity (variation) associated with a marked dilated cerebral ventricle (malformation, see following section).
At 300 and 100 mg/kg bw/day, there were no test-item related variations at fetal soft tissue examinations.
The few other soft tissue variations are common observations in this strain of rabbit and the litter incidences were not dose-related and below or close to the upper limit of the Historical Control Data. Therefore, any relationship to the test-item was considered to be unlikely.
Malformations:
At 1000 mg/kg bw/day, there were 4 fetuses from 3 different litters with at least one soft-tissue malformation:
• fetuses 4507-06 and 4507-10 with bilateral microphthalmia (small eyes) and marked dilated cerebral ventricles,
• fetuses 4507-07 and 4508-06 with bilateral microphthalmia,
• fetus 4507-09 with marked dilated cerebral ventricle/short right ureter/dilated left
ureter/malpositioned kidney,
• fetus 4508-12 with a dilated aortic arch,
• fetus 4510-08 with a core trioculare (three-chambered heart, common atrium) and a dilated aortic arch.
At 300 mg/kg bw/day, one litter had a malformed fetus:
• fetus 3502-01 with a short right ureter and a malpositioned kidney.
At 100 mg/kg/day, there were no malformations at fetal soft tissue examinations. In controls, 3 litters had a malformed fetus:
• fetus 1509-02 with a core trioculare and a dilated aortic arch;
• fetuses 1518-08 and 1521-04 with a right malpositioned kidney and a short ureter.
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- external malformations
- skeletal malformations
- visceral malformations
Fetal abnormalities
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- external: eye
- skeletal: vertebra
- other: brain
- Description (incidence and severity):
- 11 malformed fetuses at 1000 mg/kg bw/day (6 abnormal litters; 1 to 4 malformed fetuses per litter)
Overall developmental toxicity
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- no
Any other information on results incl. tables
MATERNAL TOXICITY
Pregnancy Status
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Number of time-mated females | 22 | 22 | 22 | 22 |
Pregnant females | 19 | 21 | 21 | 21 |
Females with live fetuses at termination | 19 | 21 | 21 | 21 |
Non-pregnant females | 3 | 1 | 1 | 1 |
Clinical signs
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Loud breathing | 1 |
|
|
|
Wound (neck) |
|
| 1 |
|
Scab(s) (interscapular region, ears, neck) |
|
| 1 | 2 |
Soft feces |
|
|
| 1 |
(): in brackets, period of occurrence.
Mean body weight (g)
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Body weight (g) | ||||
GD 2 (pre-treatment) | 3466 | 3435 | 3459 | 3468 |
| (-1) | (0) | (0) | |
GD 5 (pre-treatment) | 3487 | 3464 | 3537 | 3506 |
| (-1) | (+1) | (+1) | |
GD 6 | 3504 | 3468 | 3562 | 3530 |
| (-1) | (+2) | (+1) | |
GD 9 | 3531 | 3511 | 3601 | 3521 |
| (-1) | (+2) | (0) | |
GD 12 | 3578 | 3563 | 3654 | 3555 |
| (0) | (+2) | (-1) | |
GD 15 | 3650 | 3645 | 3729 | 3640 |
| (0) | (+2) | (0) | |
GD 18 | 3677 | 3655 | 3769 | 3665 |
| (-1) | (+3) | (0) | |
GD 21 | 3699 | 3698 | 3796 | 3709 |
| (0) | (+3) | (0) | |
GD 24 | 3741 | 3734 | 3843 | 3755 |
| (0) | (+3) | (0) | |
GD 27 | 3794 | 3788 | 3905 | 3812 |
| (0) | (+3) | (0) | |
GD 29 | 3868 | 3855 | 3967 | 3892 |
| (0) | (+3) | (+1) | |
( ): in brackets, percentage difference vs. controls.
No statistically significant differences vs. controls
Mean Body Weight Changes (g)
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Body weight change (g) | ||||
GD 2-5 (pre-treatment | +41 | +29 | +79 | +38 |
GD 5-6 (pre-treatment) | +18 | +4 | +25 | +25 |
GD 6-9 | +27 | +43 | +39 | -9 |
GD 9-12 | +47 | +52 | +53 | +33 |
GD 12-15 | +72 | +82 | +75 | +85 |
GD 15-18 | +27 | +10 | +40 | +25 |
GD 18-21 | +22 | +43 | +27 | +44 |
GD 21-24 | +41 | +36 | +47 | +46 |
GD 24-27 | +54 | +55 | +61 | +56 |
GD 27-29 | +74 | +66 | +62 | +80 |
GD 6-29 | +364 | +387 | +405 | +362 |
No statistically significant differences vs. controls.
Mean Food Consumption (g/day/animal)
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
GD 2-3 (pre-treatment) | 120 | 122 | 126 | 147 |
| (+2) | (+5) | (+23) | |
GD 3-4 (pre-treatment) | 152 | 155 | 184 | 164 |
| (+2) | (+21) | (+8) | |
GD 4-5 (pre-treatment) | 181 | 161 | 184 | 170 |
| (-11) | (+2) | (-6) | |
GD 5-6 (pre-treatment) | 177 | 168 | 193 | 176 |
| (-5) | (+9) | (-1) | |
GD 6-7 | 161 | 167 | 173 | 135 |
| (+4) | (+7) | (-16) | |
GD 7-8 | 173 | 160 | 183 | 120** |
| (-8) | (+6) | (-31) | |
GD 8-9 | 163 | 163 | 166 | 112# |
| (0) | (+2) | (-31) | |
GD 9-10 | 164 | 165 | 171 | 115# |
| (+1) | (+4) | (-30) | |
GD 10-11 | 160 | 172 | 173 | 128* |
| (+8) | (+8) | (-20) | |
GD 11-12 | 151 | 161 | 164 | 125 |
| (+7) | (+9) | (-17) | |
GD 12-13 | 138 | 143 | 146 | 111 |
| (+4) | (+6) | (-20) | |
GD 13-14 | 137 | 142 | 143 | 115 |
| (+4) | (+4) | (-16) | |
GD 14-15 | 142 | 144 | 150 | 120 |
| (+1) | (+6) | (-15) | |
GD 15-16 | 148 | 152 | 160 | 134 |
| (+3) | (+8) | (-9) | |
GD 16-17 | 166 | 167 | 173 | 144 |
| (+1) | (+4) | (-13) | |
GD 17-18 | 171 | 169 | 168 | 150 |
| (-1) | --2) | (-12) | |
GD 18-19 | 157 | 156 | 151 | 138 |
| (-1) | (-4) | (-12) | |
GD 19-20 | 151 | 162 | 157 | 137 |
| (+7) | (+4) | (-9) | |
GD 20-21 | 151 | 153 | 153 | 140 |
| (+1) | (+1) | (-7) | |
GD 21-22 | 141 | 137 | 138 | 131 |
| (-3) | (-2) | (-7) | |
GD 22-23 | 130 | 129 | 135 | 136 |
| (-1) | (+4) | (+5) | |
GD 23-24 | 113 | 120 | 130 | 122 |
| (+6) | (+15) | (+8) | |
GD 24-25 | 111 | 114 | 119 | 123 |
| (+3) | (+7) | (+11) | |
GD 25-26 | 113 | 126 | 127 | 123 |
| (+12) | (+12) | (+9) | |
GD 26-27 | 127 | 129 | 120 | 121 |
| (+2) | (-6) | (-5) | |
GD 27-28 | 132 | 135 | 134 | 128 |
| (+2) | (+2) | (-3) | |
GD 28-29 | 140 | 135 | 138 | 143 |
| (-4) | (-1) | (+2) |
( ): in brackets, percentage difference vs. controls;
Statistically significant differences vs. controls: *: p<0.05; **: p<0.01; #: p<0.001.
Summary of Gross Pathology Findings
| Females | |||
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 |
Number of Animals per Group | 22 | 22 | 22 | 22 |
Thymus: reddish color | 1 |
|
|
|
Stomach: colored areas |
| 1 |
|
|
Gall bladder: abnormal color |
|
|
| 1 |
Female gonads: serous cyst on connective tissue, periovarian region | 2 | 1 | 5 | 3 |
No statistically significant differences vs. controls.
Mean Carcass Weights, Net Body Weight Changes and Gravid Uterus Weights (g)
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Gravid uterus weight | 575.5 | 523.5 | 582.5 | 576.1 | [483.0; 589.5] |
|
| (-9) | (+1) | (0) |
|
Carcass weight | 3292.9 | 3331.0 | 3384.4 | 3315.8 | [3287.0; 3525.0] |
|
| (+1) | (+3) | (+1) |
|
Net body weight change from GD 6 | -211.5 | -136.6 | -178.0 | -214.4 | [-296.9; -121.7] |
| (-35) | (-16) | (+1) |
|
( ): in brackets, percentage difference vs. controls.
No statistically significant differences vs. controls.
HCD: Historical Control Data, n = 8 studies (2019 to 2021).
Hysterectomy Data
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Number of pregnant females at hysterectomy | 19 | 21 | 21 | 21 | 183 |
Number of females with live fetuses at termination | 19 | 21 | 21 | 21 | 165 |
Number of females with total resorption | 0 | 0 | 0 | 0 | 0 |
Mean number of corpora lutea | 10.9 | 10.4 | 12.0 | 11.1 | [10.8; 13.0] |
Mean number of implantation sites | 9.8 | 8.9 | 10.2 | 9.9 | [9.2; 10.6] |
Mean pre-implantation loss (%) | 9.5 | 14.4 | 13.8 | 10.8 | [9.4; 21.7] |
Mean number of live fetuses | 9.4 | 8.6 | 9.7 | 9.4 | [8.7; 9.9] |
Dead fetuses (%) | 0.0 | 0.0 | 0.4 | 0.0 | [0.00; 0.39] |
Mean number of implantation scars | 0.0 | 0.0 | 0.0 | 0.0 | / |
Mean number of early resorptions | 0.2 | 0.2 | 0.1 | 0.2 | / |
Mean number of late resorptions | 0.3 | 0.0 | 0.3 | 0.3 | / |
Mean post-implantation loss (%) | 4.4 | 3.5 | 4.4 | 4.8 | [2.6; 13.2] |
No statistically significant differences vs. controls.
HCD: Historical Control Data, n = 8 studies (2019 to 2021).
/: not available in HCD
FETAL TOXICITY
Mean Fetal Body Weights
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Mean fetal body weight (male fetuses, g) | 42.4 | 42.9 | 41.6 | 41.5 | [38.9; 43.8] |
| (+1) | (-2) | (-2) |
| |
Mean fetal body weight (female fetuses, g) | 39.5 | 40.6 | 40.0 | 39.9 | [37.0; 42.5] |
| (+3) | (+1) | (+1) |
| |
Mean fetal body weight (male and female fetuses combined, g) | 41.1 | 41.7 | 40.7 | 40.7 | [37.8; 43.3] |
| (+1) | (-1) | (-1) |
|
( ): in brackets, percentage difference vs. controls.
No statistically significant differences vs. controls.
HCD: Historical Control Data, n = 8 studies (2019 to 2021).
Mean Percentages of Male Fetuses
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Mean percentage of male fetuses (%) | 50.6 | 53.7 | 47.6 | 53.0 | [42.3; 55.3] |
| (+6) | (-6) | (+5) |
|
( ): in brackets, percentage difference vs. controls.
No statistically significant differences vs. controls
HCD: Historical Control Data, n = 8 studies (2019 to 2021).
Litter (L) and Fetal (F) incidences (%) of External Variations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 205 | 198 | 1518 |
General |
|
|
|
|
|
. local edema, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0; 0.0] |
Mouth, jaw, palate |
|
|
|
|
|
. protruding tongue, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [0.0; 0.0] |
Trunk |
|
|
|
|
|
. enlarged abdomen, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0; 4.8] |
n: number.
No statistically significant differences vs. controls.
HCD: Historical Control Data, n = 8 studies (2019 to 2021).
Litter (F) and Fetal (F) incidences (%) of External Malformations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 205 | 198 | 1518 |
Eyes |
|
|
|
|
|
. open eye, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [0.0; 4.8] |
Trunk |
|
|
|
|
|
. omphalocele, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0; 5.6] |
Litters affected, n (%) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 2 (9.5) | 7 (4.3) |
Fetuses affected, n (%) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 3 (1.5) | 8 (0.5) |
n: number.
No statistically significant differences vs. controls.
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)
Litter (F) and Fetal (F) incidences (%) of External Malformations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 205 | 198 | 1518 |
Brain |
|
|
|
|
|
. cranial cavity: liquid content, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [0.0; 0.0] |
n: number.
No statistically significant differences vs. controls.
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)
Litter (L) and Fetal (F) Incidences (%) of Soft Tissue Malformations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 204 | 198 | 1518 |
Eye |
|
|
|
|
|
. microphthalmia, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 9.5 (2.5) | [5.0] |
Brain |
|
|
|
|
|
. marked dilated cerebral ventricle, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.5) | [4.5] |
Heart |
|
|
|
|
|
. cor triloculare, L (F) | 5.3 (0.6) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [4.8] |
Kidneys |
|
|
|
|
|
. malpositioned kidney, L (F) | 10.5 (1.1) | 0.0 (0.0) | 4.8 (0.5) | 4.8 (0.5) | [0.0] |
Vessels |
|
|
|
|
|
. dilated aortic arch, L (F) | 5.3 (0.6) | 0.0 (0.0) | 0.0 (0.0) | 9.5 (1.0) | [5.6] |
Ureter |
|
|
|
|
|
. short ureter, L (F) | 10.5 (1.1) | 0.0 (0.0) | 4.8 (0.5) | 4.8 (0.5) | [0.0] |
Litters affected, n (%) | 3 (15.8) | 0 (0.0) | 1 (4.8) | 3 (14.3) | 18 (11.0) |
Fetuses affected, n (%) | 3 (1.7) | 0 (0.0) | 1 (0.5) | 7 (3.5) | 19 (1.3) |
n: number.
No statistically significant differences vs. controls
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)
Litter (L) and Fetal (F) incidences (%) of Skeletal Cartilages
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 204 | 198 | 1518 |
Head-others |
|
|
|
|
|
. cartilage of hyoid centrum: present, L (F) | 0.0 (0.0) | 4.8 (0.6) | 4.8 (0.5) | 9.5 (2.0) | [33.3] |
Thoracic vertebra(e) |
|
|
|
|
|
. cartilage of thoracic vertebra(e): fused, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [0.0] |
. cartilage of thoracic vertebra(e): absent, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [4.3] |
Caudal vertebra(e) |
|
|
|
|
|
. cartilage of caudal vertebra(e): absent, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [9.1] |
Rib |
|
|
|
|
|
. cartilage of rib(s): absent, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [4.3] |
n: number.
No statistically significant differences vs. controls
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)
Litter (L) and Fetal (F) incidences (%) of Skeletal Variations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 204 | 198 | 1518 |
Head-others |
|
|
|
|
|
. hyoid: incomplete ossification of centrum | 0.0 (0.0) | 4.8 (0.6) | 4.8 (0.5) | 9.5 (2.0) | [44.4] |
Thoracic vertebra(e) |
|
|
|
|
|
. unossified arch, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0] |
Sternebra |
|
|
|
|
|
. unossified 5th sternebra, L (F) | 52.6 (13.5) | 47.6 (15.5) | 61.9 (15.2) | 81.0 (26.8**) | [80.0] |
. unossified 6th sternebra, L (F) | 15.8 (3.4) | 9.5 (2.2) | 14.3 (1.5) | 28.6 (5.1) | [38.1] |
. incomplete ossification of 1st to 4th sternebra(e) , L (F) | 5.3 (0.6) | 4.8 (0.6) | 0.0 (0.0) | 19.0 (2.0) | [28.6] |
. extra sternebral ossification site, L (F) | 5.3 (1.1) | 4.8 (0.6) | 0.0 (0.0) | 9.5 (1.0) | [10.0] |
Rib |
|
|
|
|
|
. unossified rib(s) , L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0] |
Statistically significant difference vs. controls **: p<0.01.
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021).
Litter (L) and Fetal (F) Incidences (%) of Skeletal Malformations
Dose level (mg/kg bw/day) | 0 | 100 | 300 | 1000 | HCD [Min.; Max.] |
Litters evaluated, n | 19 | 21 | 21 | 21 | 163 |
Fetuses evaluated, n | 178 | 181 | 204 | 198 | 1518 |
Head-skull |
|
|
|
|
|
. nasal: split, L (F) | 5.3 (0.6) | 0.0 (0.0) | 4.8 (0.5) | 0.0 (0.0) | [0.0; 0.0] |
Thoracic vertebra(e) |
|
|
|
|
|
. fused, L (F) | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (1.0) | [0.0; 4.3] |
. absent hemivertebra(e) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | 4.8 (0.5) | [0.0; 4.3] |
Lumbar vertebra(e) |
|
|
|
|
|
. absent | 5.3 (0.6) | 4.8 (0.6) | 4.8 (0.5) | 0.0 (0.0) | [0.0; 9.1] |
. supernumerary | 0.0 (0.0) | 0.0 (0.0) | 0.0 (0.0) | 4.8 (0.5) | [0.0; 0.0] |
Rib |
|
|
|
|
|
. branched rib(s) | 0.0 (0.0) | 4.8 (0.6) | 0.0 (0.0) | 0.0 (0.0) | [0.0; 0.0] |
n: number.
No statistically significant differences vs. controls
HCD: Historical Control Data (litter incidences), n = 8 studies (2019 to 2021)
Applicant's summary and conclusion
- Conclusions:
- Under the test conditions of this study, the NOAEL (maternal and fetal) = 300 mg/kg bw/day.
- Executive summary:
In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, the test material diluted in PBS was administered through gavage to groups of New Zealand White time-mated rabbits (22/dose) at dose levels of 0 (vehicle control), 300 and 1000 mg/kg bw/day from Day 6 to 28 of pregnancy. The females were checked daily for mortality and clinical signs. Body weights were recorded at designated intervals. Food consumption was recorded daily. On GD 29, the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early/late resorptions and live/dead fetuses were recorded. Placentas were observed. The fetuses were weighed and sexed by internal examination of the gonads. Detailed external, soft tissue and skeletal examinations (including cartilage) were performed in the fetuses. Macroscopic lesions were collected from the dams and preserved in 10% buffered formalin.
Maternal toxicity:
There were no unscheduled deaths and no test-item related clinical signs. There were no adverse effects on mean body weight at any dose-levels [only a minimal body weight loss at 1000 mg/kg bw/day (-9 g vs. +27 g in controls on GD 6-9) with a return towards control values from
GD 9-12]. Mean food consumption was low at 1000 mg/kg/day from the beginning of the treatment period (down to -31% vs. controls on GD 7-8 and GD 8-9 with p < 0.01 and 0.001, respectively) with a return towards control values from GD 22-23. Taking into account the amplitude of the differences and the duration of the effect, this finding was considered to be test-item related and adverse. At 300 and 100 mg/kg/day, there were no effects on mean food consumption. At necropsy, there were no test-item related macroscopic findings and no effects on mean gravid uterus weight, mean carcass weight, mean net body weight change or hysterectomy data (mean pre-/post-implantation losses and mean number of live fetuses).Embryo-Fetal Toxicity:
There were no effects on mean fetal body weight or sex ratio (percentage of male fetuses). At 1000 mg/kg bw/day, 6 litters had fetuses with a series of malformations (external, soft-tissue and/or skeletal examinations) that were only observed at this dose level or with higher incidences when compared to controls or Historical Control Data. At 300 and 100 mg/kg bw/day, there were no test-item related malformations. From 100 mg/kg bw/day and when compared to controls, there were dose-related delays in ossification (i.e. variations), which were considered to represent non-adverse test item-related effects.
On the basis of the experimental conditions and results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 300 mg/kg bw/day based on the decreased mean food consumption (down to -31% controls ) at 1000 mg/kg bw/day,
- the NOAEL for embryo-fetal development was considered to be 300 mg/kg bw/day based on the increased incidence of fetal malformations at 1000 mg/kg bw/day.
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