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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:
2020-08-10 to 2020-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018-06-25
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2017-05-08
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Zirconium praseodymium yellow zircon
EC Number:
269-075-7
EC Name:
Zirconium praseodymium yellow zircon
Cas Number:
68187-15-5
Molecular formula:
Pr(x)Zr(1-x)SiO4 0,03≤x≤0,09
IUPAC Name:
Zirconium praseodymium yellow zircon
Test material form:
solid: particulate/powder
Details on test material:
- Name: Colorante amarillo
- EC Name: Zirconium Praseodymium Yellow Zircon
- Substance type: inorganic pigment
- Physical state: solid, yellow powder, odourless
- Storage condition of test material: at room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25 °C, stored dry in tightly closed containers

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7 ,97633 Sulzfeld, Germany
- Age (on day 0 of pregnancy): 55 - 62 days
- Weight (on day 0 of pregnancy): 172.4 - 260.0 g
- Housing (exception: mating period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt/Arkeburg, Germany; bedding material did not contain unacceptable high levels of hormonally active substances); environmental enrichment: one piece of wood (certified for animal use) to gnaw on and octagon-shaped red-tinted huts (polycarbonate).
- Diet (ad libitum): commercial diet ssniff® R/Z V1154 (ssniff Spezialdiäten GmbH, Soest, Germany); food did not contain unacceptable high levels of hormonally active substances
- Water (ad libitum): drinking water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55 % ± 10 % (maximum range)
- Air changes: between 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.8 % aqueous hydroxypropylmethylcellulose gel
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared on every administration day.

The test item was suspended in the vehicle to the appropriate concentrations and was administered as a single dose orally at a constant volume/kg bw. The test item was administered at approximately the same time each day. The administration formulations were continuously agitated by stirring throughout the entire administration procedure to ensure homogeneity.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.

Administration volume: 10 mL/kg bw/day

VEHICLE
- Supplier: Fagron GmbH & Co. KG, Glinde, Germany
- Lot: 19G17-B04194737
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, two aliquots of exact 5 mL each were taken at the following times and stored at -20 °C ± 10 % (only one aliquot was analysed; the second aliquot served as back-up):

1) At start of dosing:
- analysis of stability and concentration: immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature (3 sample/test item group; number of samples 3 x 3 = 9 (18)).

- homogeneity: at the start of treatment, during (middle) administration and before administration to the last animal of the test item group (3 samples/test item group; number of samples 3 x 3 = 9 (18))

2) At the end of the dosing period, at a time when the majority of the animals was dosed:

- analysis of concentration: during treatment always before administration to the last animal of the group (1 sample/test item group; number of samples: 1 x 3 = 3 (6)).

The sample containers were weighed prior to filling (empty) and after filling (filled) and the weights were provided to the test site conducting the formulation analysis.

Method:
The dry weight of undissolved test item was determined gravimetrically for each application solution after lyophilization until weight constancy. In addition, the lyophilisation residues were melted in order to measure their zirconium and praseodymium content by ICP-OES.

Results:
The results of the test item-formulation analyses for the investigated parameters are as follows:

Range of % nominal concentration:
- stability: 94.1 % - 97.4 %
- concentration (before administration of the last animal of each dose group at a time when the majority of animals was dosed): 92.9 % - 105 %
- homogeneity: 82.7% - 99.4%

For the homogeneity, the test item concentrations at start of the administration for the intermediate (84.5%) and high dose group (82.7%) were below the range of the admissible test item concentrations for suspensions (85 - 115% of the nominal test item concentration). However, all other test item concentrations (92.2 – 105.0%) in the test item formulations were within the admissible range. Therefore, the test item formulations were considered to be correctly prepared.

The measurement of zirconium and praseodymium concentrations in dissolved products of lyophilization residue melts revealed recovery rates across all samples between 91.5% and 104% for zirconium and 80.7% and 99.0% for praseodymium. These results verify the nominal concentrations of the application solutions and that the test item remains unchanged.

Based on the results, the measured actual concentrations of the test item in the test item vehicle-mixtures indicate correctly prepared, stable and homogenous formulations.
Details on mating procedure:
- Impregnation procedure: cohoused (during the dark period)

- M/F ratio per cage: 1 male / 1 female
Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner.

- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

- Male rats for mating remained untreated.

- non-pregnant rats were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Gestation day 6 to gestation day 20
Frequency of treatment:
once daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 pregnant female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected by the Sponsor based on available toxicological data.

NOTE: the current study has a joint control group with another study (Report No. 38111). This study included 13 control animals and the other study included 12 animals in order to obtain altogether 20 live litters for evaluation.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
1) clinical signs: immediately after administration, obervations were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

2) mortality: early in the morning and again in the afternoon of each working day (saturdays and sundays: final check was carried out at approx. midday)

- Cage side observations checked: clinical signs (incl. faeces), mortality, abortion, premature delivery and with special attention to signs of irritation after dosing (e.g increased salivation or redness of the oral cavity).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation day 0 followed by daily weighing (always at the same time of the day).

The body weight gain was calculated in intervals (i.e. gestation day 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 20), for the whole study (gestation day 0 - 20) and for the period after the start of dosing (gestation day 6 to gestation day 20).

The carcass weight and the net weight gain from day 6 were determined, as follows:
Net weight gain from day 6 = carcass weight minus day 6 body weight
Carcass weight = terminal body weight minus gravid uterine weight

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg bw/day) was calculated using the following formula:
Daily food consumption (g/kg bw/day) = total food intake in g/ body weight in kg

WATER CONSUMPTION: Yes (visual appraisal)
- Time schedule for examinations: daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
On the 21st day of gestation the rats were laparotomized under CO2 narcosis. Necropsy was scheduled across groups and necropsy technicians. For example, on the first day technician A processes animals of groups 1 and 2, while technician B processes animals of groups 3 and 4. The next day, technician A processes groups 2 and 3, while technician B processes groups 4 and 1. This rotation is continued over all necropsy days. The ovaries, thyroids including parathyroid and the uteri were removed. The thyroid including parathyroid and the gravid uterus including cervix were weighed. For calculation of the relative weights of the thyroids and the gravid uterus, the respective organ weights were divided by the carcass weight (carcass weight = terminal body weight minus gravid uterine weight).

In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. The thyroid (including parathyroid) and any organs with macroscopic findings of all dams were fixed in 7% neutral buffered formalin. The thyroids of all evaluated dams were examined histopathologically after preparation or hematoxylin-eosin stained paraffin sections.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight including cervix: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes, all per litter (dead or alive)
- Soft tissue examinations: Yes, half per litter
The foetuses were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.

- Skeletal examinations: Yes, half per litter
The foetuses were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).

The foetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

- External foetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations).

-Indication of incomplete testicular descent/cryptorchidism was noted in male foetuses

- Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations or abnormal appearance (e.g. size, colour, shape).
- The number of foetuses (alive and dead at the time point of sacrifice of the dams) and placentae (location in the uterus and the assignment of the foetuses) was determined.
- Sex and viability (spontaneous breathing, spontaneous movement) of foetuses were determined.
- Location of foetuses in the uterus.
- Weights of foetuses and weights of the placentae were determined (foetuses were considered as runts if their weight was less than 70% of the mean litter weight).
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis (Provantis integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the foetuses) or an internal computer program (e.g. findings during the foetal skeletal or soft tissue examination).
Indices:
- Total malformation rate (%)* = (malformed foetuses per group / foetuses per group) x 100

- Total vairiation rate (%)* = (foetuses per group with variations / foetuses per group) x 100

- Total retardation rate (%)* = (foetuses per group with retardations / foetuses per group) x 100

- Pre-implantation loss (%) = ((corpora lutea per group - implantations per group) / corpora lutea per group) x 100

- Post-implantation loss (%) = ((implantations per group - living foetuses per group) / implantations per group) x 100

- Pre-implantation loss (%)= sum of pre-implantation losses per dam in a group (%) / number of litters in a group

- Post-implantation loss (%) = sum of post-implantation losses per dam in a group (%) / number of litters in a group

* foetuses affected by several changes will be counted as one foetal incidence.
Historical control data:
Historical control data was provided by the laboratory for the following parameters (data collected from 2000 to 2017):
- general reproductive indices (laparotomy on gestation day 21, since 2016)
- foetal external malformations and variations
- foetal skeletal malformations (laparotomy on gestation day 20 or 21)
- foetal skeletal variations (laparotomy on gestation day 20 or 21)
- foetal skeletal retardations (laparotomy on gestation day 21, since 2016)
- foetal visceral malformations (laparotomy on gestation day 20 or 21)
- foetal visceral variations (laparotomy on gestation day 20 or 21)
- serum thyroid hormone levels (T3, T4 and TSH; gestation day 21)

Please also refer for historical control data to the field "Attached background material" below.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
NOTE: the current study has a joint control group with another study (Report No. 38112). This study included 13 control animals and the other study included 12 animals in order to obtain altogether 20 live litters for evaluation.

CLINICAL SIGNS:
- 0, 100, 300, and 1000 mg/kg bw/day: no changes in behaviour or the external appearance were noted in the control group and the dose groups. Also, no changes were noted for the faecal consistency and the faecal output of any dose group.

MORTALITY:
- 0, 100, 300, and 1000 mg/kg bw/day: no premature deaths were noted in the control group and the dose groups.

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences in body weight were noted between the dams of the control group and the dose groups. The difference in the body weight between the control group and the dose groups ranged from -0.5% to +3.2% during the whole study period.

- 100, 300, and 1000 mg/kg bw/day: no test item-related difference between the control group and the dose groups was noted for the body weight gain from gestation day 0 to 20 (body weight gain of +5.7 %, +3.0 %, and +4.2 % for 100, 300 and 1000 mg/kg bw/day compared to the control group, respectively) and in the dosing period from gestation day 6 to gestation day 20 (body weight gain of +5.6 %, +3.0 %, and +3.7 % for 100, 300 and 1000 mg/kg bw/day compared to the control group, respectively). There was no test item-related influence of the gravid uterus weight on the body weight gain for any group.

- 100, 300 and 1000 mg/kg bw/day: no test item-related difference was noted for the carcass weight between the control dams (258.68 g ± 18.43) and the dams of the dose groups (268.82 g ± 19.20, 268.98 g ± 18.37 and 263.94 g ± 18.93 for the 100, 300 and 1000 mg/kg bw/day dose groups, respectively). Furthermore, no test item-related differences were noted for the net body weight gain from gestation 6 to 21 between the dams of the control group and the dams of the dose groups.

- 100 and 300 mg/kg bw/day: a statistically significant increase was noted for the net body weight gain (16.57 g ± 7.11 or 18.48 g ± 10.03 of the low or intermediate dose group compared to 11.17 g ± 7.89 of the control group, p ≤ 0.05). However, no dose response relationship was noted as the net body weight gain of the high dose group was 12.78 g ± 8.16. Therefore, the increased net body weight gain in the low and intermediate dose groups was considered to be spontaneous and not test item-related.

FOOD CONSUMPTION:
- 100, 300 and 1000 mg/kg bw/day: no test item-related difference was noted between the control group and the treatment groups.

- 100 mg/kg bw/day: a statistically significantly increased food consumption was noted between gestation day 15 and gestation day 16 and between gestation day 16 and gestation day 17 (8.5% and 7.3% above the value of the control group, respectively; p ≤ 0.01). However, as no difference was noted for the 300 mg/kg bw/day and 1000 mg/kg bw/day dose group, the transiently increased food consumption was considered to be spontaneous.

WATER CONSUMPTION:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.

ENDOCRINE FINDINGS:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences were noted for the serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) in all dose groups compared to the control group. The mean values of the T3, T4 and TSH thyroid hormone concentrations were within the range of the background data.

Please also refer to the field "Attached background material" below.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences to the control group were noted for the absolute and relative thyroid weights of the dose groups. Furthermore, no test item-related differences were noted for the gravid uterus weight (absolute and relative) between the control dams and the dams of the dose groups.

GROSS PATHOLOGICAL FINDINGS:
- 100, 300 and 1000 mg/kg bw/day: no test item-related pathological changes were noted for the dams of the dose groups during the macroscopic inspection of the organs and tissues.

- 100 mg/kg bw/day: one dam was noted with a yellow focus on the right medial lobe of the liver. However, this single occurrence was considered to be not test item-related but spontaneous.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- 100, 300 and 1000 mg/kg bw/day: no test item-related morphological lesions were noted during histopathological examination of the thyroids of the dose groups.

The findings noted during evaluation of the left and right thyroids did not differ with regard to incidence and severity between the control group and the dose groups. In summary, a keratinized cyst were noted in 11 thyroids of group 1 (control), in 9 thyroids of the 100 mg/kg bw/day group and in 19 thyroids each of the 300 and 1000 mg/kg bw/day groups. With the exception of two findings in group 1 (control), one finding in the 300 mg/kg bw/day group and 3 findings in the 1000 mg/kg b.w./day group in the left or right thyroid with a mild severity, all findings were of minimal severity. The cyst(s) were noted for 9 (group 1; control), 8 (100 mg/kg b.w.), 14 (300 mg/kg b.w.) or 16 (1000 mg/kg b.w.) different animals.
As no statistically significant difference between the control group and any of the dose groups was present, the observed morphological changes were considered to be not test item-related.

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:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
NUMBER OF ABORTIONS.
- 100, 300, and 1000 mg/kg bw/day: no abortions occurred during the study. Also, no premature delivery were observed.

PRE- AND POST IMPLANTATION LOSS:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence on the index of pre- and post-implantation loss was noted between the dams of the control group and the dams of the treatment groups. Although there was an increase in the post-implantation loss in the dose groups compared to the control group, this parameter was considered to be not test item-related as the parameter was within the range of the control groups of the laboratory background data and no statistically significant changes were observed.

The values for post-implantation loss can be seen below:

Control: 2.9 %
100 mg/kg bw/day: 3.4 %
300 mg/kg bw/day: 3.6 %
1000 mg/kg bw/ day: 4.0 %
Laboratory background data (mean %):
- control animals: 3.46 % ± 1.14 (range: 1.80 % - 5.32 %)
- test animals not significantly influenced by any test item: 3.53 % ± 1.80 (range: 1.00 % - 9.68 %)

Please also refer to the field "Attached background material" below.

TOTAL LITTER LOSSES BY RESORPTION
- 100, 300, and 1000 mg/kg bw/day: no litter was totally lost to resorption. The resorption per litter ranged for the control group between 0.0 % - 18.8 % (mean number per dam: 0.5), for the 100 mg/kg bw/day dose group, between 0.0 % - 18.8 % (mean number per dam: 0.5), for the 300 mg/kg bw/day dose group between 0.0 % - 30.8 % (mean number per dam: 0.5), and for the 1000 mg/kg bw/day dose group between 0.0 % - 12.5 % (mean number per dam: 0.6). Although there was an increase in the total number of resorptions compared to the control, this parameter was considered to be not test item-related as the parameter was within the range of the control groups of the laboratory background data and no statistically significant changes were observed.

Laboratory background data (total resorption, mean number per dam):
- control animals: 0.49 ± 0.15 (range: 0.20 - 0.70)
- test animals not significantly influenced by any test item: 0.48 ± 0.76 (range: 0.20 - 1.40)

Please also refer to the field "Attached background material" below.

EARLY OR LATE RESORPTIONS:
- 100, 300, and 1000 mg/kg bw/day: no test item-related influence on the early or late resorptions was noted between the dams of the control group and the dams of the treatment groups. All values were within the laboratory background data.

DEAD FOETUSES:
- 100, 300, and 1000 mg/kg bw/day: no foetuses died during the study.
No test item-related influence on the number of live foetuses was observed. The number of live foetuses was above the background data range of the control group. As this was the result of a high number of corpora lutea that developed before start of dosing on gestation day 6 and a quit low pre-implantation loss the higher number of live foetuses in the control group was considered to be spontaneous

Please also refer to the field "Attached background material" below.

CHANGES IN NUMBER PREGNANT:
- 100, 300, and 1000 mg/kg bw/day: no test item-related influence on the number of pregnant animals was noted between the dams of the control group and the dams of the treatment groups.

CORPORA LUTEA:
- 100, 300, and 1000 mg/kg bw/day: no test item-related influence on the number of corpora lutea was noted between the dams of the control group and the dams of the treatment groups. All values were within the laboratory background data, except for the value of the control group, which was above the laboratory background data.

The values for corpora lutea can be seen below (mean number per dam):

Control: 15.6 ± 1.7
100 mg/kg bw/day: 15.3 ± 2.5
300 mg/kg bw/day: 14.9 ± 1.9
1000 mg/kg bw/ day: 15.2 ± 1.5
Laboratory background data (mean number per dam):
- control animals: 14.11 ± 0.54 (range: 13.30 - 15.20)
- test animals not significantly influenced by any test item: 14.23 ± 0.73 (range: 12.70 - 16.00)

Please also refer to the field "Attached background material" below.

IMPLANTATION SITES:
- 100, 300, and 1000 mg/kg bw/day: no test item-related influence on the number of implantation sites was observed between the dams of the control group and the dams of the treatment groups.

- 0, 100 and 1000 mg/kg bw/day: the number of implantation sites was above the background data range for the control group. No dose-response relationship was observed. As this was the result of a high number of corpora lutea that developed before start of dosing on gestation day 6 and a quite low pre-implantation loss, the high numbers of implantation sites in the control group were considered to be spontaneous.

The values for implantation sites can be seen below (mean number per dam):

Control: 15.5 ± 1.8
100 mg/kg bw/day: 14.9 ± 2.6
300 mg/kg bw/day: 14.5 ± 2.5
1000 mg/kg bw/ day: 14.9 ± 1.5
Laboratory background data (mean number per dam):
- control animals: 13.70 ± 0.67 (range: 12.50 - 15.00)
- test animals not significantly influenced by any test item: 13.81 ± 0.76 (range: 12.40 - 15.80)

Please also refer to the field "Attached background material" below.

Effect levels (maternal animals)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
FOETAL BODY WEIGHT CHANGES:
- 100, 300 and 1000 mg/kg bw/day: the foetal weights showed no test item-related differences between the control group and the dose groups.

- 100, 300 and 1000 mg/kg bw/day: the foetal weights of dose groups were below the range of the laboratory background data. However, as also the weights of the control foetuses were below the background data range, no statistically significant differences were noted between the control group and the dose groups and as no dose response-relationship was present, the foetal weights below the background data range were considered to be spontaneous.

- 100 mg/kg bw/day: no runts were noted for the low dose group.

- 0, 300 and 1000 mg/kg bw/day: in the control group and in the 1000 mg/kg bw/day dose group, one runt each was noted and in the 300 mg/kg bw/day dose group three runts were noted. Although the number of three runts was above the laboratory background data range (control animals or test animals not significantly influenced by any test item: 0 - 2 runts), the occurrence of three runts in the 300 mg/kg bw/day group was considered to be spontaneous as only one runt was noted in the 1000 mg/kg bw/day dose group.

The foetal weights (mean ± SD; g) are shown below:

- male foetuses:
control group: 5.14 ± 0.32 g
100 mg/kg bw/day: 5.30 ± 0.28 g
300 mg/kg bw/day: 5.23 ± 0.33 g
1000 mg/kg bw/day: 5.26 ± 0.33
Laboratory background data:
- control animals: 5.49 ± 0.10 g (range: 5.31 g - 5.67 g)
- test animals not significantly influenced by any test item: 5.47 ± 0.11 g (range: 5.28 g - 5.68 g)

- female foetuses:
control group: 4.83 ± 0.28 g
100 mg/kg bw/day: 4.95 ± 0.28 g
300 mg/kg bw/day: 4.91 ± 0.27 g
1000 mg/kg bw/day: 4.97 ± 0.28 g
Laboratory background data:
- control animals: 5.20 ± 0.08 g (range: 5.05 g - 5.32 g)
- test animals not significantly influenced by any test item: 5.18 g ± 0.10 g (range: 5.00 g - 5.39 g)

- male and female foetuses combined:
control group: 4.99 ± 0.31 g
100 mg/kg bw/day: 5.13 ± 0.28 g
300 mg/kg bw/day: 5.05 ± 0.29 g
1000 mg/kg bw/day: 5.11 ± 0.29 g
Laboratory background data:
- control animals: 5.35 ± 0.08 g (range: 5.21 g - 5.48 g)
- test animals not significantly influenced by any test item: 5.33 ± 0.10 g (range: 5.15 g - 5.53 g)

CHANGES IN SEX RATIO:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences between the ratio of male and female foetuses were noted between the control group and the dose groups (control group: 1.07; 100 mg/kg bw/day: 1.09; 300 mg/kg bw/day: 0.86; 1000 mg/kg bw/day: 0.97). The values are in the range of the laboratory background data (control animals: 1.00 ± 0.14 (range: 0.77 - 1.40); test animals not significantly influenced by any test item: 1.02 ± 0.10 (range: 0.75 - 1.28)).

ANOGENITAL DISTANCE:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences to the control group were noted for the foetal ano-genital distance of the dose groups.

- 100 and 1000 mg/kg bw/day: a slight but statistically significantly reduced absolute and/or relative ano-genital distances were noted for the male and female foetuses at the low and high dose level. As no dose-response relationship was noted, the differences were considered to be spontaneous.

The results of the anogenital distance (absolute and relative values) can be seen below:

- male foetsues (absolute anogenital distance; ocular units; mean ± SD)
control group: 27.0 ± 2.4
100 mg/kg bw/day: 25.5 ± 2.0
300 mg/kg bw/day: 25.9 ± 1.5
1000 mg/kg bw/day: 25.4 ± 1.6 (p ≤ 0.05)

- male foetuses (relative anogenital distance; ocular unit/ g bw; mean ± SD)
control group: 15.63 ± 1.18
100 mg/kg bw/day: 14.65 ± 1.17 (p ≤ 0.01)
300 mg/kg bw/day: 14.96 ± 0.84
1000 mg/kg bw/day: 14.59 ± 0.87 (p ≤ 0.01)

- female foetuses (absolute anogenital distance; ocular units; mean ± SD)
control group: 12.9 ± 1.9
100 mg/kg bw/day: 11.6 ± 1.7 (p ≤ 0.05)
300 mg/kg bw/day: 12.2 ± 1.7
1000 mg/kg bw/day: 11.4 ± 1.3 (p ≤ 0.05)

- female foetuses (relative anogenital distance; ocular unit/ g bw; mean ± SD)
control group: 7.62 ± 1.06
100 mg/kg bw/day: 6.84 ± 1.02 (p ≤ 0.05)
300 mg/kg bw/day: 7.18 ± 1.00
1000 mg/kg bw/day: 6.67 ± 0.78 (p ≤ 0.01)

EXTERNAL MALFORMATION:
- 0, 100 and 300 mg/kg bw/day: no macroscopically visible external changes were noted for the foetuses of the control group and the low and intermediate dose groups during the external inspection at laparotomy.

- 1000 mg/kg bw/day: one foetus was noted with multiple malformations in form of a short abdomen and tail and a misshapen head (back region of the head protruded) and a variation in form of the flexure of the hind-limbs. As the single occurrence of one fetus with multiple malformations and a variation was within the background data range, the malformations and the variation noted for the foetus were considered to be spontaneous.

SKELETAL MALFORMATION:
- 100, 300 and 1000 mg/kg bw/day: no skeletal malformations were noted for the foetuses of the control group and the test item-treated groups during the skeletal examination according to DAWSON.

Skeletal variations:
- 100, 300 and 1000 mg/kg bw/day: skeletal variations were noted for the ribs (accessory 14th rib (control group 0.7%/low dose group 0.0%/intermediate dose group 0.0%/high dose group 0.0% of the foetuses affected), less than 13 ribs ossified (0.7/0.7%/0.7%/0.0%) or short (0.7%/0.0%/0.0%/0.7%)), the sternum (fused (0.0%/0.7%/0.0%/0.0%) or misaligned to a slight degree (1.3%/2.8%/5.7%/1.4%)), the lumbar vertebral bodies (fused (0.0%/0.0%/0.0%/0.7%)) and the thoracic vertebral bodies (misaligned (0.7%/0.0%/0.0%/0.0%)).

No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the foetuses of the treatment groups.

A statistically significantly increased incidence of misaligned sternebrae was noted for the 300 mg/kg bw/day dose group. However, the incidence was within the laboratory background data range and no dose response relationship was present. Therefore, the increased incidence of misaligned sternebrae in the intermediate dose group was considered to be not test item-related.

All values for the incidences of the skeletal variations for the dose groups were within the range of the LPT background data.

Laboratory background data (foetal incidence %):
Sternebrae (misaligned)
- control animals: 1.39 % ± 1.69 (range: 0.00 % - 9.40 %)
- test animals not significantly influenced by any test item: 1.41 % ± 1.57 (range: 0.00 % - 8.50 %)

Sternebrae (minor fusion)
- control animals: 0.04 % ± 0.18 (range: 0.00 % - 0.80 %)
- test animals not significantly influenced by any test item: 0.04 % ± 0.26 (range: 0.00 % - 3.00 %)

Thoracic vertebral body/bodies (misaligned)
- control animals: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)
- test animals not significantly influenced by any test item: 0.02 % ± 0.11 (range: 0.00 % - 0.90 %)

Ribs (less than 13 rib(s) ossified)
- control animals: 0.11 % ± 0.48 % (range: 0.00 % - 2.90 %)
- test animals not significantly influenced by any test item: 0.17 % ± 0.74 (range: 0.00 % - 5.60 %)

Ribs (accessory 14 rib(s))
- control animals: 0.02 % ± 0.11 (range: 0.00 % - 0.80 %)
- test animals not significantly influenced by any test item: 0.09 % ± 0.35 (range: 0.00 % - 2.10 %)

Ribs (shortened)
- control animals: 0.11 % ± 0.35 (range: 0.00 % - 1.60 %)
- test animals not significantly influenced by any test item: 0.18 % ± 0.41 (range: 0.00 % - 2.20 %)

Skeletal retardations:
- 0, 100, 300 and 1000 mg/kg bw/day: retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas (control group 4.6%/low dose group 0.7%/intermediate dose group 7.1%/high dose group 6.3% of the foetuses affected), the hyoid (unossified (25.8%/41.0%/27.1%/35.7%)), the sternum (sternebra(e) incompletely ossified (0.7%/4.9%/1.4%/2.1%), reduced in size (57.6%/47.2%/57.1%/49.7%) or unossified (2.0%/0.7%/0.7%/2.8%)), the thoracic vertebral bodies (bipartite (2.6%/6.3%/5.0%/5.6%), dumbbell-shaped (5.3%/17.4%/9.3%/13.3%), reduced in size (0.7%/0.0%/0.0%/0.0%) or unossified (0.0%/0.0%/0.0%/0.7%)), the lumbar vertebral bodies (bipartite (0.7%/0.0%/0.0%/0.0%)) the caudal vertebral bodies (all bodies unossified (0.0%/0.0%/0.0%/0.7%)), the cervical vertebral bodies (all bodies unossified (0.0%/0.0%/0.0%/0.7%)), the os ischii (unossified (0.0%/0.0%/0.0%/0.7%)), os pubis (incompletely ossified (0.0%/0.7%/1.4%/0.0%) or unossified (0.0%/0.7%/0.0%/0.7%)) the metacarpalia (absence of ossification in metacarpalia 2 to 5 (0.7%/0.0%/0.7%/0.7%)) and the metatarsalia (absence of ossification in metatarsalia 2 to 5 (0.0%/0.7%/0.7%/0.0%)).

No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg test item/kg bw/day was noted during skeletal examination according to DAWSON.

A statistically significant increased incidence of unossified hyoid was noted for the 100 mg/kg bw/day and 1000 mg/kg bw/day dose group. However, this finding was considered to be spontaneous as the incidences were within the laboratory background data range and no dose dependence relationship noted.

Furthermore, a statistically significant decreased incidence of incomplete ossification of the skull was observed for the 100 mg/kg bw/day dose group. This finding was considered to be spontaneous as the incidence was onyl slightly below the laboratory background data range and the incidence was decreased.

In addition, a statistically significant increased incidence of throracic vertebral bodies (dumbbell-shaped) was observed for the 100 mg/kg bw/day and 1000 mg/kg bw/day dose group. However, this finding was considered to be spontaneous as the incidences were within the laboratory background data range and no dose dependence relationship noted.

Also, an increased incidence of cervical vertebral bodies (all bodies unossified) was observed for the 1000 mg/kg bw/day dose group. The single observation of cervical vertebral bodies being unossified for one foetus of the high dose group was considered to be spontaneous.

Lastly, an increased incidence of thoracic vertebral (bodies unossified) was observed for the 1000 mg/kg bw/day dose group. The single observation of thoracic vertebral bodies being unossified for one foetus of the high dose group was considered to be spontaneous.

All other skeletal retardations observed were within the range of the laboratory background data and also did not display a statistically significant difference between the control group and the dose groups.

Laboratory background data (foetal incidence %):

Skull (incompletely ossified)
- control animals: 17.89 % ± 12.57 (range: 1.70 % - 43.90 %)
- test animals not significantly influenced by any test item: 17.56 % ± 10.93 (range: 0.80 % - 45.60 %)

Hyoid bone (unossified)
- control animals: 54.18 % ± 16.30 (range: 24.30 % - 77.60 %)
- test animals not significantly influenced by any test item: 52.57 % ± 15.04 (range: 17.30 % - 79.20 %)

Sternebrae (unossified)
- control animals: 6.71 % ± 3.24 (range: 2.30 % - 13.60 %)
- test animals not significantly influenced by any test item: 7.48 % ± 4.50 (range: 0.70 % - 21.40 %)

Sternebrae (incompletely ossified)
- control animals: 10.28 % ± 6.09 (range: 3.40 % - 26.70 %)
- test animals not significantly influenced by any test item: 8.38 % ± 5.23 (range: 0.00 % - 25.90 %)

Sternebrae (reduced in size)
- control animals: 60.66 % ± 10.29 (range: 42.90 % - 78.00 %)
- test animals not significantly influenced by any test item: 61.64 % ± 9.21 (range: 38.90 % - 81.30 %)

Cervical vertrebral body/bodies (unossified)
- control animals: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)
- test animals not significantly influenced by any test item: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)

Thoracic vertebral body/bodies (unossified)
- control animals: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)
- test animals not significantly influenced by any test item: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)

Thoracic vertebral body/bodies (reduced in size)
- control animals: 0.05 % ± 0.21 (range: 0.00 % - 0.80 %)
- test animals not significantly influenced by any test item: 0.05 % ± 0.19 (range: 0.00 % - 0.80 %)

Thoracic vertebral body/bodies (dumbbell-shaped)
- control animals: 8.40 % ± 3.62 (range: 2.30 % - 14.10 %)
- test animals not significantly influenced by any test item: 9.29 % ± 5.18 (range: 0.70 % - 19.00 %)

Thoracic vertebral body/bodies (bipartite)
- control animals: 2.55 % ± 1.49 (range: 0.70 % - 5.10 %)
- test animals not significantly influenced by any test item: 2.51 % ± 1.77 (range: 0.00 % - 7.00 %)

Lumbar vertebral body/bodies (bipartite)
- control animals: 0.05 % ± 0.21 (range: 0.00 % - 0.80 %)
- test animals not significantly influenced by any test item: 0.04 % ± 0.17 (range: 0.00 % - 0.80 %)

Caudal vertebral body/bodies (all bodies unossified)
- control animals: 0.24 % ± 0.59 (range: 0.00 % - 2.20 %)
- test animals not significantly influenced by any test item: 0.30 % ± 0.62 (range: 0.00 % - 2.80 %)

Os ischii (unossified)
- control animals: 0.29 % ± 0.47 (range: 0.00 % - 1.50 %)
- test animals not significantly influenced by any test item: 0.13 % ± 0.31 (range: 0.00 % - 1.40 %)

Os pubis (unossified)
- control animals: 0.20 % ± 0.53 (range: 0.00 % - 1.50 %)
- test animals not significantly influenced by any test item: 0.16 % ± 0.41 (range: 0.00 % - 1.50 %)

Os pubis (incompletely ossified)
- control animals: 1.70 % ± 2.30 (range: 0.00 % - 8.20 %)
- test animals not significantly influenced by any test item: 1.80 % ± 2.26 (range: 0.00 % - 7.00 %)

Forelimbs (absence of ossification in metacarpalia 2 to 5)
- control animals: 3.59 % ± 3.96 (range: 0.00 % - 15.00 %)
- test animals not significantly influenced by any test item: 4.70 % ± 6.40 (range: 0.00 % - 32.80 %)

Hindlimbs (absence of ossification in metatarsalia 2 to 5)
- control animals: 1.20 % ± 1.81 (range: 0.00 % - 4.60 %)
- test animals not significantly influenced by any test item: 1.26 % ± 2.38 (range: 0.00 % - 12.20 %)

VISCERAL MALFORMATION:
- 0, 100, 300, and 1000 mg/kg bw/day: the macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the foetuses of the control group and the foetuses of the dose groups. One foetus of the 1000 mg/kg bw/day dose group was noted with multiple malformations in form of a short abdomen and tail and a misshapen head (domed) and a variation in form of the flexure of the hind-limbs. The single occurrence of one foetus with malformations and a variation was considered to be spontaneous.

- 0, 100 and 1000 mg/kg bw/day: no malformations were noted for the foetuses of the control group and the foetuses of the 100 and 1000 mg/kg bw/day dose groups during the soft tissue examination according to WILSON.

- 300 mg/kg bw/day: one foetus was noted with a malformation in form of a cleft palate (foetal incidence: 0.7%). However, the occurrence of one foetus with a cleft palate was within the laboratory background data range of the dose groups (0.0% - 0.7%) and no observation of a cleft palate was noted for the high dose group. Therefore, the single observation of one cleft palate in the 300 mg/kg bw/day dose group was considered to be spontaneous.

Visceral variations:
- 0, 100, 300 and 1000 mg/kg bw/day: during the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle (control group 4.7%/low dose group 2.8%/intermediate dose group 2.9%/high dose group 3.5%)), the kidneys (uni- or bilateral dilatation of the renal pelvis (4.0%/6.9%/4.3%/4.2%) or malpositioned (1.3%/0.0%/1.4%/2.1%)) and the liver (haemorrhagic focus/foci (0.7%/2.1%/2.9%/2.8%)).

No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups. All incidences were within the laboratory background data.

Laboratory background data:
Cerebral ventricle: dilatation
- control animals: 2.27 % ± 2.85 (range: 0.0 % - 13.2 %)
- test animals not significantly influenced by any test item: 2.48 % ± 2.39 (range: 0.0 % - 12.9 %)

Renal pelvis: dilatation
- control animals: 4.66 % ± 3.46 (range: 0.0 % - 12.6 %)
- test animals not significantly influenced by any test item: 4.42 % ± 3.66 (range: 0.0 % - 12.8 %)

Kidney (malpositioned)
- control animals: 0.18 % ± 0.11 (range: 0.0 % - 1.7 %)
- test animals not significantly influenced by any test item: 0.38 % ± 0.61 (range: 0.0 % - 3.7 %)

Liver (haemorrhagic focus/foci)
- control animals: 1.63 % ± 1.43 (range: 0.0 % - 5.6 %)
- test animals not significantly influenced by any test item: 1.65 % ± 1.40 (range: 0.0 % - 6.7 %)

PLACENTAL WEIGHT:
- 100, 300, and 1000 mg/kg bw/day: the placental weights showed no test item-related differences between the control group and the dose groups.

- 100, 300 and 1000 mg/kg bw/day: for the placental weights, dose-dependently increased values were noted for the placentae of the male foetuses, the female foetuses and accordingly for the male and female foetuses combined. In addition, the values for the 300 mg/kg bw/day (male foetuses only) and 1000 mg/kg bw/day dose group were above the range of the laboratory background data. However, no statistically significant difference to the control group was noted and the values were only marginally above the background data range. Therefore, the placental weights above the background data range for the intermediate and high dose group were considered to be spontaneous.

The placental weights (mean ± SD; g) are shown below:

- male foetuses:
control group: 0.546 ± 0.056 g
100 mg/kg bw/day: 0.546 ± 0.050 g
300 mg/kg bw/day: 0.573 ± 0.094 g
1000 mg/kg bw/day: 0.577 ± 0.075
Laboratory background data:
- control animals: 0.525 ± 0.017 g (range: 0.497 g - 0.555 g)
- test animals not significantly influenced by any test item: 0.521 ± 0.023 g (range: 0.471 g - 0.572 g)

- female foetuses:
control group: 0.524 ± 0.054 g
100 mg/kg bw/day: 0.527 ± 0.054 g
300 mg/kg bw/day: 0.542 ± 0.050 g
1000 mg/kg bw/day: 0.554 ± 0.057
Laboratory background data:
- control animals: 0.506 ± 0.021 g (range: 0.469 g - 0.551 g)
- test animals not significantly influenced by any test item: 0.502 ± 0.022 g (range: 0.446 g - 0.557 g)

- male and female foetuses:
control group: 0.536 ± 0.050 g
100 mg/kg bw/day: 0.537 ± 0.050 g
300 mg/kg bw/day: 0.558 ± 0.064 g
1000 mg/kg bw/day: 0.565 ± 0.063
Laboratory background data:
- control animals: 0.517 ± 0.019 g (range: 0.483 g - 0.552 g)
- test animals not significantly influenced by any test item: 0.511 ± 0.020 g (range: 0.460 g - 0.560 g)

TESTICULAR DEVELOPMENT:
- 100, 300 and 1000 mg/kg bw/day: no cryptorchidism and no testicular malposition were noted during assessment of the testicular development of the male foetuses of the control group and the dose groups.

UNCLASSIFIED OBSERVATIONS:
0, 100, 300 and 1000 mg/kg bw/day: unclassified observations in form of a thoracic cavity filled with blood were noted for one foetus each of the control group and the intermediate dose group and for two foetuses each of the low dose group and the high dose group. These observations were considered to be preparation-induced artefacts.

Effect levels (fetuses)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the current prenatal developmental toxicity study, zirconium praseodymium yellow zircon in 0.8% aqueous hydroxypropylmethyl-cellulose gel was administered via gavage to groups of pregnant female Crl:CD (SD) rats (n = 20) at dose levels of 100, 300, and 1000 mg/kg bw/day. The administration occurred once daily from gestation day 6 to gestation day 20. A vehicle control group was run concurrently.

During the observation of the dams , no test item-related effects were observed for mortality, clinical signs, body weight, body weight gain, gravid uterus weight, carcass weight, food consumption, water consumption, gross pathology, thyroid weights, thyroid hormone concentrations (triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH)), and histopathology of the thyroid. Furthermore, no test item-related effect was noted on the reproductive parameters (number of corpora lutea, implanation sites, resorptions (total, early and late) and foetuses (dead and alive) as well as the index of pre- and post implantation loss).

In addition, the examination of the foetuses reveal that no foetal deaths occurred and no test item-related effects were observed for body weight, placental weight, and foetal developmental parameters (anogenital distance or testicular developmental of the male foetuses). Also, no test item-related malformations or variations were noted during the macroscopic inspection at laparotomy (including external inspection and a gross inspection of the organs), the skeletal examination according to Dawson and the soft tissue examination according to Wilson. Lastly, no test item-related retardations (delay in ossification) were noted in any of the treatment groups.

Based on the results, the NOAEL for maternal toxicity and developmental toxicity is consider to be greater than 1000 mg/kg bw/day.