Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The in-vitro and in-vivo experiments described in the dataset are in very good agreement with regards to the negligible level of bioavailability of the elements Cr and Fe contained in the pigment, indicating a lack of any concern for toxicity to reproduction properties.

(1) no signs of local toxicity in an acute inhalation toxicity test at the limit dose of 5.01 mg/L. The study has been performed according to OECD TG 436 and which shows no signs of acute toxicity after inhalation exposure to the pigment chromium iron oxide, indicating a LC50 > 5.01 mg/L. No mortality occurred.

(2) In in-vitro dissolution experiments in five different artificial physiological media, dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of 0.018 %.

(3) In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Cr and Fe plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of < 0.0025 % (m/f) were found in the terminal 24-h urine collection period. 

(4) In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 89.1% Cr, and 94.1 % Fe of the dose were excreted via faeces within 3 days, with only <0.0024% of the dose being excreted via urine at the same time.

(5) In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated 0.07 % (Cr) in relation to a soluble Cr3+compound (Cr3(OH)2(CH3COO)7) injected i.v..

Comparing the findings of in-vitro dissolution testing (1) with in-vivo results (2-4), the in-vivo data consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Chromium iron oxide" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution experiment in five different artificial physiological media.

A rounded value of <0.01 % for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.008 % for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.00009 % for Cr, and <0.0024 % for Fe).

It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Cr and Fe plasma concentrations were observed, and only a minor fraction (<0.002 %) of the total administered dose of Cr and Fe was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

Based on the information from a pre-natal developmental toxicity study via gavage in rats (according to OECD 414, GLP) it is concluded that maternal toxicity up to the highest dose level of 1000 mg/kg bw/day, and reproduction data of dams were not affected by treatment. Furthermore, there was no embryo-toxicity and no effect on the development of foetuses. No malformations or variations were noted during macroscopic inspection at laparotomy (including an external inspection and gross inspection of the organs), the skeletal examination according to Dawson and the soft tissue examination according to Wilson. No incidences of skeletal retardations were observed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-30 to 2020-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
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 - 64 days
- Weight (on day 0 of pregnancy): 181.6 - 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
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 samples of 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 chromium and iron content by ICP-OES.

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

Range of % nominal concentration:
- stability: 88.0 % - 99.4 %
- concentration (before administration of the last animal of each dose group at a time when the majority of animals was dosed): 92.0 % - 97.2 %
- homogeneity: 91.5% - 98.8%

The measurement of chromium and iron concentrations in digested lyophilisation residues revealed recovery rates across all samples between 92.8% and 99.3% for chromium and between 95.7% and 103% for iron. These results verify the nominal concentrations of the application solutions and that the test item remains unchanged.

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

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

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

- Male rats for mating remained untreated.

- non-pregnant rats were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Gestation day 6 to gestation day 20
Frequency of treatment:
once daily
Duration of test:
21 days
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. 38109). This study included 12 control animals and the other study included 13 animals in order to obtain altogether 20 live litters for evaluation.
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) 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 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.
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. 38109). 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.

- 1000 mg/kg bw/day: pultaceous faeces were noted for one dam on gestation day 4. As the observation of pultacous faeces was noted before start of dosing on gestation day 6, this single observation was considered to be spontaneous.

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 +1.9% to +4.9% 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 group animals was noted for the body weight gain from gestation day 0 to 20 (body weight gain of +2.9 %, +0.7 %, and +5.1 % 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 +1.7 %, -2.7 %, and +4.9 % 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, 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 and the dams of the dose groups.

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.
Between gestation day 0 and gestation day 1, statistically significant increases were noted for the groups dosed with 300 or 1000 mg test item/kg bw/day (14.4% or 21.8% above the value of the control group, statistically significant at p ≤ 0.05 or 0.01). However, this transient increase for the food consumption was noted before start of dosing on gestation 6. Therefore, the increased food consumption between gestation day 0 and gestation day 1 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 any dose groups.

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

- 300 mg/kg bw/day: a statistically significantly increased level of T4 (dose group: 16.9334 ± 2.6915 nmol/L; control group: 14.3042 ± 2.5578 nmol/L; 18.4% above the value of the control group, p ≤ 0.05) and a statistically significantly decreased level of TSH (dose group: 0.6172 ± 0.6290 ng/mL; control group: 1.1607 ± 0.5417 ng/mL; 45.9% below the value of the control group, p ≤ 0.05) were noted. However, no statistically significant differences in the T4 or TSH serum levels were noted for the 1000 mg/kg bw/day dose group. Therefore, the increased T4 serum level and the decreased TSH serum level of the 300 mg/kg bw/day dose group were considered to be not test item-related. Furthermore, the T4 and TSH serum levels were within the range of the laboratory background data.

Laboratory background data:
- T4 (control animals): 19.82 ± 2.60 nmol/L (range: 11.82 nmol/L - 37.82 nmol/L)
- T4 (test animals not significantly influenced by any test item): 20.14 ± 3.47 nmol/L (range: 9.61 nmol/L - 52.03 nmol/L);
- TSH (control animals): 1.07 ± 0.53 ng/mL (range: 0.08 ng/mL - 4.75 ng/mL)
- TSH (test animals not significantly influenced by any test item): 1.14 ± 0.64 ng/mL (range: 0.08 ng/mL - 5.35 ng/mL).

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODYWEIGHT 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 11 thyroids of the 100 mg/kg b.w./day dose group, in 7 thyroids of the 300 mg/kg b.w./day dose group and in 9 thyroids of the 1000 mg/kg b.w./day dose group. With the exception of one finding each in the 100, 300, and 1000 mg/kg b.w./day dose groups with a mild severity, all findings were of minimal severity. Cyst(s) were noted for 5 (control group), 6 (300 mg/kg b.w/day dose group) or 9 (100 and 1000 mg/kg b.w./day dose groups) different animals. 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.
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 bewteen 0.0 % - 7.1 % (mean number per dam: 0.3 ± 0.5), for the 300 mg/kg bw/day dose group between 0.0 % - 11.8 % (mean number per dam: 0.4 ± 0.6), and for the 1000 mg/kg bw/day dose group between 0.0 % - 12.5 % (mean number per dam: 0.3 ± 0.6). All values were within the laboratory background data.

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

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

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

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
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. The mean values of the foetal weights of the control and treatment groups were slightly below the laboratory background data and were considered not to be test item-related due to biological variance.

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

- 0, 100 and 1000 mg/kg bw/day: in the control group, two runts were noted. In the low and high dose group (100 or 1000 mg test item/kg bw/day), one runt each was noted. The occurrences of one runt each in the low 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.

- male foetuses (mean ± SD):
control group: 5.26 ± 0.40 g
100 mg/kg bw/day: 5.05 ± 0.43 g
300 mg/kg bw/day: 5.15 ± 0.43 g
1000 mg/kg bw/day: 5.11 ± 0.25 g
Laboratory background data (mean ± SD):
- 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 (mean ± SD):
control group: 4.92 ± 0.39 g
100 mg/kg bw/day: 4.81 ± 0.35 g
300 mg/kg bw/day: 4.85 ± 0.33 g
1000 mg/kg bw/day: 4.85 ± 0.27 g
Laboratory background data:
- control animals: 5.20 ± 0.08 g (range: 5.05 g - 5.32 g)
- test animals not significantly influenced by any test item: 5.18 ± 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: 4.94 ± 0.40 g
300 mg/kg bw/day: 5.02 ± 0.38 g
1000 mg/kg bw/day: 4.97 ± 0.24 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: 0.96; 100 mg/kg bw/day: 1.23; 300 mg/kg bw/day: 1.22; 1000 mg/kg bw/day: 1.08).
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: 1.023 ± 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.

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

- 1000 mg/kg bw/day: one foetus was noted with a malformation in form of a generalized oedema. This is slightly different to the laboratory background data (control animals: 0.00 % ± 0.00 (range: 0.0 % - 0.0 %); test animals not significantly influenced by any test item: 0.01%± 0.08 (range: 0.0 % - 0.8 %). However, the occurrence of one foetus with this malformation was considered to be spontaneous and not test item-related.

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

Skeletal variations:
- 100, 300, and 1000 mg/kg bw/day: skeletal variations were noted for the ribs (less than 13 rib(s) ossified: control 1.4%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected or wavy: control 0.7%, low dose group 1.4%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected or short: control 0.0%, low dose group 0.0%, intermediate dose group 0.7% and high dose group 0.0% of the animals affected) or the sternum (bipartite: control 0.0%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.7% of the animals affected or misaligned to a slight degree: control 1.4%, low dose group 2.7%, intermediate dose group 2.1% and high dose group 2.7% of the animals affected or misshapen: control 0.7%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.0% of the animals affected). Misshapen sternum and less than 13 ribs ossified were only observed in one and two control animals, respectively.

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. The incidences of skeletal variations were within the laboratory background data.

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

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

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

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 %)

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

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

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

Skeletal retardations:
- 100, 300, and 1000 mg/kg bw/day: retardations (delayed ossifications) were related to the skull (incomplete ossification of parietal, interparietal and/or supraoccipital areas: control 5.7%, low dose group 4.8%, intermediate dose group 4.1% and high dose group 5.3% of the animals affected), the hyoid (unossified: control 22.7%, low dose group 41.1%, intermediate dose group 32.9% and high dose group 34.7% of the animals affected), the sternum (sternebrae€ unossified: control 6.4%, low dose group 3.4%, intermediate dose group 0.7% and high dose group 3.3% of the animals affected; sternebra(e) incompletely ossified: control 0.7%, low dose group 2.1%, intermediate dose group 0.0% and high dose group 2.0% of the animals affected; reduced in size: control 54.6%, low dose group 52.1%, intermediate dose group 57.5% and high dose group 47.3% of the animals affected), the caudal vertebral bodies (only one body ossified: control 0.7%, low dose group 0.0%, intermediate dose group 0.0% and high dose group 0.7% of the animals affected), the thoracic vertebral bodies (bipartite: control 2.8%, low dose group 4.8%, intermediate dose group 2.7% and high dose group 3.3% of the animals affected or dumbbell-shaped: control 9.9%, low dose group 2.1%, intermediate dose group 4.8% and high dose group 5.3% of the animals affected), and the metacarpalia (absence of ossification in metacarpalia 2 to 5: control 2.1%, low dose group 0.0%, intermediate dose group 0.7% and high dose group 0.7% of the animals affected).

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

A statistically significant (p ≤ 0.01 or p ≤ 0.05) increased incidence of the hyoid being unossified was noted for all dose groups. However, as the incidences of all dose groups were within the range of the laboratory background data, the increased incidences were considered to be spontaneous and therefore without any influence on the results. Statistically significant (p ≤ 0.01 or p ≤ 0.05) decreased incidences of skeletal retardations were noted for the observations of sternebra(e) unossified of the intermediate dose group, for thoracic vertebral body (bodies) dumbbell-shaped of the low dose group and for the total number of retardations of the high dose group. However, decreased incidences were considered to be spontaneous and therefore without any influence on the results.

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

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

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

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

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

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

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

Caudal ertebral bodies (only one body ossified)
- 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 %)

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

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

VISCERAL MALFORMATION:
- 100, 300, and 1000 mg/kg bw/day: the macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the foetuses of the control group and the foetuses of the dose groups. Furthermore, no malformations were noted for the foetuses of the control group and the foetuses of the dose groups during the soft tissue examination according to WILSON. All values were in the range of the laboratory background data.

Visceral variations:
- 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 2.1%, low dose group 4.1%, intermediate dose group 3.4% and high dose group 4.0% of the animals affected), the kidneys (uni- or bilateral dilatation of the renal pelvis: control 6.4%, low dose group 5.5%, intermediate dose group 4.1% and high dose group 3.4% of the animals affected) or malpositioned: control 0.0%, low dose group 1.4%, intermediate dose group 0.0% and high dose group 0.7% of the animals affected) ) and the liver (hemorrhagic focus/foci: control 1.4%, low dose group 1.4%, intermediate dose group 1.4% and high dose group 1.3% of the animals affected). 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. Furthermore, all values were in the range of the laboratory background data.

Laboratory background data (foetal incidence % per group):
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 treatment groups. The mean values of the placental weights of the treatment groups were in the range of the laboratory background data. The mean values of the placental weights of the control group were slightly above the laboratory background data.

- male foetuses:
control group: 0.576 ± 0.056 g
Laboratory background data (mean ± SD):
- control animals: 0.525 ± 0.017 g (range: 0.497 g - 0.555 g)

- female foetuses:
control group: 0.554 ± 0.102 g
Laboratory background data (mean ± SD):
- control animals: 0.506 ± 0.021 g (range: 0.469 g - 0.551 g)

- male and female foetuses combined:
control group: 0.565 ± 0.077 g
Laboratory background data (mean ± SD):
- control animals: 0.517 ± 0.019 g (range: 0.483 g - 0.552 g)

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

UNCLASSIFIED OBSERVATIONS:
- 0, 100, 300, and 1000 mg/kg bw/day: no unclassified observations were noted for the control group. Unclassified observations in form of a thoracic cavity filled with blood were noted for one foetus of the 100 mg/kg bw/day dose group, for two foetuses of the 300 mg/kg bw/day dose group and for one foetus of the 1000 mg/kg b/day dose group. These observations were considered to be preparation-induced artefacts.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In the current prenatal developmental toxicity study, chromium iron oxide 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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One guideline and GLP-conform study available, the overall quality of the database is therefore high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

One GLP and guideline-compliant prenatal developmental toxicity study (OECD 414) in rats is available. Chromium iron oxide 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.

Justification for classification or non-classification

Effects on fertility:


It is concluded that the pigment was well tolerated and that no signs of systemic toxicity whatsoever were observed in rats when administered at a dose of 1000 mg/kg bw/day for up to 28 days. Either no or only marginal increases in Cr and Fe plasma concentrations were observed, and only a minor fraction (<0.002%) of the total administered dose of Cr and Fe was collected via urine, documenting the lack of bioavailability of this pigment. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.No classification for toxicity to reproduction according to EC Regulation No. 1272/2008 is anticipated.


Effects on developmental toxicity:


In one guideline and GLP compliant study, chromium iron oxide did not show any effects on the developing foetuses of rats when administered up to the limit dose of 1000 mg/kg bw/day. In conclusion, based on the data observed for chromium iron oxide in one developmental toxicity study in rats, a classification as reproductive toxicant according to EC Regulation No. 1272/2008 is not justified due to the complete absence of any adverse effects.

Additional information