Hematite, chromium green black

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-10 to 2020-09-24

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:

Manganese alumina pink corundum

EC Number:

269-061-0

EC Name:

Manganese alumina pink corundum

Cas Number:

68186-99-2

Molecular formula:

Mn(x)Al(2-x)O3 0,01≤x≤0,25

IUPAC Name:

Manganese aluminium corundum

Test material form:

solid: particulate/powder

Details on test material:

- Chemical description: Manganese Alumina Pink Corundum
- Substance type: inorganic pigment
- Physical state: solid, pink powder, odourless, Hematite-corundum structure
- 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 - 63 days
- Weight (on day 0 of pregnancy): 167.4 - 260.0 g
- Housing (exception: mating period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt/Arkeburg, Germany; bedding material did not contain unacceptable high levels of hormonally active substances); environmental enrichment: one piece of wood (certified for animal use) to gnaw on and octagon-shaped red-tinted huts (polycarbonate).
- Diet (ad libitum): commercial diet ssniff® R/Z V1154 (ssniff Spezialdiäten GmbH, Soest, Germany); food did not contain unacceptable high levels of hormonally active substances
- Water (ad libitum): drinking water
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.8 % aqueous hydroxypropylmethylcellulose gel

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared on every administration day.

The test item was suspended in the vehicle to the appropriate concentrations and was administered as a single dose orally at a constant volume/kg bw. The test item was administered at approximately the same time each day. The administration 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

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 manganese and aluminium 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: 90.2 % - 97.2 %
- concentration (before administration of the last animal of each dose group at a time when the majority of animals was dosed): 97.6 % - 98.1 %
- homogeneity: 88.5% - 97.3%

The measurement of aluminium and manganese concentrations in digested lyophilization residues revealed recovery rates across all samples between 95.8% and 101% for aluminium and 93.5% and 97.6% for manganese. 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 have been selected by the Sponsor based on available toxicological data.

NOTE: the current study has a joint control group with another study (Report No. 38112). This study included 12 control animals and the other study included 13 animals in order to obtain altogether 20 live litters for evaluation.

Examinations

Maternal examinations:

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

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

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

DETAILED CLINICAL OBSERVATIONS: No

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

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

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

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

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

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

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

In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. The thyroid (including parathyroid) and any organs with macroscopic findings of all dams (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, values below the LOD were set to 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 of sacrifice of the dams) and placentae (location in the uterus and the assignment of the foetuses) was determined.
- Sex and viability (spontaneous breathing, spontaneous movement) of foetuses were determined.
- Location of foetuses in the uterus.
- Weights of foetuses and weights of the placentae were determined (foetuses were considered as runts if their weight was less than 70% of the mean litter weight).

Statistics:

Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis (Provantis integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

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

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the foetuses) or an internal computer program (e.g. findings during the foetal skeletal or soft tissue examination).

Indices:

- Total malformation rate (%)* = (malformed foetuses per group / foetuses per group) x 100

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

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

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

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

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

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

* foetuses affected by several changes will be counted as one foetal incidence.

Historical control data:

Historical control data was provided by the laboratory for the following parameters (data collected from 2000 to 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:

NOTE: the current study has a joint control group with another study (Report No. 38112). This study included 12 control animals and the other study included 13 animals in order to obtain altogether 20 live litters for evaluation.

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

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

BODY WEIGHT AND WEIGHT CHANGES:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences in body weight were noted between the dams of the control group and the dose groups. The difference in the body weight between the control group and the dose groups ranged from -2.3% to +2.5% 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 +1.6 %, +6.0 %, and +5.0 % 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 -1.1 %, +4.4 %, and +1.5 % 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 for the carcass weight between the control dams (258.68 g) and the dams of the dose groups (261.49, 263.42 and 264.82 g for the 100, 300 and 1000 mg/kg bw/day dose groups, respectively). Furthermore, no test item-related differences were noted for the net body weight gain from gestation day 6 to 21 between the dams of the control group (11.17 g) and the dams of the dose groups (14.02 g, 15.99 g and 15.17 g for 100, 300 and 1000 mg/kg bw/day dose groups, respectively).

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.

- 300 mg/kg bw/day: a statistically significantly decreased food consumption was noted between gestation day 16 and gestation day 17 (6.8% below the value of the control group, p ≤ 0.05). However, as no difference was noted for the 1000 mg/kg bw/day dose group and the food consumption recovered after gestation day 17, the transiently reduced consumption was considered to be spontaneous.

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

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

- 300 and 1000 mg/kg bw/day: a statistically significant decrease were noted for the T4 values of the intermediate and high dose group (45.1% or 42.9% below the value of the control group for the intermediate and high dose group, respectively; p ≤ 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 decreased T4 levels for the dose groups were considered to be not test item-related.

The mean values of the T4 and TSH thyroid hormone concentrations were below the range of the background data. This is regarded to be spontaneous .

The results for the T4 and TSH values (mean per group) can be seen below:

control group: 15.5293 ± 5.5669 nmol/L
100 mg/kg bw/day: 15.2992 ± 3.2048 nmol/L
300 mg/kg bw/day: 8.5332 ± 3.3853 nmol/L (p ≤ 0.01)
1000 mg/kg bw/day: 8.8633 ± 4.8186 nmol/L (p ≤ 0.01)
Laboratory background data (mean per group):
- 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: 9.61 nmol/L - 52.03 nmol/L)

control group: 0.9638 ± 1.2514 ng/mL
100 mg/kg bw/day: 0.3188 ± 0.5640 ng/L
300 mg/kg bw/day: 0.5164 ± 0.8876 ng/L
1000 mg/kg bw/day: 0.6188 ± 0.8198 ng/L
Laboratory background data (mean per group):
- control animals: 1.07 ± 0.53 nmol/L (range: 0.08 ng/L - 4.75 ng/L)
- test animals not significantly influenced by any test item: 1.14 ± 0.64 ng/L (range: 0.08 nmol/L - 5.35 ng/L)

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

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

- 100 and 300 mg/kg bw/day: one dam of the 100 mg/kg bw/day dose group was noted with enlarged and black-green discoloured placentae and one dam of the 300 mg/kg bw/day dose group was observed with some dark-red foci for the heart. However, these single occurrences were considered to be not test item-related but spontaneous.

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

The findings noted during evaluation of the left and right thyroids did not differ with regard to incidence and severity between the control group and the dose groups. In summary, a keratinized cyst were noted in 11 thyroids of group 1 (control) and the 100 mg/kg bw/day dose group, in 15 thyroids of the 300 mg/kg bw/day dose group and in 10 thyroids of the 1000 mg/kg bw/day dose group. With the exception of two findings each in group 1 (control), the 100 mg/kg b.w./day dose group and the 1000 mg/kg bw/day dose group and one finding in the 300 mg/kg bw/day dose group in the right thyroid with a mild severity, all findings were of minimal severity. The cyst(s) were noted for 9 (group 1 (control) and 1000 mg/kg bw/day), 10 (100 mg/kg bw/day) or 11 (300 mg/kg bw/day) different animals.
In addition, a minimal focal lymphocytic infiltration was observed for the left thyroid of one animal of the 300 mg/kg bw/day dose group and a mild chronic focal inflammation was observed for the right thyroid of one animal of the 100 mg/kg bw/day dose group. The single occurrences of one focal lymphocytic infiltration and one chronic inflammation were 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 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. All values of the index of pre- and post-implantation loss were with the laboratory background data, except for the pre-implantation value of the 100 mg/kg bw/day dose group, which was above the laboratory background data. This incidence was considered to be spontaneous as no dose dependence-relationship was present.

The values for pre-implantation loss (%; mean per dam; mean ± SD) can be seen below:

Control: 0.67 % ± 2.07
100 mg/kg bw/day: 7.89 ± 16.82 %
300 mg/kg bw/day: 3.53 ± 5.90 %
1000 mg/kg bw/ day: 5.28 ± 11.39 %
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 % - 18.8 % (mean number per dam: 0.5), for the 100 mg/kg bw/day dose group, between 0.0 % - 31.3 % (mean number per dam: 0.9), for the 300 mg/kg bw/day dose group between 0.0 % - 20.0 % (mean number per dam: 0.4), and for the 1000 mg/kg bw/day dose group between 0.0 % - 15.4 % (mean number per dam: 0.9). All values were within the laboratory background data.

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

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

EARLY OR LATE RESORPTIONS:
- 100, 300, and 1000 mg/kg bw/day: no test item-related influence on the early or late resorptions was noted between the dams of the control group and the dams of the treatment groups. All values were within the laboratory background data, except for the value of late resorption for the 1000 mg/kg bw/day dose group, which was above the laboratory background data. This incidence was consider to be spontaneous as the value was marginally above the background data range.

The values for late resorption (mean number per dam, mean ± SD) can be seen below:

Control: 0.1 ± 0.4
100 mg/kg bw/day: 0.1 ± 0.3
300 mg/kg bw/day: 0.0 ± 0.0
1000 mg/kg bw/ day: 0.3 ± 0.6
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 foetuses died during the study.
No test item-related influence on the number of live foetuses was 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. The values of the number of corpora lutea were above the laboratory background data for the control group as well as for the 100 and 1000 mg/kg bw/day dose groups. However, the numbers of corpora lutea above the background data range were considered to be spontaneous, as also the control group was above the range o the background data.

The values for corpora (mean number per dam, mean ± SD) can be seen below:

Control: 15.6 ± 1.7
100 mg/kg bw/day: 16.2 ± 4.8
300 mg/kg bw/day: 15.2 ± 1.4
1000 mg/kg bw/ day: 15.7 ± 2.4
Laboratory background data (mean number per dam):
- control animals: 14.1 ± 0.5 (range: 13.30 - 15.2)
- test animals not significantly influenced by any test item: 14.2 ± 0.73 (range: 12.7 - 16.0)

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. The values of the control group were above the range of the laboratory control data. This was considered to be a spontaneous finding.

The results for implantation sites (mean number per dam; mean ± SD) can be seen below:

control group: 15.5 ± 1.8
100 mg/kg bw/day: 14.6 ± 2.9
300 mg/kg bw/day: 14.6 ± 1.5
1000 mg/kg bw/day: 15.0 ± 2.8
Laboratory background data (mean number per dam):
- control animals: 13.7 ± 0.67 (range: 12.50 - 15.0)
- test animals not significantly influenced by any test item: 13.8 ± 0.76 (range: 12.4 - 15.8)


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: foetal weights showed no test item-related differences between the control group and the dose groups.
The weights of the foetuses of the low and high dose groups were below the range of the laboratory background data. However, also the weights of the control foetuses were below the background data range. Therefore, and as no statistically significant differences were noted between the control group and the dose groups, the foetal weights below the background data range were considered to be spontaneous.

- 0, 100, 300 and 1000 mg/kg bw/day: no runts were noted for the dose groups. However, one runt was noted in the control group. The single occurrence of one runt in the control group is within the normal range of biological variability (laboratory background data (control animals or test animals not significantly influenced by any test item): 0 - 2).

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

- male foetuses:
control group: 5.14 ± 0.32 g
100 mg/kg bw/day: 5.21 ± 0.31 g
300 mg/kg bw/day: 5.31 ± 0.19 g
1000 mg/kg bw/day: 5.26 ± 0.37
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: 4.83 ± 0.28 g
100 mg/kg bw/day: 4.89 ± 0.32 g
300 mg/kg bw/day: 5.02 ± 0.17 g
1000 mg/kg bw/day: 4.99 ± 0.24 g
Laboratory background data:
- control animals: 5.20 ± 0.08 g (range: 5.07 g - 5.32 g)
- test animals not significantly influenced by any test item: 5.20 g ± 0.10 g (range: 5.05 g - 5.39 g)

- male and female foetuses combined:
control group: 4.99 ± 0.31 g
100 mg/kg bw/day: 5.04 ± 0.30 g
300 mg/kg bw/day: 5.15 ± 0.17 g
1000 mg/kg bw/day: 5.13 ± 0.27 g
Laboratory background data:
- control animals: 5.35 ± 0.08 g (range: 5.21 g - 5.48 g)
- test animals not significantly influenced by any test item: 5.33 ± 0.10 g (range: 5.15 g - 5.53 g)

CHANGES IN SEX RATIO:
- 100, 300 and 1000 mg/kg bw/day: no test item-related differences between the ratio of male and female foetuses were noted between the control group and the dose groups (control group: 1.07; 100 mg/kg bw/day: 0.89; 300 mg/kg bw/day: 0.85; 1000 mg/kg bw/day: 1.09). 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.
A statistically significantly increased absolute and relative ano-genital distance of the male low dose foetuses (6.0% or 5.6% above the value of the control group, p ≤ 0.05 or 0.01) was observed. However, as no dose response-relationship was present, the slightly increased ano-genital distance of the male low dose foetuses is considered to be not test item-related.

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

- male foetsues (absolute anogenital distance; ocular units; mean ± SD)
control group: 27.0 ± 2.4
100 mg/kg bw/day: 28.6 ± 2.9 (p ≤ 0.05)
300 mg/kg bw/day: 28.2 ± 2.5
1000 mg/kg bw/day: 28.5 ± 2.2

- male foetuses (relative anogenital distance; ocular unit/ g bw; mean ± SD)
control group: 15.63 ± 1.18
100 mg/kg bw/day: 16.51 ± 1.63 (p ≤ 0.01)
300 mg/kg bw/day: 16.17 ± 1.42
1000 mg/kg bw/day: 16.39 ± 1.30

- female foetuses (absolute anogenital distance; ocular units; mean ± SD)
control group: 12.9 ± 1.9
100 mg/kg bw/day: 13.7 ± 1.5
300 mg/kg bw/day: 13.3 ± 1.1
1000 mg/kg bw/day: 13.3 ± 0.9

- female foetuses (relative anogenital distance; ocular unit/ g bw; mean ± SD)
control group: 7.62 ± 1.06
100 mg/kg bw/day: 8.07 ± 0.91
300 mg/kg bw/day: 7.79 ± 0.69
1000 mg/kg bw/day: 7.81 ± 0.57

EXTERNAL MALFORMATION:
- 100, 300 and 1000 mg/kg bw/day: no macroscopically visible external changes were noted for the foetuses of the control group and the dose groups during the external inspection at laparotomy.

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

Skeletal variations:
- 0, 100, 300 and 1000 mg/kg bw/day: skeletal variations were noted for the ribs (accessory 14th rib (control group 0.7%/low dose group 0.0%/intermediate dose group 0.0%/high dose group 0.0% of the foetuses affected), less than 13 ribs ossified (0.7%/0.0%/0.7%/0.0%) or ribs short (0.7%/0.0%/0.0%/0.0%)), the sternum (fused (0.0%/0.0%/0.7%/0.0%) or misaligned to a slight degree, (1.3%/1.5%/1.4%/0.0%)) and the thoracic vertebral bodies (misaligned (0.7%/0.0%/0.0%/0.0%)).

No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the foetuses of the treatment groups (100, 300 or 1000 mg test item/kg bw/day).

A decreased incidence of total skeletal variations was observed (control group 3.3%/low dose group 1.5%/intermediate dose group 2.1%/high dose group 0.0% of the foetuses affected). The decrease was statistically significant in the 1000 mg/kg bw/day dose group (p ≤ 0.05), but it was considered to be spontaneous as incidences were within the laboratory background data range and the incidence was decreased.

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

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

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

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

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

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

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

Total skeletal variations
- 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:
- 0, 100, 300 and 1000 mg/kg bw/day: retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal and/or interparietal areas (control group 4.6%/low dose group 1.5%/intermediate dose group 2.1%/high dose group 1.4% of the foetuses affected)), the hyoid (unossified (25.8%/16.8%/20.3%17.7%), the sternum (sternebra(e) incompletely ossified (0.7%/0.7%/2.1%/2.1%), reduced in size (57.6%/57.7%/65.7%/60.3%) or unossified (2.0%/5.1%/5.6%/2.8%)), the thoracic vertebral bodies (bipartite (2.6%/2.2%/2.8%/0.0%), dumbbell-shaped (5.3%/8.8%/2.1%/2.8%), reduced in size (0.7%/0.0%/0.0%/0.0%) or unossified (0.0%/0.7%/0.0%/0.0%)), the lumbar vertebral bodies (bipartite (0.7%/0.0%/0.0%/0.0%)) the caudal vertebral bodies (all bodies unossified (0.0%/0.7%/0.0%/0.0%) and the metacarpalia (absence of ossification in metacarpalia 2 to 5 (0.7%/0.0%/0.0%/0.7%)).

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

- 100 and 1000 mg/kg bw/day: a statistically significant decreased incidence (p ≤ 0.05) was noted for the hyoid (unossified) for the low dose group and for the thoracic vertebral bodies (bipartite) for the high dose group. However, as the incidences of all dose groups were within or marginally below the range of the laboratory background data, the decreased incidences were considered to be spontaneous and therefore without any influence on the results. Except for the findings of the thoracic vertebral bodies being unossified and for the total skeletal retardations, all incidences of the dose groups were within the range of the laboratory background data.

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

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

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

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

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

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

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

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

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

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

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

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:
- 0, 100, 300, and 1000 mg/kg bw/day: the macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the foetuses of the control group and the foetuses of the dose groups. 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 4.7%/low dose group 6.6%/intermediate dose group 5.6%/high dose group 3.5% of the foetuses affected)), the kidneys (uni- or bilateral dilatation of the renal pelvis (4.0%/6.6%/4.2%/5.0%) or malpositioned (1.3%/0.7%/0.0%/0.7%)) and the liver (haemorrhagic focus/foci (0.7%/1.5%/3.5%2.8%)). No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). All incidences were within the laboratory background data.

Laboratory background data (foetal incidence %):
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: placental weights showed no test item-related differences between the control group and the dose groups. All values were within the laboratory background data, except for values of the 100 mg/kg bw/day dose group (placental weights (males and combined sexes). This incidence was considered to be spontaneous as no dose dependence-relationship was present and the values were above the background data range.

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

- male foetuses:
control group: 0.546 ± 0.056 g
100 mg/kg bw/day: 0.579 ± 0.157 g
300 mg/kg bw/day: 0.565 ± 0.050 g
1000 mg/kg bw/day: 0.526 ± 0.054
Laboratory background data:
- control animals: 0.525 ± 0.02 g (range: 0.497 g - 0.555 g)
- test animals not significantly influenced by any test item: 0.521 ± 0.02 g (range: 0.471 g - 0.572 g)

- female foetuses:
control group: 0.524 ± 0.054 g
100 mg/kg bw/day: 0.548 ± 0.116 g
300 mg/kg bw/day: 0.544 ± 0.034 g
1000 mg/kg bw/day: 0.506 ± 0.055
Laboratory background data:
- control animals: 0.506 ± 0.021 g (range: 0.469 g - 0.551 g)
- test animals not significantly influenced by any test item: 0.502 ± 0.022 g (range: 0.446 g - 0.557 g)

- male and female foetuses:
control group: 0.536 ± 0.050 g
100 mg/kg bw/day: 0.562 ± 0.129 g
300 mg/kg bw/day: 0.551 ± 0.036 g
1000 mg/kg bw/day: 0.517 ± 0.049
Laboratory background data:
- control animals: 0.517 ± 0.02 g (range: 0.483 g - 0.552 g)
- test animals not significantly influenced by any test item: 0.511 ± 0.02 g (range: 0.460 g - 0.560 g)

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

UNCLASSIFIED OBSERVATIONS:
- 1000 mg/kg bw/day: no unclassified observations were noted.

- 0, 100 and 300 mg/kg bw/day: unclassified observations in form of a thoracic cavity filled with blood were noted for one control foetus and for two foetuses each of the 100 mg test item/kg bw/day dose group and the 300 mg test item/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, manganese alumina pink corundum 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.