High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-22 to 2020-07-21

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

Test material information

Constituent 1

Reference substance name:

oxo[(oxoferrio)oxy]iron; silanedione

EC Number:

701-304-2

Cas Number:

1353091-50-5

Molecular formula:

(SiO2)(Fe2O3)

IUPAC Name:

oxo[(oxoferrio)oxy]iron; silanedione

Test material form:

solid: particulate/powder

Details on test material:

- Name: Colorante Rojo
- OLD EC Name: Reaction mass of fumes, silica and diiron trioxide
- NEW EC Name: High-temperature calcination products of diiron trioxide and amorphous silica resulting in a glassy silica matrix
- Substance type: inorganic pigment
- Physical state: solid, red 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): 172.0 - 255.9 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 Services B.V, Uitgest, The Netherlands
- Lot: 18D04-B03-355 311

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 digested by a microwave procedure in order to measure their iron 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: 95.0 % - 101 %
- concentration (before administration of the last animal of each dose group at a time when the majority of animals was dosed): 93.0 % - 98.8 %
- homogeneity: 95.8% - 102%

The measurement of iron concentrations in digested lyophilisation residues revealed recovery rates across all samples between 97.4% and 103% for iron. 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 / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

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 were selected by the Sponsor based on available toxicological data.

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.

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 (including prematurely deceased or prematurely sacrificed animals) 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

Blood sampling:

- Plasma: No
- Serum: Yes, for thyroid hormone determination (triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH))

In order to obtain approximately 2 aliquots of 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight following a randomisation scheme on the day of necropsy. Blood samples were taken always at the same time of day within 3 hours (approximately from 6:00 a.m. to 9:00 a.m.).

The samples were stored at -20 °C ± 10 %.

The commercial kits for T3, T4 and TSH ELISA were provided by IBL International GmbH (instrument: Tecan Sunrise). The QC certificates can be found in the field "Attached backgrund material" below.

The LODs (limit of detection) and LLOQs (lowest standard value) are as follows:

T3:
- LOD = 0.1 ng/mL
- LLOQ = 0.5 ng/mL

T4:
- LOD = 8.0 nmol/L
- LLOQ = 25.0 nmol/L

TSH:
- LOD = 0.081 ng/mL
- LLOQ = 2.5 ng/mL

In order to calculate the mean values, the following formula was used:
If < LOD than LOD/square root of 2.

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 dams in a group

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

Endocrine findings:

no effects observed

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:

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.

- 1000 mg/kg bw/day: 17 of 20 animals displayed occasionally red discoloured faeces during the treatment period. However, the red discolouration was considered to be due to the red colour of the test item administered and therefore, regarded to be of no toxicological relevance.

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 -3.7 % to +0.1 % 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 -6.4 %, -2.7 %, and -1.8 % 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 -7.3 %, -1.9 %, and -2.6 % for 100, 300 and 1000 mg/kg bw/day compared to the control group, respectively). There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups.

- 100, 300 and 1000 mg/kg bw/day: no test item-related differences were noted between the carcass weight of the control dams and the dams of the treatment groups. Furthermore, no test item-related difference was noted for the net body weight gain between the control group and the dams dosed with the test item.

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.The food consumption of the dose groups ranged between -9.7% and +6.3% of the value of the control group.

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.

- 300 and 1000 mg/kg bw/day: for the thyroid hormone T4, statistically significantly increased levels were noted for the intermediate and high dose group (40.5% and 28.2% above the value of the control group, respectively; p ≤ 0.01). However, no dose response-relationship was present, no differences were noted for the thyroid weights and no pathological changes were noted for the thyroids. Furthermore, the control group values were low as well as outside the historical control data and the statistically significant values of the intermediate and high dose group were a result of the low control group values. Taken together, the increased T4 levels for the dose groups were considered to be not biologically relevant or test item-related.

Lastly, the T4 level of the 100 mg/kg b.w./day dose group was also outside the range of the laboratory background data, whereas all remaining values (T3, T4, TSH) of all dose groups were within the range of the laboratory background data

T4 hormone level (mean ± SD)
control group: 10.65 ± 1.96 nmol/L
100 mg/kg bw/day: 10.17 ± 2.97 nmol/L
300 mg/kg bw/day: 14.97 ± 3.67 nmol/L (p ≤ 0.01)
1000 mg/kg bw/day: 13.66 ± 3.37 nmol/L (p ≤ 0.01)

Laboratory background data (T4 hormone level; mean ± SD):
- control animals: 19.82 ± 2.60 nmol/L (range: 11.82 nmol/L - 37.82 nmol/L)
- test animals not significantly influenced by any test item: 20.14 ± 3.47 nmol/L (range: 9.61nmol/L - 52.03 nmol/L)

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 statistically significant differences to the control group were noted for the thyroid weights (absolute and relative) of the dose groups. Furthermore, no test item-related differences were noted between the gravid uterus weight (absolute and relative) of the control dams and the dams of the treatment groups

GROSS PATHOLOGICAL FINDINGS:
- 0, 100 and 300 mg/kg bw/day: no pathological changes were noted for the dams of the control group and the dams of the low and intermediate dose groups during the macroscopic inspection of the organs and tissues.

- 1000 mg/kg bw/day: one dam was noted with the left half of the uterus being filled with approximately 3 mL of red-brown jellied liquid. However, the occurrence a fluid-filled uterus in one dam was considered to be not test item-related.

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, single or multiple keratinized cyst(s) were noted in 11 thyroids of control group and the 100 mg/kg b.w./day dose group, in 17 thyroids of the 300 mg/kg bw/day dose group and in 13 thyroids in the 1000 mg/kg bw/day dose group. With the exception of two findings each in the control group and 300 mg/kg bw/day dose group and one finding in the 1000 mg/kg bw/day dose group with a mild severity, all findings were of minimal severity. The cyst(s) were noted for 9 (control group), 11 (100 mg/kg bw/day), 12 (300 mg/kg bw/day) or 10 (1000 mg/kg bw/day) different animals.

In addition, a mild focal lymphocytic infiltration was observed for the left thyroid of one animal of the 1000 mg/kg bw/day dose group. The single occurrence of one focal lymphocytic infiltration was considered to be coincidental.

As no statistically significant difference between the control group and any of the dose groups was present, and no difference was noted for the severity, the observed morphological changes were considered to be coincidental and 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. No dose response relationship was noted.

- 100 and 1000 mg/kg bw/day: the pre-implantation loss was noted being below the background data range of the control groups. However, a decreased pre-implantation loss was considered to be of no toxicological relevance.

The results of the pre-implantation loss (%; mean per dam) are shown below:

control group: 1.31 % ± 3.46
100 mg/kg bw/day: 0.29 % ± 1.32
300 mg/kg bw/day: 2.57 % ± 4.53
1000 mg/kg bw/day: 0.36 % ± 1.60

Laboratory background data (mean %):
- control animals: 2.91 % ± 1.44 (range: 0.61 % - 5.00 %)
- test animals not significantly influenced by any test item: 2.87 % ± 1.64 (range: 0.25 % - 7.00 %)

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 % - 18.2 % (mean number per dam: 0.7 ± 1.0), for the 100 mg/kg bw/day dose group, between 0.0 % - 13.3 % (mean number per dam: 0.4 ± 0.7), for the 300 mg/kg bw/day dose group between 0.0 % - 27.3 % (mean number per dam: 0.6 ± 0.9), and for the 1000 mg/kg bw/day dose group between 0.0 % - 7.7 % (mean number per dam: 0.3 ± 0.4). No dose response relationship was noted. All values were within the laboratory background data.

Laboratory background data (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. No dose response relationship was noted.

- 1000 mg/kg bw/day: the number of early resorptions was noted to be below the background data range of the controls. However, a decrease in the number of early resorptions was considered to be not toxicologically relevant. Furthermore, values above the upper limit of the laboratory background data range of the control groups were observed for the numbers of late resorptions.
The numbers of late resorptions are regarded to be spontaneous.

The results of the early and late resorption (mean number per dam) are shown below:

Early resorptions (mean number per dam)
control group: 0.7 ± 0.1
100 mg/kg bw/day: 0.4 ± 0.7
300 mg/kg bw/day: 0.5 ± 0.9
1000 mg/kg bw/day: 0.1± 0.3

Laboratory background data (mean number per dam):
- control animals: 0.45 ± 0.16 (range: 0.20 - 0.70)
- test animals not significantly influenced by any test item: 0.41 ± 0.22 (range: 0.10 - 1.30)

Late resorptions (mean number per dam)
control group: 0.0 ± 0.0
100 mg/kg bw/day: 0.0 ± 0.0
300 mg/kg bw/day: 0.1 ± 0.3
1000 mg/kg bw/day: 0.2 ± 0.4

Laboratory background data (mean number per dam):
- control animals: 0.05 ± 0.06 (range: 0.00 - 0.20)
- test animals not significantly influenced by any test item: 0.09 ± 0.06 (range: 0.00 - 0.20)

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

DEAD FOETUSES:
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence on the number of dead foetuses was noted between the dams of the control group and the dams of the treatment groups. No dose response relationship was noted.

- 100 and 300 mg/kg bw/day: no dead foetuses were observed.

- 0 and 1000 mg/kg bw/day: the number of dead foetuses of the control group ( 2 dead foetuses) and the high dose group (2 dead foetuses) were above the laboratory background data. However, the 2 dead foetuses in the high dose group were regarded to be spontaneous as also 2 dead foetuses were noted for the control group. Therefore, the high number of dead foetuses noted in the high dose group were considered to be spontaeous and not test item related.

The laboratory background data for dead foetuses is shown below

Laboratory background data (number at laparotomy):
- control animals (range): 0 - 0
- test animals not significantly influenced by any test item (range): 0 - 1

Laboratory background data (mean number per dam):
- 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)

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

LIVE FOETUESES AND FOETUESES COMBINED (ALIVE AND DEAD)
- 100, 300 and 1000 mg/kg bw/day: no test item-related influence on the number of live foetuses were observed. Furthermore, no test item-related influence on the number of live and dead foetuses combined were noted. No dose response relationship was noted.

The results of alive and dead foetueses combined (mean number per dam) are shown below:

control group: 14.4 ± 1.5
100 mg/kg bw/day: 13.9 ± 1.4
300 mg/kg bw/day: 13.5 ± 3.0
1000 mg/kg bw/day: 14.5 ± 1.7

Laboratory background data (mean number per dam)
- control animals: 13.23 ± 0.68 (range: 11.90 - 14.60)
- test animals not significantly influenced by any test item: 13.35 ± 0.77 (range: 11.70 - 15.40)

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. The values were within the range of the background data.

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

Control: 15.2 ± 2.1
100 mg/kg bw/day: 14.3 ± 1.5
300 mg/kg bw/day: 14.5 ± 2.8
1000 mg/kg bw/ day: 14.8 ± 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. No dose response relationship was observed. The values were within the range of the background data.

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

Control: 15.0 ± 2.2
100 mg/kg bw/day: 14.2 ± 1.4
300 mg/kg bw/day: 14.1 ± 2.7
1000 mg/kg bw/ day: 14.7 ± 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

Anogenital distance of all rodent fetuses:

no effects observed

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 treatment groups.

- 0 and 100 mg/kg bw/day: foetal weights were noted below the lower limit of the laboratory background data range. However, as there was no statistically significant difference between the foetal weights of the control group and those of the low dose group, the lower foetal weights were considered to be due to the relatively high number of foetuses per litter.

- 100 and 300 mg/kg bw/day: no runts were noted for the low and intermediate dose groups.

- 0 and 1000 mg/kg bw/day: in the control group and in the high dose group, two runts each were noted. The occurrences of two runts per group are within the normal range of biological variability (historical control value (control animals or test animals not significantly influenced by any test item): 0 - 2) and therefore, were considered to be not test item-related in particular as also two runts were noted for the control group.

The foetal weights are shown below:

- male foetuses:
control group: 5.28 ± 0.34 g
100 mg/kg bw/day: 5.23 ± 0.26 g
300 mg/kg bw/day: 5.35 ± 0.3 g
1000 mg/kg bw/day: 5.30 ± 0.27 g
Laboratory background data:
- control animals: 5.49 ± 0.10 g (range: 5.31g - 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: 4.92 ± 0.41 g
100 mg/kg bw/day: 4.93 ± 0.20 g
300 mg/kg bw/day: 5.09 ± 0.25 g
1000 mg/kg bw/day: 5.06 ± 0.31 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: 5.11 ± 0.33 g
100 mg/kg bw/day: 5.09 ± 0.23 g
300 mg/kg bw/day: 5.22 ± 0.24 g
1000 mg/kg bw/day: 5.17 ± 0.28 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: 1.05; 100 mg/kg bw/day: 1.11; 300 mg/kg bw/day: 0.94; 1000 mg/kg bw/day: 0.88). 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 statistically significant decrease was noted for the absolute ano-genital distance of the female low dose foetuses (11.3% below the value of the control group, p ≤ 0.01). In addition, the relative ano-genital distance was decreased for the female foetuses of the low and high dose groups (11.7% or 8.4% below the value of the control group, p ≤ 0.01 or 0.05). However, as no dose-dependence relationship was present, the decreased absolute and/or relative ano-genital distances were considered to be not test item-related. No similar effects were noted for the males

The results of the anogenital distance can be seen below:

- male foetsues (absolute anogenital distance; ocular units)
control group: 29.5 ± 3.1
100 mg/kg bw/day: 27.7 ± 3.1
300 mg/kg bw/day: 30.9 ± 3.4
1000 mg/kg bw/day: 27.9 ± 3.9

- male foetuses (relative anogenital distance; ocular unit/ g bw)
control group: 16.94 ± 1.78
100 mg/kg bw/day: 15.95 ± 1.72
300 mg/kg bw/day: 17.65 ± 1.78
1000 mg/kg bw/day: 15.99 ± 2.28

- female foetuses (absolute anogenital distance; ocular units)
control group: 15.1 ± 1.2
100 mg/kg bw/day: 13.4 ± 1.8 (p ≤ 0.01)
300 mg/kg bw/day: 15.7 ± 1.8
1000 mg/kg bw/day: 14.0 ± 1.5

- female foetuses (relative anogenital distance; ocular unit/ g bw)
control group: 8.94 ± 0.82
100 mg/kg bw/day: 7.89 ± 1.01 (p ≤ 0.01)
300 mg/kg bw/day: 9.10 ± 1.04
1000 mg/kg bw/day: 8.18 ± 0.91 (p ≤ 0.05)

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

- 1000 mg/kg bw/day: one foetus was noted with a malformation in form of a missing scull cap (cranioschisis), with the meninges being intact. However, the single occurrence of one foetus with a malformation was considered to be spontaneous and not test item-related.

SKELETAL MALFORMATION:
- 0, 100 and 300 mg/kg bw/day: no skeletal malformations were noted for the foetuses of the control group and the low and intermediate dose groups during the skeletal examination according to DAWSON.

- 1000 mg/kg bw/day: the malformation that was noted during the external examination for the foetus was also noted during the skeletal examination in form of an absent cranium. However, the occurrence of one malformation in the high dose group was considered to be spontaneous and not test item-related.

Skeletal variations:
- 0, 100, 300 and 1000 mg/kg bw/day: skeletal variations were noted for the ribs (less than 13 rib(s) ossified or short: control 0.0%, low dose group 0.7%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected or wavy: control 0.7%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected) or the sternum (bipartite: control 0.7%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected or misaligned to a slight degree: control 1.4%, low dose group 0.0%, intermediate dose group 1.5% and high dose group 0.7% of the animals affected). 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.

The findings were within the range of the laboratory background data.

Laboratory background data:
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 (bipartite)
- control animals: 0.47 % ± 0.75 (range: 0.00 % - 3.20 %)
- test animals not significantly influenced by any test item: 0.47 % ± 0.88 (range: 0.00 % - 5.60 %)

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 (wavy)
- control animals: 2.59 % ± 4.31 % (range: 0.00 % - 17.60 %)
- test animals not significantly influenced by any test item: 2.23 % ± 3.92 (range: 0.00 % - 24.10 %)

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 parietal, interparietal and/or supraoccipital areas: control 1.4%, low dose group 0.7%, intermediate dose group 0.7% and high dose group 2.1% of the animals affected), the hyoid (unossified: control 12.5%, low dose group 10.8%, intermediate dose group 8.9% and high dose group 11.7% of the animals affected), the sternum (sternebra(e) incompletely ossified: control 0.7%, low dose group 0.7%, intermediate dose group 0.7% and high dose group 0.7% of the animals affected; reduced in size: control 56.9%, low dose group 66.2%, intermediate dose group 63.0% and high dose group 55.2% of the animals affected), the thoracic vertebral bodies (bipartite: control 1.4%, low dose group 0.7%, intermediate dose group 0.7% and high dose group 0.7% of the animals affected or dumbbell-shaped: control 9.0%, low dose group 6.5%, intermediate dose group 8.1% and high dose group 4.8% of the animals affected), and the metacarpalia (absence of ossification in metacarpalia 2 to 5: control 0.0%, low dose group 0.7%, intermediate dose group 0.0% and high dose group 0.7% of the animals affected). No test item-related difference in the incidence of skeletal retardations was noted during skeletal examination according to DAWSON.

Statistically significantly decreased incidences were observed for sternebra(e) (unossified) and total skeletal retardations during skeletal examination of the foetuses. However, decreased incidences were not considered to be test item-related.

The results for sternebra(e) unossified and total skeletal retardations can be seen below:

Sternebra(e) unossified (foetal incidence in % per group)
control group: 9.0 %
100 mg/kg bw/day: 1.4 % (p ≤ 0.01)
300 mg/kg bw/day: 1.5 % (p ≤ 0.01)
1000 mg/kg bw/day: 2.1 % (p ≤ 0.01)

Total skeletal retardations (foetal incidence in % per group)
control group: 71.5 %
100 mg/kg bw/day: 74.8 %
300 mg/kg bw/day: 68.1 %
1000 mg/kg bw/day: 61.4 % (p ≤ 0.05)

Furthermore, incidences that were below the laboratory background data range were noted for the findings of 'hyoid unossified' and 'sternebra(e) incompletely ossified' for the control group and all dose groups, as well as for 'skull incompletely ossified' for all dose groups. However, skeletal retardations with incidences below the background data range were considered to be of no toxicological relevance but coincidental findings. The remaining parameters were within the laboratory background data range (absence of ossification in metacarpalia; sternebrae reduced in size; thoracic vertebral bodies (bipartite or dumbbell-shaped)).

Laboratory background data (foetal incidence in % per group):
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 %)

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 %)

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 %)

Total skeletal retardations
- control animals: 86.78 % ± 7.67 (range: 72.90 % - 96.20 %)
- test animals not significantly influenced by any test item: 88.31 % ± 6.56 (range: 71.30 % - 97.70 %)

VISCERAL MALFORMATION:
- 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. Furthermore, no malformations were noted for the foetuses of the control group and the foetuses of the dose groups during the soft tissue examination according to WILSON.

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 4.9%, low dose group 4.3%, intermediate dose group 3.7% and high dose group 7.6% of the animals affected), the kidneys (uni- or bilateral dilatation of the renal pelvis: control 7.0%, low dose group 5.8%, intermediate dose group 3.7% and high dose group 6.9% of the animals affected) or malpositioned: control 0.7%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.7% of the animals affected) ) and the liver (hemorrhagic focus/foci: control 1.4%, low dose group 1.4%, intermediate dose group 3.0% and high dose group 2.8% of the animals affected). No test item-related differences in the incidences of the observed variations were noted between the control group and the treatment groups. All incidences for soft tissue variations noted in the control group and the dose groups were within the range of 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 treatment groups.

The results of the placental weights are shown below:

- male foetuses:
control group: 0.553 ± 0.052 g
100 mg/kg bw/day: 0.508 ± 0.045 g
300 mg/kg bw/day: 0.564 ± 0.175 g
1000 mg/kg bw/day: 0.543 ± 0.055
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: 0.517 ± 0.048 g
100 mg/kg bw/day: 0.492 ± 0.049 g
300 mg/kg bw/day: 0.541 ± 0.174 g
1000 mg/kg bw/day: 0.507 ± 0.053
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: 0.537 ± 0.046 g
100 mg/kg bw/day: 0.500 ± 0.042 g
300 mg/kg bw/day: 0.552 ± 0.170 g
1000 mg/kg bw/day: 0.521 ± 0.051
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 was noted during assessment of the testicular development of the male foetuses of the control group and the dose groups.

UNCLASSIFIED OBSERVATIONS:
- 0 and 100 mg/kg bw/day: no unclassified observations were noted for the control group and the low dose group.

- 300 and 1000 mg/kg bw/day: unclassified observations in form of a thoracic cavity filled with blood were noted for 3 foetuses of the 300 mg/kg bw/day dose group and for 4 foetuses of the 1000 mg/kg bw/day 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, the test item "high-temperature calcination products of diiron trioxide and amorphous silica resulting in glassy silica matrix" 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.