Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-04-27 to 2020-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
Cobalt zinc aluminate blue spinel
EC Number:
269-049-5
EC Name:
Cobalt zinc aluminate blue spinel
Cas Number:
68186-87-8
Molecular formula:
Co(x)Zn(1-x)Al2O4 0,1≤x≤0,9
IUPAC Name:
Cobalt zinc aluminate blue spinel
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Cobalt zinc aluminate blue spinel
- Physical state: Solid, blue powder, odourless
- Structure: spinel
- Storage condition of test material: Kept dry in closed containers
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:
Remarks:
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 - 64 days
- Weight (on day 0 of pregnancy): 180.0 - 261.3 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 formulation was 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)).


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 cobalt and zinc 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: 88.4 % - 97.7 %
- concentration (before administration of the last animal of each dose group at a time when the majority of animals was dosed): 93.1 % - 96.6 %
- homogeneity: 86.7% - 98.1%

The measurement of cobalt and zinc concentrations in digested lyophilization residues revealed recovery rates across all samples between 100% and 104% for cobalt and 95.2% and 99.6% for zinc. 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 (LPT No. 38108). 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. The absolute and relative weight were provided for the organs. The relative weight was calculated using the carcass 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 (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
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 of foetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
- 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 2004 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
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 (LPT No. 38108). 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 and the faeces were noted in the control group and the dose groups.

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 dose groups. The difference in the body weight between the control group and the dose groups ranged from -2.5 % to +2.7 % 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 0 to 20 (body weight gain of +5.3 %, +7.8 %, and +6.1 % 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 +3.3 %, +6.4 %, and +6.3 % 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 differences were noted between the carcass weight of the control dams and the dams of the treatment groups. Furthermore, for the net body weight gain, statistically significant increased values were noted for the animals dosed with 100 or 300 mg test item/kg bw/day (20.9 g or 20.6 g net body weight gain in the low and intermediate dose group compared to 11.2 g in the control group, p ≤ 0.05). However, as no statistically significant difference was noted in the high dose group (17.97 g net body weight gain), the increased net body weight gain was considered to be not test item-related. In addition, an increased body weight gain was not considered to be a toxic effect.

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

- 100 mg/kg bw/day: between gestation 7 and gestation day 8, a statistically significant decreased food consumption was noted for the animals (5.1% below the value of the control group, statistically significant at p ≤ 0.05). However, the transient decrease for the food consumption in the low dose group was considered to be spontaneous as no dose dependence relationship was present.

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.
For the thyroid hormone T4, statistically significantly increased levels were noted in all dose groups (27.4%, 33.0% or 26.3% above the value of the control group for the low, intermediate and high dose group, respectively; p ≤ 0.05 or 0.01). However, as no dose response-relationship was present, no differences were noted for the thyroid weights and no pathologic changes were noted for the thyroids, the increased T4 levels for the dose groups were considered to be not test item-related. Furthermore, the values were within the range of the laboratory background data (18.23 ± 5.09 nmol/L, 19.03 ± 5.80 nmol/L and 18.07 ± 4.41 nmol/L for the 100, 300 and 1000 mg/kg bw/day dose levels; laboratory background data (control animals): 19.82 ± 2.60 nmol/L (range: 11.82 nmol/L - 37.82 nmol/L); laboratory background data (test animals not significantly influenced by any test item): 20.14 ± 3.47 nmol/L (range: 9.61 nmol/L - 52.03 nmol/L))

Lastly, all values for thyroid hormone concentrations (T3, T4, and TSH) were within the range of the laboratory background data.

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 between the gravid uterus weight (absolute and relative) of the control dams and the dams of the treatment groups

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

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 6 thyroids of the control group, in 15 thyroids of 100 mg/kg bw/day dose group, in 11 thyroids of 300 mg/kg bw/day dose group and in 10 thyroids of 1000 mg/kg bw/day dose group. The severity was minimal for all groups with the exception of one mild finding each in the 100 and 300 mg/kg bw/day dose groups and two mild findings in 1000 mg/kg bw/day dose group. The cyst(s) were noted for 5 (control), 13 (100 mg/kg bw/day), 9 (300 mg/kg bw/day) or 8 (1000 mg/kg bw/day) different animals. In addition, one animal of the 100 mg/kg bw/day dose group and two animals of 300 mg/kg bw/day dose group were noted with a minimal focal lymphocytic infiltration of the left or right thyroid. 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 was 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. All values were within the laboratory background data.

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 % - 25.0 % (mean number per dam: 0.5 ± 0.7), for the 100 mg/kg bw/day dose group, between 0.0 % - 14.3 % (mean number per dam: 0.4 ± 0.6), for the 300 mg/kg bw/day dose group between 0.0 % - 13.3 % (mean number per dam: 0.3 ± 0.6), and for the 1000 mg/kg bw/day dose group between 0.0 % - 17.6 % (mean number per dam: 0.4 ± 0.8). All values were within the laboratory background data.

Laboratory background data (resorption, mean number per dam):
- control animals: 0.49 ± 0.17 (range: 0.20 - 0.70)
- test animals not significantly influenced by any test item: 0.47 ± 0.67 (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.

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

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 were observed.

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.

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. All values were within the laboratory background data.

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: foetal weights showed no test item-related differences between the control group and the treatment groups.
Statistically significantly increased body weights were noted for the foetuses of all dose groups (6.8%, 6.7% or 9.3% above the value of the control group for the male and female fetuses combined for the low, intermediate and high dose groups, p ≤ 0.01). However, an increased foetal body weight was not considered to be toxicologically relevant. Furthermore, the control group values of the foetal body weights were slightly below the values of the background data and the statistically significant values of the intermediate and high dose group were a result of the low control group values. Furthermore, all values of the foetal weight of the low and intermediate dose group are within the laboratory background data, however male foetal weight and males and females combined foetal weight of the high dose group were above the laboratory background data, which is of no toxicological relevance.

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

- 300 and 1000 mg/kg bw/day: in the control group, two runts were noted. In the 300 or 1000 mg test item/kg bw/day dose groups, one runt each was noted. The occurrences of one runt each in the intermediate and high dose 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 (mean ± SD):
control group: 5.26 ± 0.40 g
100 mg/kg bw/day: 5.60 ± 0.27 g (p ≤ 0.01)
300 mg/kg bw/day: 5.55 ± 0.31 g (p ≤ 0.05)
1000 mg/kg bw/day: 5.73 ± 0.26 (p ≤ 0.01)
Laboratory background data:
- control animals: 5.50 ± 0.10 g (range: 5.31g - 5.67 g)
- test animals not significantly influenced by any test item: 5.46 ± 0.11 g (range: 5.28 g - 5.68 g)

- female foetuses (mean ± SD):
control group: 4.92 ± 0.39 g
100 mg/kg bw/day: 5.25 ± 0.26 g (p ≤ 0.01)
300 mg/kg bw/day: 5.26 ± 0.29 g (p ≤ 0.01)
1000 mg/kg bw/day: 5.34 ± 0.28 g (p ≤ 0.01)
Laboratory background data:
- control animals: 5.21 ± 0.08 g (range: 5.07 g - 5.32 g)
- test animals not significantly influenced by any test item: 5.16 g ± 0.10 g (range: 5.00 g - 5.39 g)

- male and female foetuses combined (mean ± SD):
control group: 5.08 ± 0.37 g
100 mg/kg bw/day: 5.42 ± 0.25 g (p ≤ 0.01)
300 mg/kg bw/day: 5.42 ± 0.27 g (p ≤ 0.01)
1000 mg/kg bw/day: 5.55 ± 0.20 g (p ≤ 0.01)
Laboratory background data:
- control animals: 5.35 ± 0.09 g (range: 5.21 g - 5.48 g)
- test animals not significantly influenced by any test item: 5.31 ± 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: 0.96; 100 mg/kg bw/day: 1.00; 300 mg/kg bw/day: 0.99; 1000 mg/kg bw/day: 1.00).
The values are in the range of the laboratory background data (control animals: 1.01 ± 0.15 (range: 0.77 - 1.40); test animals not significantly influenced by any test item: 1.03 ± 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.
A statistically significant decrease was noted for the relative ano-genital distance of the male low and intermediate dose foetuses (6.4% or 7.0% below the value of the control group, p ≤ 0.05). However, no dose-dependence relationship was present and no statistically significant difference was noted for the absolute ano-genital distance. Therefore, the decreased ano-genital distance was considered to be not test item-related but partly due to the increased foetal body weight. No similar effect was noted for the females.

The results of the anogenital distance can be seen below:

- male foetsues (absolute anogenital distance; ocular units)
control group: 25.5 ± 2.4
100 mg/kg bw/day: 24.5 ± 1.8
300 mg/kg bw/day: 24.2 ± 1.9
1000 mg/kg bw/day: 25.1 ± 1.8

- male foetuses (relative anogenital distance; ocular unit/ g bw)
control group: 14.72 ± 1.50
100 mg/kg bw/day: 13.78 ± 1.06 (p ≤ 0.05)
300 mg/kg bw/day: 13.68 ± 1.17 (p ≤ 0.05)
1000 mg/kg bw/day: 14.02 ± 0.96

- female foetsues (absolute anogenital distance; ocular units)
control group: 14.2 ± 2.5
100 mg/kg bw/day: 13.2 ± 2.1
300 mg/kg bw/day: 13.9 ± 1.6
1000 mg/kg bw/day: 13.6 ± 1.2

- female foetuses (relative anogenital distance; ocular unit/ g bw)
control group: 8.38 ± 1.48
100 mg/kg bw/day: 7.62 ± 1.21
300 mg/kg bw/day: 8.00 ± 0.95
1000 mg/kg bw/day: 7.76 ± 0.66

EXTERNAL MALFORMATION:
- 300 and 1000 mg/kg bw/day: no macroscopically visible external changes were noted for the foetuses during the external inspection at laparotomy.

- 100 mg/kg bw/day: one foetus was noted with a malformation in form of a generalized oedema. However, the occurrence of one foetus with a malformation was considered to be spontaneous and not test item-related, even if the finding is outside the historical control data (control animals: 0.00 % ± 0.00 (range: 0.0 % - 0.0 5); test animals not significantly influenced by any test item: 0.01 % ± 0.08 (range: 0.0 % - 0.8 %).

SKELETAL MALFORMATION:
- 0, 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:
Skeletal variations were noted for the skull (fontanelle enlarged), the rib(s) (less than 13 ribs ossified or wavy) the sternum (bipartite, fused, misshapen or misaligned to a slight degree), the thoracic vertebral arches (fused) or the thoracic vertebral bodies (misaligned). However, 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 (100, 300 or 1000 mg test item/kg bw/day).

In the 1000 mg/kg bw/day dose group a statistically significant decrease was noted for the foetal incidence of the total skeletal variations (0.7% in the 1000 mg/kg bw/day dose group in comparison to 4.3% in the control group, p ≤ 0.05). However, this small decrease was considered to be not test item-related but spontaneous as no dose dependence relationship was present.

All values of skeletal variations were in the range of the historical control data.

The following results are given in foetal incidence in % per group:

- Skull (fontanelle enlarged)
Control: 0.0 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.7 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- control animals: 0.03 % ± 0.22 (range: 0.00 % - 1.60 %)
- test animals not significantly influenced by any test item: 0.01 % ± 0.17 (range: 0.00 % - 2.10 %)

- Ribs (less than 13 rib(s) ossified)
Control: 1.4 %
100 mg/kg bw/day: 1.5 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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: 0.7 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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 %)

- Sternum (bipartite)
Control: 0.0 %
100 mg/kg bw/day: 0.7 %
300 mg/kg bw/day: 0.7 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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 %)

- Sternum (fused)
Control: 0.0 %
100 mg/kg bw/day: 0.7 %
300 mg/kg bw/day: 0.7 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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 % - 5.60 %)

- Sternum (misshapen)
Control: 0.7 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.0%
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- control animals: 0.13 % ± 0.55 (range: 0.00 % - 3.70 %)
- test animals not significantly influenced by any test item: 0.15 % ± 0.48 (range: 0.00 % - 3.30 %)

- Sternum (misaligned)
Control: 1.4 %
100 mg/kg bw/day: 1.5 %
300 mg/kg bw/day: 1.4 %
1000 mg/kg bw/day: 0.7 %
Laboratory background data:
- 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 %)

- Thoracic vertebral arches (fused)
Control: 0.0 %
100 mg/kg bw/day: 0.7 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- control animals: 0.00 % ± 0.00 (range: 0.00 % - 0.00 %)
- test animals not significantly influenced by any test item: 0.01 % ± 0.06 (range: 0.00 % - 0.80 %)

- Thoracic vertebral bodies (misaligned)
Control: 0.0 %
100 mg/kg bw/day: 0.7 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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 %)

- Total skeletal variations
Control: 4.3 %
100 mg/kg bw/day: 2.2 %
300 mg/kg bw/day: 2.8 %
1000 mg/kg bw/day: 0.7 % (p ≤ 0.05)
Laboratory background data:
- control animals: 5.06 % ± 5.50 (range: 0.00 % - 28.80 %)
- test animals not significantly influenced by any test item: 4.95 % ± 4.83 (range: 0.00 % - 28.70 %)

Skeletal retardations:
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped), the caudal vertebral bodies (only one body ossified) and the metacarpalia (absence of ossification in metacarpalia 2 to 5). However, no test item-related difference 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.

Statistically significantly decreased incidences for some of the retardations were noted during skeletal examination of the foetuses, as can be seen below:

The results are given as foetal incidence in % per group.

- Hyoid (unossified)
Control: 22.7 %
100 mg/kg bw/day: 21.2 %
300 mg/kg bw/day: 10.4 % (p ≤ 0.01)
1000 mg/kg bw/day: 24.5 %
Laboratory background data:
- 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 %)

- Skull (incompletely ossified)
Control: 5.7 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.7 % (p ≤ 0.05)
Laboratory background data:
- 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 %)

- Sternebra(e) (reduced in size)
Control: 54.6 %
100 mg/kg bw/day: 54.0 %
300 mg/kg bw/day: 44.4 %
1000 mg/kg bw/day: 41.3 % (p ≤ 0.05)
Laboratory background data:
- 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 %)

- Sternebra(e) (unossified)
Control: 6.4 %
100 mg/kg bw/day: 1.5 % (p ≤ 0.05)
300 mg/kg bw/day: 0.7 % (p ≤ 0.01)
1000 mg/kg bw/day: 0.7 % (p ≤ 0.01)
Laboratory background data:
- 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 %)

- Thoracic vertebral body/bodies (bipartite)
Control: 2.8 %
100 mg/kg bw/day: 0.0 % (p ≤ 0.05)
300 mg/kg bw/day: 2.8 %
1000 mg/kg bw/day: 0.7 %
Laboratory background data:
- 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 %)

- Thoracic vertebral body/bodies (dumbbell-shaped)
Control: 9.9 %
100 mg/kg bw/day: 9.5 %
300 mg/kg bw/day: 4.2 % (p ≤ 0.05)
1000 mg/kg bw/day: 5.6 %
Laboratory background data:
- 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 %)

- Total skeletal retardations
Control: 75.9 %
100 mg/kg bw/day: 67.9 %
300 mg/kg bw/day: 50.7 % (p ≤ 0.01)
1000 mg/kg bw/day: 58.7 % (p ≤ 0.01)
Laboratory background data:
- 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 %)

Although, some of the values observed are outside the historical control data (hyoid unossified in control group and 100 and 300 mg/kg bw/day dose groups, skull incompletely ossified in the 100, 300 and 1000 m/kg bw/da dose groups, sternum reduced in size in the 1000 mg/kg bw/day dose group, sternum unossified in the 100, 300 and 1000 m/kg bw/da dose groups and thoracic vertebral bodys bipartite in the 100 mg/kg bw/day dose group), decreased incidences were not considered to be test item-related. Furthermore, no dose-dependency was observed.

Statisticall not significant skeletal retardations can be found below (foetal incidence in % per group):

- Sternum (incompletely ossified)
Control: 0.7 %
100 mg/kg bw/day: 0.7 %
300 mg/kg bw/day: 1.4 %
1000 mg/kg bw/day: 0.7 %
Laboratory background data:
- 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 %)

- Caudal vertebral bodies (only one body ossified)
Control: 0.7 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- control animals: 0.98 % ± 2.05 (range: 0.00 % - 7.90 %)
- test animals not significantly influenced by any test item: 1.01 % ± 1.82 (range: 0.00 % - 7.60 %)

- Metacarpalia (absence of ossification in metacarpalia 2 to 5)
Control: 2.1 %
100 mg/kg bw/day: 0.0 %
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 0.0 %
Laboratory background data:
- 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 %)

VISCERAL MALFORMATION:
- 0, 100, 300, and 1000 mg/kg bw/day: 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 300 or 1000 mg test item/kg bw/day dose groups during the soft tissue examination according to WILSON. In the 100 mg test item/kg bw/day dose group, one foetus was noted with a malformation in form of a generalized oedema confirming the external observation (0.7 % of foetal incidence; laboratoy background data: control animals: 0.00 % ± 0.00 (range: 0.0 % - 0.0 %); test animals not significantly influenced by any test item: 0.01 % ± 0.06 (range: 0.0 % - 0.8 %)). However, as no malformations were noted in the 1000 mg/kg bw/day dose group, the occurrence of one foetus with malformations in the low dose group was considered to be spontaneous and not test item-related.

- Visceral variations:
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (hemorrhagic focus/foci). However, no test item-related differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg bw/day).
In the low dose group, statistically significantly increases were observed for the incidence of malpositioned kidney(s) (4.4% in comparison to 0.0% in the control group, p ≤ 0.01) and for the total number of soft tissue variations (19.0% in comparison to 9.9% in the control group, p ≤ 0.05). However, as no dose-response relationship was present, increased incidences of foetal soft tissue variations in the low dose group were considered to be not test item-related.

The results are given as foetal incidence in % per group.

- brain (dilatation of the 4th cerebral ventricle)
Control: 2.1 %
100 mg/kg bw/day: 5.1 %
300 mg/kg bw/day: 2.1 %
1000 mg/kg bw/day: 2.8 %
Laboratory background data:
- 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 %)

- kidney (dilatation of renal pelvis)
Control: 6.4 %
100 mg/kg bw/day: 9.5 %
300 mg/kg bw/day: 6.3 %
1000 mg/kg bw/day: 7.0 %
Laboratory background data:
- 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(s) (malpositioned)
Control: 0.0 %
100 mg/kg bw/day: 4.4 % (p ≤ 0.01)
300 mg/kg bw/day: 0.0 %
1000 mg/kg bw/day: 1.4 %
Laboratory background data:
- 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: 1.4 %
100 mg/kg bw/day: 3.6 %
300 mg/kg bw/day: 1.4 %
1000 mg/kg bw/day: 2.8 %
Laboratory background data:
- 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 %)

- total foetal soft tissue variations
Control: 9.9 %
100 mg/kg bw/day: 19.0 % (p ≤ 0.05)
300 mg/kg bw/day: 9.8 %
1000 mg/kg bw/day: 11.9 %
Laboratory background data:
- control animals: 9.04 % ± 5.29 (range: 0.70 % - 19.10 %)
- test animals not significantly influenced by any test item: 9.46 % ± 5.59 (range: 0.0 % - 23.10 %)

All values are within the range of the laboratory background data, except for the values of malpositioned kidneys (above background data).

PLACENTAL WEIGHT:
- 100, 300, and 1000 mg/kg bw/day: placental weights showed no test item-related differences between the control group and the treatment groups. The placentae weights of the control group and of the high dose group combined were slightly above the values of the background data, but not statstically significant.

- placental weight (mean ± SD; male foetuses):
control group: 0.576 ± 0.056 g
100 mg/kg bw/day: 0.560 ± 0.069 g
300 mg/kg bw/day: 0.553 ± 0.071 g
1000 mg/kg bw/day: 0.572 ± 0.053
Laboratory background data:
- control animals: 0.523 ± 0.016 g (range: 0.497 g - 0.554 g)
- test animals not significantly influenced by any test item: 0.520 ± 0.025 g (range: 0.471 g - 0.572 g)

- placental weight (mean ± SD; female foetuses):
control group: 0.554 ± 0.102 g
100 mg/kg bw/day: 0.542 ± 0.069 g
300 mg/kg bw/day: 0.527 ± 0.055 g
1000 mg/kg bw/day: 0.548 ± 0.064 g
Laboratory background data:
- control animals: 0.505 ± 0.022 g (range: 0.469 g - 0.551 g)
- test animals not significantly influenced by any test item: 0.501 g ± 0.024 g (range: 0.446 g - 0.557 g)

- placental weight (mean ± SD; male and female foetuses combined):
control group: 0.565 ± 0.077 g
100 mg/kg bw/day: 0.550 ± 0.068 g
300 mg/kg bw/day: 0.541 ± 0.059 g
1000 mg/kg bw/day: 0.562 ± 0.054 g
Laboratory background data:
- control animals: 0.514 ± 0.019 g (range: 0.483 g - 0.552 g)
- test animals not significantly influenced by any test item: 0.510 ± 0.022 g (range: 0.460 g - 0.560 g)

TESTICULAR DEVELOPMENT:
- 0, 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:
- 300 and 1000 mg/kg bw/day: no unclassified observations were noted for the control group and the 300 or 1000 mg test item/kg bw/day dose groups.

- 100 mg/kg bw/day: an unclassified observation in form of a thoracic cavity filled with blood was noted for one foetus of the 100 mg test item/kg bw/day dose group. This observation was considered to be a preparation-induced artefact.

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, cobalt zinc aluminate blue spinel 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.