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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 december 2012 to June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 414 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: flakes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Number: 96 female rats were received at CiToxLAB France between 07 and 21 December 2012.
- Strain and sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Breeder: Charles River Laboratories Italia, Calco, Italy.
- Age/Weight: at the beginning of the treatment period, the females were 10-11 weeks old and had a mean body weight of 265 g (range: 224 g to 323 g). The females were sexually mature and primigravid.
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2, 48 cm x 26.5 cm x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen because it is preferable for pregnant animals.
Each cage contained an object for the environmental enrichment of the animals (rat hut).
The cages were placed in numerical order on the racks.
- Food and water: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2537604 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly. The diet formula is presented in Appendix 3.
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation: the animals were acclimated to the conditions of the study for a period of 4 or 5 days before the beginning of the treatment period (arrival of the females on day 1 or 2 p.c.).
- Allocation to study: before the beginning of the treatment period, the animals were allocated to the groups, according to a stratification procedure based on body weight recorded on day 2 p.c., to ensure comparatively similar mean body weights of the groups.

- Identification: each animal was individually identified by an ear tattoo (unique CiToxLAB France identity number).

ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 12 cycles/hour of filtered, non-recycled air.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Dose formulation preparation
The test item was administered as a solution in the vehicle.
Before preparing the dose formulations, the test item was ground, using a mortar and pestle. Then, the required quantities were mixed progressively with the vehicle in order to obtain the desired concentration.
After addition of the vehicle, the dose formulations were kept under magnetic stirring for at least 30 minutes to ensure effective solubilization of the test item.
The test item dose formulations were prepared for up to 11 days, stored at room temperature and protected from light and delivered in brown flasks.

VEHICLE
- Lot/batch no. (if required): The vehicle was drinking water treated by reverse osmosis using ELIX 5 (Millipore SA).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 39417 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
Details on mating procedure:
- Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).
Duration of treatment / exposure:
once daily, from days 6 to 20 p.c., inclusive, to time-mated female
Frequency of treatment:
Daily
Duration of test:
Two months and a half
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 mated female per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected by the Sponsor, following the results of a previous study: Combined repeated dose toxicity with the reproduction/developmental toxicity screening test, according to the OECD 422 guideline (report B96142, Sept. 2009).

Examinations

Maternal examinations:
Morbidity and mortality
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

Clinical signs
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

Body weight
The body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

Food consumption
The quantity of food consumed by each female was recorded for the following intervals:
- days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- Other organs examined: ovaries, uterus
Any macroscopic lesions observed were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative).
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.

Uterine horns without visible implantation sites were immersed in an aqueous solution of ammonium sulphide (Salewski, 1964) to reveal the presence of uterine scars, which were counted.

A gross evaluation of placentas was also undertaken.

Body weight of fetuses
The body weight of each live fetus was recorded.

Sex of fetuses
The sex of each fetus was determined at the time of hysterectomy.
The sex of fetuses was determined by visual assessment of anogenital distance and was confirmed by examination of sexual organs at detailed dissection of the soft tissues or at evisceration.
Fetal examinations:
Fetal examination was conducted for all litters where the female had at least one live fetus.

External examination
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

Soft tissue examination
As soon as possible after sacrifice, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head.

Skeletal examination
The remaining live fetuses per litter was eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed from 100 mg/kg/day with few animals affected. This effect was considered to be related to the treatment with the test item, but of minor toxicological significance. This effect was not considered as adverse. Hypoactivity and half-closed eyes were also observed from 100 mg/kg/day in some animals (14/24 for hypoactivity and 12/24 for half-closed eyes). These effect are transient. Plioerection, locomotory difficulties and sedation were observed from 200 mg/kg/day. Round back and tonic contraction were observed at 400 mg/kg/day. All these clinical signs were recorded with dose-related increased incidence/severity and were considered to be test treatment-related from 100 mg/kg/day and of toxicological significant and adverse from 200 mg/kg/day. These effects were considered to be very severe at the dose level of 400 mg/kg/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day and when compared with controls, there were a statistically significant minimal decrease in mean body weight (- 6.7% on day 9 p.c., p<0.05) which resulted in a statistically significant lower mean body weight gain (+3 g vs. +16 g in controls, p<0.001).
At 400 mg/kg/day and when compared with control values, there was a marked mean body weight loss (-19 g) over the period of days 6 to 9 p.c., thereafter mean body weight gain returned toward control values and decreased from day 15 to 21 p.c.. During all the treatment period and at the dose of 400 mg/kg/day, there were statistically significant decreases in mean body weight (up to -15.8% on day 21 p.c. p<0.01). All these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicological significant at 400 mg/kg/day.

There were no effect on mean body weight or mean body weight change at 100 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day and when compared with controls, there were a statistically significant but minimal decrease in mean food consumption at initiation of the treatment period (-11.1% on days 6-9 p.c., p<0.05) which returned to control values thereafter.
At 200 mg/kg/day and when compared with controls, there were a statistically significant and marked decrease in mean food consumption at initiation of the treatment period (-33.3% on days 6-9 p.c., p<0.05) which increased thereafter but remained lower than controls at the end of the treatment period (-9.7% on days 18-21 p.c., p<0.01).
At 400 mg/kg/day and when compared with controls, there were a statistically significant and severe decrease in mean food consumption at initiation of the treatment period (-63.0% on days 6-9 p.c., p<0.001) which never returned to control values (-19.4% on days 18-21 p.c., p<0.01).
All these findings were considered to be test item treatment-related from 100 mg/kg/day and toxicologically significant from 200 mg/kg/day.
Food efficiency:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, there was a statistically significant decrease in mean uterus weight at 400 mg/kg/day (-18.7%, p<0.05). Taking into account the amplitudes of the changes, this finding was considered to be toxicologically significant.
When compared with controls, there were statistically significant decreases in carcass weight at 200 and 400 mg/kg/day (-7.7%, p<0.25 and -14.8%, p<0.01; respectively), resulting in dose related decreased mean body weight changes.
Taking into account the amplitude of the changes, these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 400 mg/kg/day, one female (A25766) showed a dilatation of pelvis (kidneys) and ureter. These findings are commonly observed in this species and strain; therefore, a test item treatment-related effect was considered unlikely.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean implantation loss (19.9% vs. 2.2, p<0.01) corresponding mainly to early resorptions (females A25765 and A25766 had 100% post-implantation losses and female A25770 had 66.7% post-implantation loss) which resulted in decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05). All these findings were observed only at the dose level of 400 mg/kg/day and were considered to be toxicologically significant.
At the two other treated doses (100 and 200 mg/kg/day) and when compared with controls, there was no change in mean post-implantation loss.
When compared with controls, there was no change in mean pre-implantation loss whatever the treated doses.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean implantation loss (19.9% vs. 2.2, p<0.01) corresponding mainly to early resorptions (females A25765 and A25766 had 100% post-implantation losses and female A25770 had 66.7% post-implantation loss) which resulted in decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05). All these findings were observed only at the dose level of 400 mg/kg/day and were considered to be toxicologically significant. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day.

At the two other treated doses (100 and 200 mg/kg/day) and when compared with controls, there was no increase in the mean number of resorptions.

Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean number of early resorptions. There was no change in mean number of early resorptions at the two other tested doses (100 and 200 mg/kg/day). When compared with controls, there was no change in mean number of late resorptions whatever the tested doses.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant decrease in mean number of live fetuses per litter (11.1% vs. 13.8, p<0.05) corresponding mainly to early resorptions.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
At termination on day 21 p.c., there were 22, 23, 21 and 18 rats with live fetuses and 2, 1, 3 and 4 non-pregnant females in the groups treated at 0, 100, 200 and 400 mg/kg/day, respectively. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day. At termination on day 21 p.c., there were 22, 23, 21 and 18 rats with live fetuses and 2, 1, 3 and 4 non-pregnant females in the groups treated at 0, 100, 200 and 400 mg/kg/day, respectively. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PREGNANCY STATUS: At termination on day 21 p.c., there were 22, 23, 21 and 18 rats with live fetuses and 2, 1, 3 and 4 non-pregnant females in the groups treated at 0, 100, 200 and 400 mg/kg/day, respectively. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day.

MORTALITY: There were no unscheduled deaths.

CLINICAL SIGNS (Table 1): Ptyalism was considered to be related to the treatment with the test item, but of minor toxicological significance.
Hypoactivity and half-closed eyes were observed from 100 mg/kg/day in some animals (transient effects), piloerection, locomotory difficulties and sedation from 200 mg/kg/day and, round back and tonic contraction at 400 mg/kg/day. All these clinical signs were recorded with dose-related increased incidence/severity and were considered to be test item treatment-related from 100 mg/kg/day and of toxicological signifance from 200 mg/kg/day.

BODY WEIGHT: At 200 mg/kg/day and when compared with controls, there were a statistically significant minimal decrease in mean body weight (-6.7%, on day 9 p.c., p<0.05) which resulted in a statistically significant lower mean body weight gain (+3 g vs. +16 g in controls, p<0.001).
At 400 mg/kg/day and when compared with control values, there was a marked mean body weight loss (-19 g) over the period of days 6 to 9 p.c., thereafter mean body weight gain returned toward control values and decreased from day 15 to 21 p.c.. During all the treatment period, there were statistically significant decreases in mean body weight (up to -15.8% on day 21 p.c., p<0.01).
All these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day.

FOOD CONSUMPTION: At 100 mg/kg/day and when compared with controls, there were a statistically significant but minimal decrease in mean food consumption at initiation of the treatment period (-11.1% on days 6-9 p.c., p<0.05) which returned to control values thereafter.
At 200 mg/kg/day and when compared with controls, there were a statistically significant and marked decrease in mean food consumption at initiation of the treatment period (-33.3% on days 6-9 p.c., p<0.05) which increased thereafter but remained lower than controls at the end of the treatment period (-9.7% on days 18-21 p.c., p<0.01).
At 400 mg/kg/day and when compared with controls, there were a statistically significant and severe decrease in mean food consumption at initiation of the treatment period (-63% on days 6-9 p.c., p<0.001) which never returned to control values (-19.4% on days 18-21 p.c., p<0.01).
All these findings were considered to be test item treatment-related from 100 mg/kg/day and toxicologically significant from 200 mg/kg/day.

MACROSCOPIC post-mortem EXAMINATION: At 400 mg/kg/day, one female (A25766) showed a dilatation of pelvis (kidneys) and ureter. These findings are commonly observed in this species and strain; therefore, a test item treatment-related effect was considered unlikely.

NET BODY WEIGHT CHANGE: When compared with controls, there was a statistically significant decrease in mean uterus weight at 400 mg/kg/day (-18.7%, p<0.05). Taking into account the amplitudes of the changes, this finding was considered to be toxicologically significant.
When compared with controls, there were statistically significant decreases in carcass weight at 200 and 400 mg/kg/day (-7.7%, p<0.25 and -14.8%, p<0.01; respectively), resulting in dose related decreased mean body weight changes.
Taking into account the amplitude of the changes, these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day.

HYSTERECTOMY DATA (Table 2): At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean implantation loss (19.9% vs. 2.2, p<0.01) corresponding mainly to early resorptions (females A25765 and A25766 had 100% post-implantation losses and female A25770 had 66.7% post-implantation loss) which resulted in decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05). All these findings were considered to be toxicologically significant. No effect were observed at the two other tested doses (100 and 200 mg/kg/day).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, there was a dose-related decrease in mean fetal body weight from 100 mg/kg/day which resulted in a statistically significant difference at 400 mg/kg/day (-15.9%, p<0.01). At 200 mg/kg/day, mean fetal body weight was below the lower limit of the Historical Control Data (5.35 g vs. 5.5 g, respectively). However, taking into account the amplitude of the changes this finding was considered to be toxicologically significant only at 400 mg/kg/day.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
There is a decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05) due to early resorptions in two females at 400 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (mean percentage of male fetuses).
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
There is a decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05) due to early resorptions in two females at 400 mg/kg/day.
External malformations:
effects observed, treatment-related
Description (incidence and severity):

Exencephaly was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.

However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day (Tera, Anasarca, Cleft lip, Cleft palate, Omphalocele, short trunk and short tail) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

The increase fetal incidence of external malformation was statistically significant for the following external malformations: Cleft lip, Cleft palate and Omphalocele.

Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Absent lumbar vertebra(e) was recorded at comparable incidence in the control, 100 and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.

However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day (Vertebra(e), multiple malformations ; Interparietal, unossified ; Skullcap, absent ; Palate, split ; Mandible, fused ; Cervical vertebra(e), fused arches ; Cervical vertebra(e), absent ; Thoracic vertebrae(e), absent ; Supernumerary lumbar vertebra ; Sacral vertebra(e), absent ; Caudal vertebra(e), absent ; Fused sternebrae ; Misshapen sternum ; Misshapen rib ; Absent rib ; Masshapen rib cage and Fused ribs) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

The increase in fetal incidence of each skeletal malformation was not statistically significant. However, the increase in the number of fetus affected at the higher tested dose (400 mg/kg/day) was statistically significant.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Soft tissues malformations:
Misshapen cerebrum was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.

However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day (Cleft palate, Anophthalmia, and Misshapen inner ear) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data. The increase in fetal incidence was statistically significant for Anophthalmia.

Soft tissues variations:
When compared with the control group or Historical Control Data, there were no test item treatment-related soft tissues variations.

External variations:
Malrotated limb, curled tail and domed head were not previously recorded in contemporaneous Historical Control Data. When compared with the control group or to the Historical Control Data, the increased litter and fetal incidences of external variations at 400 mg/kg/day were considered to be related to the test item treatment. The increase was not statistically significant.

Skeletal variations:
Forepaw(s) with unossified distal phalanx, unossified 1st metatarsal, hindpaw(s) with unossified distal phalanx, were observed at comparable litter and/or fetal incidences across groups, therefore a test item treatment-related effect was considered unlikely for these observations. However, when compared with the control group or to the Historical Control Data, the increased litter and fetal incidences in all other skeletal variations at 400 mg/kg/day (Frontal, incomplete ossification ; Fontanel, enlarged ; Maxilla, incomplete ossification ; Premaxilla, incomplete ossification and Metacarpal(s), unossified) were considered to represent significant ossifications delay and to be related to the test item treatment. The increase in fetal incidence was statistically significant for the following skeletal variations: Frontal, incomplete ossification ; Fontanel, enlarged ; Maxilla, incomplete ossification ; Premaxilla, incomplete ossification and Metacarpal(s), unossified.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHT AND SEX RATIO: When compared with controls, there was a dose-related decrease in mean fetal body weight from 100 mg/kg/day which resulted in a statistically significant difference at 400 mg/kg/day (-15.9%, p<0.01). At 200 mg/kg/day, mean fetal body weight was below the lower limit of the Historical Control Data (5.35 g vs. 5.5 g, respectively). However, taking into account the amplitude of the changes this finding was considered to be toxicologically significant only at 400 mg/kg/day.
There were no effects on sex ratio (mean percentage of male fetuses).

EXTERNAL EXAMINATION (Tables 3 and 4): Malrotated limb, curled tail and domed head were not previously recorded in contemporaneous Historical Control Data. When compared with the control group or to the Historical Control Data, the increased litter and fetal incidences of external variations at 400 mg/kg/day were considered to be related to the test item treatment.
Exencephaly was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

SOFT TISSUE EXAMINATION (Tables 5 and 6): When compared with the control group or Historical Control Data, there were no test item treatment-related soft tissues variations.
Misshapen cerebrum was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

CARTILAGE AND SKELETAL EXAMINATION (Tables 7 and 8): In control, 100 and 200 mg/kg/day group, there were no toxicologically significant findings at cartilage examination.
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in fused cartilage of cervical vertebra(e) [11.1 (3.5 *) vs. 0.0 (0.0) in terms of litter (fetal) incidences, p<0.05) and two fetuses from the same litter (A25763) with fused cartilage of ribs. Taking into account the findings described below, these observations were considered to be related to the test item treatment.
Forepaw(s) with unossified distal phalanx, unossified 1st metatarsal, hindpaw(s) with unossified distal phalanx, were observed at comparable litter and/or fetal incidences across groups, therefore a test item treatment-related effect was considered unlikely for these observations.
However, when compared with the control group or to the Historical Control Data, the increased litter and fetal incidences in all other skeletal variations at 400 mg/kg/day were considered to represent significant ossifications delay and to be related to the test item treatment.

Absent lumbar vertebra(e) was recorded at comparable incidence in the control, 100 and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

DISTRIBUTION OF FETAL MALFORMATION (table 9): Taking into account both litter and fetal incidences, there was a marked increase of malformations at 400 mg/kg/day. There was no effect at the two other tested doses 100 and 200 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
Development
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
skeletal malformations
other: Soft tissues malformations, external and skeletal malformations.
Remarks on result:
other:
Remarks:
All the effects observed on fetuses: increase in number of live fetuses, decrease in mean fetal body weight, increases in variations (external and skeletal) and increase in malformations (external, soft tissues and skeletal) were observed only at the dose level of 400 mg/kg/day in a context of very marked (excessive) maternal toxicity.

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
external: face
external: tail
external: trunk
skeletal: skull
skeletal: sternum
skeletal: rib
skeletal: vertebra
visceral/soft tissue: gastrointestinal tract
Description (incidence and severity):
for more details see tables 3, 4, 5, 6, 7, 8 and 9.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

MATERNAL DATA

Table 1: Maternal clinical signs

Dose-level (mg/kg/day)

0

100

200

400

Piloerection

/

/

9

(from day 7 up to 20 p.c.)

10

(from day 7 up to 20 p.c.)

Round back

/

/

/

1

(from day 7 to 9 p.c.)

Tonic contraction

/

/

/

13

(from day 6 up to 13 p.c.)

Hypoactivity

/

14

(from day 6 up to 20 p.c.)

24

(from day 6 up to 20 p.c.)

23

(from day 6 up to 20 p.c.)

Locomotory difficulties

/

/

9

(from day 6 up to 18 p.c.)

17

(from day 6 up to 20 p.c.)

Sedation

/

/

11

(from day 6 up to 20 p.c.)

19

(from day 6 up to 13 p.c.)

Ptyalism

/

1

(from day 18 up
to 20 p.c.)

3

(from day 15 up to 18 p.c.)

3

(from day 16 up to 20 p.c.)

Half-closed eyes

 

12

(from day 6 up
 to 20 p.c.)

24

(from day 6 up to 20 p.c.)

23

(from day 6 up to 20 p.c.)

Number of affected animals

0/24

15/24

24/24

24/24

( ): in brackets, days of first and last occurrence of the clinical signs.

Table 2: Hysterectomy data

Dose-level (mg/kg/day)

0

100

200

400

HCD
[min.-max.]

Number of females with live fetuses

22

23

21

18

150(a)

Mean number ofcorpora luteaper animal

15.6

15.4

16.4

14.8

14.0 - 15.5

Mean number of implantations per animal

14.1

13.6

14.8

13.2

12.8 - 14.0

Mean pre-implantation loss (%)

9.6

12.8

10.1

11.4

7.2 - 13.9

Mean number of live fetuses per litter

13.8

13.3

14.1

11.1*

12.0 - 13.2

Mean number of early resorptions

0.3

0.3

0.6

2.0**

/

Mean number of late resorptions

0

0

0

0.1

/

Mean post-implantation loss (%)

2.2

1.6

4.7

19.9**

2.0 - 8.7

Statistical significance:*: p<0.05, **: p<0.01.

HCD: Historical Control Data.

(a): control data collected from seven studies covering a period ranging from February 2008 to March 2012.

/: not listed.

FETUS DATA

Table 3: Litter (L) and Fetal (F) incidences of external variations

Dose-level (mg/kg/day)

0

100

200

400

HCD

Dams with live fetuses, n

22

23

21

18

143(a)

Fetuses examined, n

303

307

296

222

1813

Malrotated limb, L(F) %

 

 

 

5.6 (0.5)

-

Curled tail, L(F) %

 

 

 

5.6 (0.5)

-

Domed head, L(F) %

 

 

 

5.6 (0.5)

-

Litters affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (11.1)

3 (2.1)

Fetus affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

3 (1.4)

16 (0.9)

Table 4: Litter (L) and Fetal (F) incidences of external malformations

Dose-level (mg/kg/day)

0

100

200

400

HCD

Dams with live fetuses, n

22

23

21

18

143(a)

Fetuses examined, n

303

307

296

222

1813

Tera (b), L(F) %

 

 

 

5.6 (0.5)

-

Anasarca, L(F) %

 

 

 

5.6 (0.5)

-

Cleft lip, L(F) %

 

 

 

11.1 (2.3*)

-

Cleft palate, L(F) %

 

 

 

11.1 (2.3*)

-

Omphalocele, L(F) %

 

 

 

11.1 (3.2**)

-

Short trunk, L(F) %

 

 

 

5.6 (0.5)

-

Short tail, L(F) %

 

 

 

5.6 (0.5)

-

Exencephaly, L(F) %

4.5 (0.3)

 

 

5.6 (1.4)

-

Litters affected, n (%)

1 (4.5)

0 (0.0)

0 (0.0)

4 (22.2)

1 (0.7)

Fetus affected, n (%)

1 (0.3)

0 (0.0)

0 (0.0)

10** (4.5)

1 (0.1)

Statistical significance:*: p<0.05, **: p<0.01.

HCD: Historical Control Data.

(a): control data collected from seven studies covering a period ranging from February 2008 to March 2012.

/: not listed.

Applicant's summary and conclusion

Conclusions:
Up to 200 mg/kg/day, the test item elicited no developmental toxicity in a context of moderate to marked maternal toxicity as demonstrated by adverse clinical signs (piloerection, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption.

At 400 mg/kg/day these findings were more severe and associated with increased post-implantation losses (mainly as a consequence of increased early resorptions). At this dose level only and in such a context, developmental delays (reduced affecting fetal body weight and ossification associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded. The developmental effects (variations and malformations) and the resorptions were only observed at the higher tested dose (400 mg/kg/day) in a context of very marked (excessive) maternal toxicity. These effects seem to be indirect effects as a secondary non-specific consequence of maternal toxicity effects (maternal toxicity too excessive at 400 mg/kg/day).

While the study was conducted according to the guideline and under GLP conditions, the high dose level was exceeded the guideline and therefore fetal endpoints at the 400 mg/kg/day dose level are not considered relevant for evaluation in this study (see more explanation in the endpoint summary 'Toxicity to reproduction').
Executive summary:

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, Paramethoxyphenol, once daily from day 6 to day 20 p.c., by gavage at dosages of 100, 200 or 400 mg/kg/day. An additional group of 24 time-mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as the control group. A dose volume of 12 mL/kg/day was used.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

Results

Clinical signs: Ptyalism was observed in all test item-treated groups and considered to be related to the treatment with the test item, but of minor toxicological significance. Hypoactivity and half-closed eyes were observed from 100 mg/kg/day, piloerection, locomotory difficulties and sedation from 200 mg/kg/day and, round back and tonic contraction at 400 mg/kg/day. All these clinical signs were recorded with dose-related increased incidence/severity and were considered to be test item treatment-related from 100 mg/kg/day and of toxicological significance from 200 mg/kg/day.

Mean body weight and mean body weight changes: At 200 mg/kg/day, there were a minimal decrease in mean body weight which resulted in a lower mean body weight gain. At 400 mg/kg/day, there was a marked mean body weight loss over the period of days 6 to 9 p.c., thereafter mean body weight gain returned toward control values and decreased again from days 15 to 20 p.c.. During all the treatment period, there were decreases in mean body weight. All these findings were considered to be test item treatment-related from 200 mg/kg/day and of toxicological significance at 400 mg/kg/day.

Mean food consumption: At 100 mg/kg/day, there was a minimal decrease in mean food consumption at initiation of the treatment period which returned to control values thereafter. At 200 mg/kg/day, there was a marked decrease in mean food consumption at initiation of the treatment period which returned to control values thereafter. At 400 mg/kg/day, there was a severe decrease in mean food consumption at initiation of the treatment period which never returned to control values. All these findings were considered to be test item treatment-related from 100 mg/kg/day and of toxicological significance from 200 mg/kg/day.

Necropsy and hysterectomy data: At necropsy, there were no test item treatment-related effects at macroscopic examination. At 400 mg/kg/day, there was a decrease in mean uterus weight and from 200 mg/kg/day a

decrease in mean carcass weight. These findings were considered to be treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day. At 400 mg/kg/day, there were increased mean implantation loss (corresponding mainly to early resorptions) and decreased mean number of fetuses per litter. All these findings were considered to be toxicologically significant.

Fetal examination: There were no effects on sex-ratio. At 400 mg/kg/day, there were marked decreases in mean fetal body weight and in mean number of live fetuses per litter. At fetal examination, there were increased litter and fetal incidences of variations (external and skeletal) and malformations (external, soft tissues and skeletal).

In conclusion, up to 200 mg/kg/day, the test item elicited no developmental toxicity in a context of moderate to marked maternal toxicity as demonstrated by adverse clinical signs (piloerection, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption. At 400 mg/kg/day these findings were more severe and associated with increased post-implantation losses (mainly as a consequence of increased early resorptions). At this dose level only and in such a context of very severe (excessive) maternal toxicity, developmental delays (reduced affecting fetal body weight and ossifictaion associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded. At 400 mg/kg/day the maternal toxicity was very severe and beyond the 'High dose' as intended in OECD guideline 414. Therefore fetal observations at this dose level should not be considered for assessment.

While the study was conducted according to the guideline and under GLP conditions, the high dose level exceeded the guideline and therefore fetal endpoints at the 400 mg/kg/day dose level are not considered relevant for evaluation in this study (see more explanation in the endpoint summary 'Toxicity to reproduction').

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse clinical signs (piloerection, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption from 200 mg/kg/day and increased post-implantation losses (mainly as a consequence of increased early resorptions) at 400 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 200 mg/kg/day, based upon no findings at this dose level. As noted earlier fetal observations at 400 mg/kg/day are not considered relevant for classification and labelling purposes in this study due to the excessive maternal toxicity in this dose level.