Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No indication of genetic toxicity, based on in vitro test result: Ames test (OECD TG 471). This result is supported by Ames' data on two analogous substances Cerium and iron oxide isostearates (Active matter of DPX10 and Active matter of DPX11).

No indication of genetic toxicity, by analogy with Cerium and iron oxide isostearate (Active matter of DPX10) based on in vitro test results: mammalian chromosome aberration test (OECD TG 473), and gene mutation assay in mammalian cells (OECD TG 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2 June 2005 to 12 September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: Without S9-mix: 4-nitro-o-phenylene-diamine (10-50 µg/plate, TA1537, TA98) // With S9-mix: 2-aminoanthracene (2.5-10 µg/plate, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for experiment I, preincubation method for experiment II

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: three plates/dose-level
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
On the day of the experiment, the test item ISOSTEARATE D’OXYDE DE FER was suspended in ethanol. Precipitation in the overlay agar was observed from 33 µg/plate up to 5000 µg/plate with and without metabolic activation.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A reduction in the number of revertants was observed in experiment I without metabolic activation in strain TA 1537 at 3 µg/plate and 333 µg/plate. Since there is no dose dependent reduction and the values are still in the range of the historical control data, this effect is not judged as a real toxic effect.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with iron oxide isostearate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data in the negative (exp. I, with S9 mix) and solvent control (exp. II, with S9 mix) of strain WP2 uvrA were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Table 1 : Pre-Experiment/Experiment I

Test group

Dose level (µg/plate)

Revertant colony counts (Mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without metabolic activation

Ethanol

 

14±4

15±1

44±8

136±21

58±1

Untreated

 

13±6

12±6

31±4

164±14

48±7

Iron oxide isostearate

3

10

33

100

333

1000

2500

5000

12±4

14±5

16±3 p

15±1 p

13±0 p

12±2 p

12±3 p

12±4 p

6±3

9±1

12±5 p

11±3 p

6±1 p

9±1 p

7±1 p

10±5 p

48±27

36±6

24±3 p

25±9 p

28±5 p

39±9 p

28±6 p

31±9 p

138±9

191±69

142±9 p

232±168 p

109±10 p

133±8 p

111±12 p

130±15 p

55±3

59±11

71±11 p

66±1 p

84±59 p

69±2 p

59±2 p

66±8 p

NaN3

10

1305±67

 

 

1919±24

 

4-NOPD

10

 

 

356±8

 

 

4-NOPD

50

 

92±12

 

 

1551±34

MMS

4

 

 

 

 

 

With metabolic activation

Ethanol

 

21±7

18±7

44±8

157±9

63±5

Untreated

 

21±3

12±6

39±5

171±7

61±5

Iron oxide isostearate

3

10

33

100

333

1000

2500

5000

19±5

18±6

22±4 p

18±5 p

20±2 p

14±5 p

25±8 p

19±6 p

15±3

11±3

13±3 p

17±5 p

10±4 p

13±4 p

11±2 p

16±4 p

40±4

47±8

34±4 p

49±12 p

38±11 p

38±4 p

39±3 p

31±7 p

170±12

164±12

161±3 p

147±11 p

176±11 p

167±9 p

156±8 p

133±14 p

63±5

60±5

71±15 p

75±10 p

64±12 p

69±10 p

64±15 p

70±13 p

2-AA

2.5

409±7

434±23

2371±173

2724±154

 

2-AA

10

 

 

 

 

433±30

Table 2 : Experiment II

Test group

Dose level (µg/plate)

Revertant colony counts (Mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without metabolic activation

Ethanol

 

19±3

14±6

38±2

143±14

55±12

Untreated

 

17±4

17±2

30±3

157±12

57±3

Iron oxide isostearate

33

100

333

1000

2500

5000

15±2 p

18±5 p

21±5 p

14±2 p

17±2 p

19±7 p

12±7 p

10±3 p

12±3 p

10±5 p

8±2 p

11±2 p

34±5 p

32±4 p

39±1 p

36±7 p

35±8 p

34±4 p

148±6 p

128±11 p

124±27 p

102±7 p

99±3 p

94±11 p

63±15 p

64±3 p

62±2 p

54±9 p

60±15 p

69±6 p

NaN3

10

1138±84

 

 

1855±61

 

4-NOPD

10

 

 

336±28

 

 

4-NOPD

50

 

103±9

 

 

 

MMS

4

 

 

 

 

274±152

With metabolic activation

Ethanol

 

20±4

14±4

45±5

166±4

67±7

Untreated

 

24±3

17±5

45±5

179±16

58±8

Iron oxide isostearate

33

100

333

1000

2500

5000

18±4 p

22±6 p

23±5 p

17±4 p

19±4 p

24±7 p

14±1 p

11±3 p

16±6 p

13±3 p

16±2 p

12±4 p

54±10 p

46±6 p

54±7 p

46±5 p

46±4 p

52±5 p

166±39 p

149±16 p

161±26 p

145±29 p

169±15 p

175±6 p

57±8 p

64±5 p

74±9 p

53±3 p

67±3 p

73±16 p

2-AA

2.5

357±4

401±19

2254±163

2507±139

 

2-AA

10

 

 

 

 

501±26

NaN3: sodium azide

2-AA : 2-aminoanthracene

4-NOPD : 4-nitro-o-phenylene-diamine

MMS : methyl methane sulfonate

P : Precipitate

Conclusions:
Under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The mutagenic potential of Iron oxide isostearate was tested in a bacterial reverse mutation test according to OECD guideline 471 and GLP (RCC report No.A13961). The test substance was applied on four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one strain of Escherichia coli (WP2 uvrA), using both plate incorporation and preincubation methods, at concentrations between 0 and 5000 µg/plate in ethanol, with or without metabolic activation. The appropriate positive controls were included and responded adequately.

On the day of the experiment, the test Iron oxide isostearate was suspended in ethanol. Precipitation in the overlay agar was observed from 33 µg/plate up to 5000 µg/plate with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A reduction in the number of revertants was observed in experiment I without metabolic activation in strain TA 1537 at 3 µg/plate and 333 µg/plate. Since there is no dose dependent reduction and the values are still in the range of the historical control data, this effect is not judged as a real toxic effect.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with iron oxide isostearate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Therefore, Iron oxide isostearate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

This study is classified as acceptable. It is compliant with the OECD 471 guideline requirements on bacterial reverse mutation test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from September 2001 to 15 January 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human lymphocytes (primary culture)
Details on mammalian cell type (if applicable):
the cells were prepared from whole blood samples obtained from two healthy donors and collected into heparinised sterile tubes.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 4.3, 8.5, 17, 34.1, 68.2, 136.4, 272.7 and 545.45 µg/mL (up to precipitating concentrations)
Second experiment: 8.5, 17, 34.1, 68.2, 136.4 and 272.7 µg/mL (up to precipitating concentrations)
Vehicle / solvent:
- Vehicle used: tetrahydrofuran
- Justification for choice of solvent/vehicle: the test substance was freely soluble in the vehicle at 200 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 20 or 44 hours
- Expression time (cells in growth medium): 0, 20, 44 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18.5 and 42.5 hours
(see details in tables below)

SPINDLE INHIBITOR: colcemid solution (10 µg/mL)
STAIN: Giemsa

NUMBER OF REPLICATIONS: two plates/dose-level

NUMBER OF CELLS EVALUATED: 100 metaphase per culture were scored for cytogenetic damage (200 metaphaes per dose-level).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of cells in mitosis/1000 cells examined)

OTHER:
The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication.
Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation if the findings.
Statistics:
The frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the Chi square test, in which p = 0.05 was used as the lowest level of significance.
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes, but not clearly reproducible nor dose-related
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
In the culture medium, the dose level of 545.45 µg/mL showed a marked precipitate. At the dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture.
A slight to marked precipitate was observed at the end of the treatment period, generally at dose-levels ≥ 17 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The study was considered valid since the following criteria were met :
- the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with the historical data,
- the frequency of the cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and consistent with the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S9 mix: except for some slight decreases in the mitotic index, without any clear evidence of a dose-relationship, no noteworthy toxicity was induced after 3, 20 and 44 hours treatments.
With S9 mix, some decreases in the mitotic index, which were neither reproducible nor clearly dose-related, were sometimes noted.

Table 2: Results of chromosome analysis without metabolic activation

 

Exp.

Preparation

Test item

Polyploid

Mitotic indices

Aberrant cells

 

interval

concentration

cells

in %

in %

 

 

in µg/mL

in %

of control

incl. gaps*

excl. gaps*

Exposure period 3 hrs without S9 mix

I

20 hrs

Solvent control

0

100

1

1

 

 

Positive control (MMC 3µg/ml)

0

48

46

46 ***

 

 

 68.2

0

91

1

1

 

 

136.4

0

108

1.5

1.5

 

 

272.7

0

93

0.5

0.5

Exposure period 20 hrs without S9 mix

II

20 hrs

Solvent control

0

100

1.0

1.0

 

 

Positive control (MMC 0.2 µg /ml)

0

37

16.0

15.0 ***

 

 

 68.2

0

59

2.5

2.0

 

 

136.4

0

77

1.5

1.5

 

 

272.7

0

88

0.5

0.5

Exposure period 44 hrs without S9 mix

II

44 hrs

Solvent control

0

100

1.0

0.0

 

 

272.7

0

85

1.5

0.5

 

Table 3: Results of chromosome analysis with metabolic activation

 

Exp.

Preparation

Test item

Polyploid

Mitotic indices

Aberrant cells

 

interval

concentration

cells

in %

in %

 

 

in µg/mL

in %

of control

incl. gaps*

excl. gaps*

Exposure period 3 hrs with S9 mix

I

20 hrs

Solvent control

0

100

1.5

1.5

 

 

Positive control (CPA 25 µg/ml)

0

31

23

22 ***

 

 

 68.2

0

52

1.0

1.0

 

 

136.4

0

56

2.0

2.0

 

 

272.7

0

64

2.0

2.0

Exposure period 3 hrs with S9 mix

II

20 hrs

Solvent control

0

100

2.0

1.5

 

 

Positive control (CPA 25 µg/ml)

0

53

28.0

23.0 ***

 

 

 68.2

0

116

4.0

1.5

 

 

136.4

0

116

4.0

3.0

 

 

272.7

0

93

3.5

2.5

Exposure period 3 hrs with S9 mix

II

44 hrs

Solvent control

0

100

1.5

1.0

 

 

136.4

0

78

0.5

0.0

*** = X2 test p < 0.001

Solvent control: tetrahydrofuran

MMC: mitomycin C

CPA: cyclophosphamide

Conclusions:
Negative with and without metabolic activation.
No significant increase in the frequency of cells with structural chromosome aberrations was noted in both experiments with and without metabolic activation and at both harvest times.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chomosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay) performed according to OECD guideline 473 and GLP and scored as validity 1 according to Klimisch criteria (CIT report No. 22632 MLH), primary lymphocyte cultures were exposed to Cerium and iron oxide isostearate in tetrahydrofuran, at concentrations of 4.3 to 545.45 µg/mL with and without metabolic activation. Cerium and iron oxide isostearate was tested up to precipitating concentrations and the dose-levels selected for metaphase analysis were 68.2, 136.4 and 272.7 µg/mL.

A slight to marked precipitate was observed at the end of the treatment period, generally at dose-levels >= 14 µg/mL

Positive controls induced the appropriate response.
There was no evidence of chromosome aberration induced over background. No clear cytotoxic effect was observed up to precipitating concentration.

Therefore, Cerium and iron oxide isostearate is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.


This study is classified as acceptable. This study satisfies the requirement for OECD guideline 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from February 2008 to 2 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: the cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide) at -80°C or in liquid nitrogen.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
7.81, 15.6, 31.3, 62.5, 125 and 250 μg/mL (both experiments with and without S9 mix) (up to precipitating concentrations)
Vehicle / solvent:
- Vehicle used: tetrahydrofuran
- Justification for choice of solvent/vehicle: the test item was soluble in the tetrahydrofuran at 500 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs (1st experiment with and without S9 mix and 2nd experiment with S9 mix) or 24 hrs (2nd experiment without S9 mix)
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): at the end of this expression period, 11-12 days of incubation in the presence of 4 μg TFT/mL of culture medium

SELECTION AGENT: trifluorothymidine (4 μg TFT/mL of culture medium)

NUMBER OF REPLICATIONS: two cultures per dose-level

NUMBER OF CELLS EVALUATED: 2000 cells/well (four 96-well plates/culture = eight plates/dose-level) to select the TFTr (trifluorothymidine resistant) mutant cells, differientiating small (< 25 % of the diameter of the well),and large (> 25 % of the diameter of the well),colonies.

DETERMINATION OF CYTOTOXICITY
- adjusted relative total growth (Adj. RTG), relative suspension growth (Adj. RSG) as well as cloning efficiency following the expression time.
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result which should fulfill the following criteria:
- at least at one dose-level the Mutation Frequency minus the Mutation Frequency of the vehicle control equals or exceeds the global evaluation factor (126 x 10-6 for the microtiter method),
- and a dose-related trend is demonstrated by a statistically significant trend test.
Unless considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the Mutation Frequency observed only at high levels of cytotoxicity (Relative Total Growth lower than 10%), but with no evidence of mutagenicity at dose-levels with RTG between 10 and 20%, is considered as positive result.

A test item may be determined to be non-mutagenic when there is no culture showing an Adjusted RTG value between 10-20% if:
- there is at least one negative data point between 20 and 25% Adjusted RTG and no evidence on mutagenicity in a series of data points between 100 to 20% Adjusted RTG,
- there is no evidence of mutagenicity in a series of data points between 100 to 25% and there is also a negative data point between 10 and 1% Adjusted RTG.

Comments:
Suspension Growth (SG) = Daily growth on day 1 x daily growth on day 2
CE2 = cloning efficiency at the end of the expression period
Adjusted RSG = Cell count factor x (SG treated / SG vehicle control) x 100
Adjusted RTG = Adjusted RSG x RCE2 / 100
Statistics:
not applicable
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to precipitating concentrations, but a slight toxicity has been observed in some cases.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Using a treatment volume of 20 μL/20 mL of culture medium, the final dose-level of 500 μg/mL showed a marked precipitate. At this dose-level, the pH was approximately 7.1 (as for the vehicle control) and the osmolality equal to 311 mOsm/kg H2O (319 for the vehicle control).

RANGE-FINDING/SCREENING STUDIES:
To assess the cytotoxicity of the test item, Cerium and iron oxide isostearate was tested at dose levels of 1, 10, 50, 100, 250 and 500 μg/mL, both with and without metabolic activation.
A treatment of 3 hours (with and without S9 mix) and 24 hours (without S9 mix) was performed, using same experimental conditions as described for the main experiments. Approximately one week after incubation at 37°C, the clones were counted on the plates.

At the end of the treatment periods, a precipitate was noted in the culture medium at dose-levels ≥ 50 μg/mL.
Following the 3-hour treatment without S9 mix, a moderate toxicity was induced at the dose-level of 50 μg/mL, as shown by 52% decrease in adjusted relative total growth (Adj. RTG).
Following the 24-hour treatment without S9 mix, a slight to moderate toxicity was observed at dose-levels ≥ 250 μg/mL, as shown by 26-48% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, no noteworthy toxicity was induced at any dose-level.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix: following the 3-hour treatment, a slight toxicity was induced at dose-levels ≥ 15.6 μg/mL (without any clear evidence of a dose-response relationship), as shown by 22-36% decrease in Adj. RTG. Following the 24-hour treatment, a slight toxicity was induced at 250 μg/mL, as shown by 29% decrease in Adj. RTG.
Experiments with S9 mix: in the first experiment, no noteworthy toxicity was noted at any dose-level. In the second experiment, a slight toxicity was induced at the dose-level of 31.3 μg/mL, as shown by 27% decrease in Adj. RTG.

Table 1: Mutagenicity results

Doses µg/mL

Adj. RTG %

CE2

MF x 10-6

R

First experiment without S9 mix, 3-hour treatment (mean value of 2 cultures)

0

100

0.8

125

1.0

7.81

79

0.7

118

0.9

15.6

70

0.7

110

0.9

31.3

69

0.8

126

1.0

62.5

64

0.8

108

0.9

125

78

0.9

119

1.0

250

72

0.7

137

1.1

MMS (25 µg/mL)

30

0.4

959

7.7

Second experiment without S9 mix, 24-hour treatment (mean value of 2 cultures)

0

100

0.7

158

1.0

7.81

102

0.7

98

0.6

15.6

81

0.6

125

0.8

31.3

82

0.6

121

0.8

62.5

93

0.8

97

0.6

125

79

0.8

99

0.6

250

71

0.8

107

0.7

MMS (5 µg/mL)

77

0.5

833

5.3

First experiment with S9 mix, 3-hour treatment (mean value of 2 cultures)

0

100

0.7

115

1.0

7.81

100

0.7

77

0.7

15.6

99

0.8

103

0.9

31.3

120

1.0

96

0.8

62.5

121

0.7

120

1.0

125

108

0.7

91

0.8

250

121

0.6

103

0.9

CPA (3 µg/mL)

18

0.3

1485

12.9

Second experiment with S9 mix, 3-hour treatment (mean value of 2 cultures)

0

100

0.7

102

1.0

7.81

83

0.6

117

1.2

15.6

83

0.6

118

1.2

31.3

73

0.6

115

1.1

62.5

88

0.8

109

1.1

125

79

0.6

109

1.1

250

101

0.7

112

1.1

CPA (3 µg/mL)

23

0.3

1598

15.7

0: vehicle control (tetrahydrofuran)

Adj. RTG: adjusted relative total growth

MF: mutation frequency

R: ratio between Mutation Frequency of treated cells/Mutation Frequency of control cells

MMS: methylmethane sulfonate

CPA: Cyclophosphamide

Conclusions:
Negative with and without metabolic activation.
No noteworthy increase in the Mutation Frequency was noted in comparison to the vehicle control in both experiments with and without S9 mix.
The test item did not show any mutagenic activity in the mouse lymphoma assay.
Executive summary:

In a mammalian cell gene mutation assay at the TK (Thymidine Kinase) locus(CIT report No. 34518 MLY), L5178Y mouse lymphoma cells cultured in vitro were exposed to Cerium and iron oxide isostearate in tetrahydrofuran at concentrations of 7.81, 15.6, 31.3, 62.5, 125 and 250 μg/mL for both experiments with and without S9 mix.

The test item was tested up to precipitating concentrations.

At the end of the treatment periods a precipitate was noted in the culture medium at dose-levels ≥ 15.6μg/mL. 

Experiments without S9 mix:

Cytotoxicity: following the 3-hour treatment, a slight toxicity was induced at dose-levels ≥ 15.6 μg/mL (without any clear evidence of a dose-response relationship), as shown by 22-36% decrease in Adj. RTG. Following the 24-hour treatment, a slight toxicity was induced at 250 μg/mL, as shown by 29% decrease in Adj. RTG.

Mutagenicity: following the 3 and 24-hour treatments, no noteworthy increase in the Mutation Frequency was noted in comparison to the vehicle control.

Experiments with S9 mix:

Cytotoxicity: in the first experiment, no noteworthy toxicity was noted at any dose-level. In the second experiment, a slight toxicity was induced at the dose-level of 31.3 μg/mL, as shown by 27% decrease in Adj. RTG.

Mutagenicity: in either experiment, no noteworthy increase in the Mutation Frequency was noted in comparison to the vehicle control.

The positive controls did induce the appropriate response.

 

Under the experimental conditions, the test item Cerium and iron oxide isostearate did not show any mutagenic activity in the mouse lymphoma assay.

 

This study is classified as acceptable as it is performed according to OECD guideline and GLP, and scored as validity 1 according to Klimisch criteria. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007).
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes, but not clearly reproducible nor dose-related
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
This result is considered relevant for iron oxide isostearate too. The justification for read across is attached to IUCLID Section 13.
Conclusions:
According to the experimental conditions of this study, cerium and iron oxide isosterate, an analogue of iron oxide isostearate did not induced any clastogenic effect in the presence or absence of metabolic activation. Based on the stuctural similarities between target and sources substances, the same conclusion is assumed for iron oxide isostearate.
Executive summary:

In a mammalian cell cytogenetic assay (chromosome aberration assay) performed according to OECD guideline 473 and GLP (CIT report No. MLH 22632, 2002), primary human lymphocyte cultures were exposed to Cerium and iron oxide isostearate, an structural analogue of iron oxide isostearate, in tetrahydrofuran, at concentrations of 4.3 to 545.45 µg/mL (up to precipitating concentration) with and without metabolic activation in two independent experiments.

There was no evidence of chromosome aberration induced by the test item over background in the two experiments in absence or presence of metabolic activation. No clear cytotoxic effect was observed up to precipitating concentration.

Therefore, Cerium and iron oxide isostearate is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results are assumed for iron oxide isostearate.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from cerium and iron oxide isosterate, a substance that presents structural similarities with iron oxide isostearate, was used to cover this endpoint. See the Read-across justification document (Justification for analogue approach) attached in IUCLID Section 13.2 for the justification of the read-across.
See also the original letters from the French Competent Authorities requiring the read across to be done with Cerium and iron oxide isostearate substances, attached in Section 13.2 as well (French CA testing program July 2005, and French CA testing program Sep 2007).
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to precipitating concentrations, but a slight toxicity has been observed in some cases.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
This result is considered relevant for iron oxide isostearate too. The justification for read across is attached to IUCLID Section 13.
Conclusions:
According to the experimental conditions of this study cerium and iron oxide isosterate, an analogue of iron oxide isostearate, did not induced any mutagenic effect in mammalian cells in the presence or absence of metabolic activation. Based on the stuctural similarities between target and sources substances, the same conclusions is assumed for iron oxide isostearate.
Executive summary:

In a mammalian cell gene mutation assay at the TK (Thymidine Kinase) locus(CIT report No. 34518 MLY), L5178Y mouse lymphoma cells cultured in vitro were exposed to Cerium and iron oxide isostearate, a structural analogue of iron oxide isostearate, in tetrahydrofuran at concentrations of 7.81, 15.6, 31.3, 62.5, 125 and 250 μg/mL for both experiments with and without S9 mix. The test item was tested up to precipitating concentrations.

At the end of the treatment periods a precipitate was noted in the culture medium at dose-levels ≥ 15.6μg/mL. 

Experiments without S9 mix:

Cytotoxicity: following the 3-hour treatment, a slight toxicity was induced at dose-levels ≥ 15.6 μg/mL (without any clear evidence of a dose-response relationship), as shown by 22-36% decrease in Adj. RTG. Following the 24-hour treatment, a slight toxicity was induced at 250 μg/mL, as shown by 29% decrease in Adj. RTG.

Mutagenicity: following the 3 and 24-hour treatments, no noteworthy increase in the Mutation Frequency was noted in comparison to the vehicle control.

Experiments with S9 mix:

Cytotoxicity: in the first experiment, no noteworthy toxicity was noted at any dose-level. In the second experiment, a slight toxicity was induced at the dose-level of 31.3 μg/mL, as shown by 27% decrease in Adj. RTG.

Mutagenicity: in either experiment, no noteworthy increase in the Mutation Frequency was noted in comparison to the vehicle control. The positive controls did induce the appropriate response.

 

Under the experimental conditions, the test item Cerium and iron oxide isostearate did not show any mutagenic activity in the mouse lymphoma assay.

Due to the structural similarities between cerium and iron oxide isostearate and iron oxide isostearate, same results are assumed for iron oxide isostearate.

This study is classified as acceptable as it is performed according to OECD guideline and GLP, and scored as validity 1 according to Klimisch criteria. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The ability of Iron oxide isostearate to induce genetic damage was assessed using several in vitro studies performed either with the test item or analogous substances: cerium and iron oxide isostearates (Active matter of DPX10 and Active matter of DPX11) in accordance with the testing program established by the Competent Authorities in July 2005 and September 2007 (See letters and Read-across justification document, both attached in Section 13.2). All these studies performed according to OECD guidelines and in compliance with GLP, were scored as validity 1 according to Klimisch criteria and were thus considered as Key studies.

 

In a bacterial reverse mutation test performed according to OECD guideline 471 and GLP (RCC report No. A13961, 2005), Iron oxide isostearate was applied on four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one strain of Escherichia coli (WP2 uvrA), using both plate incorporation and preincubation methods, at concentrations between 0 and 5000 µg/plate in ethanol, with or without metabolic activation in 2 independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the treated plates with and without metabolic activation No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with iron oxide isostearate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Therefore, Iron oxide isostearate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

This result is supported by negative results obtained with similar substances:

In a bacterial reverse mutation test performed according to OECD guideline 471 and GLP (CIT report No. 27881 MMT, 2004), Cerium and iron oxide isostearate (active matter of DPX11) was applied on five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA 102) and one strain of Escherichia coli (WP2 uvrA), using both plate incorporation and preincubation methods, at concentrations between 0 and 1000 µg/plate in tetrahydrofuran, with or without metabolic activation in 2 independent experiments.

No toxicity was noted towards all the strains used, in both experiments with and without S9 mix. The test substance did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six tester strains.

From the results obtained, it can be concluded that under the experimental conditions, the test item Active matter of DPX11 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

 

In a bacterial reverse mutation test performed according to OECD guideline 471 and GLP (CIT report No. 22323 MMT, 2001), Cerium and iron oxide isostearate (Active matter of DPX10, CAS 753480-32-9) was applied on five strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA 102) and one strain of Escherichia coli (WP2 uvrA), using both plate incorporation and preincubation methods, at concentrations between 0 and 500 µg/plate in tetrahydrofuran, with or without metabolic activation in 2 independent experiments.

Except for a slight toxicity noted in the TA 100 and TA 102 strains in the second experiment without S9 mix, no noteworthy toxicity was induced towards all the strains used, both with and without S9 mix. The test substance did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six tester strains.

From the results obtained, it can be concluded that under the experimental conditions, the test item Active matter of DPX10 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

 

Furthermore in a mammalian cell cytogenetic assay (chromosome aberration assay) performed according to OECD guideline 473 and GLP (CIT report No. MLH 22632, 2002), primary human lymphocyte cultures were exposed to Cerium and iron oxide isostearate (Active matter of DPX10, CAS 753480-32-9) in tetrahydrofuran, at concentrations of 4.3 to 545.45 µg/mL (up to precipitating concentration) with and without metabolic activation in two independent experiments.

There was no evidence of chromosome aberration induced by the test item over background in the two experiments in absence or presence of metabolic activation. No clear cytotoxic effect was observed up to precipitating concentration.

Therefore, Cerium and iron oxide isostearate is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations.

 

In a mammalian cell gene mutation assay at the TK (Thymidine Kinase) locus performed according to the OECD guideline 476 and in compliance with GLP (CIT report No. 34518 MLY, 2009), L5178Y mouse lymphoma cells cultured in vitro were exposed to Cerium and iron oxide isostearate (Active matter of DPX10, CAS 753480-32-9) in tetrahydrofuran at concentrations of 7.81, 15.6, 31.3, 62.5, 125 and 250μg/mL (up to precipitating concentration) with and without S9 mix in 2 independent experiments.

A slight toxicity was sometime observed at dose-levels ≥ 15.6μg/mL but not in all experiments and without any clear evidence of a dose-response relationship.

In both experiments, no noteworthy increase in the Mutation Frequency was noted after treatment with the test item in comparison with the vehicle control in the absence or in the presence of metabolic activation..

Under the experimental conditions, the test item Cerium and iron oxide isostearate (Active matter of DPX10) did not show any mutagenic activity in the mouse lymphoma assay.

 

From the results described above, it can be concluded that Iron oxide isostearate and similar substances did not induce any mutation or chromosome aberration in in vitro studies and therefore should not be classified for genetic toxicity. (see Justification for analogue approach in IUCLID 13)

 

Genetic toxicity in vivo

As no indication of genetic toxicity was observed in in vitro bacterial reverse mutation assay performed with Iron oxide isostearate and in in vitro studies (Ames test, mammalian chromosome aberration test, and gene mutation assay in mammalian cells) performed on analogous substances showing similar chemical structure and similar properties, therefore no in vivo genetic toxicity assay should be performed in accordance with column 2 adaptation of REACH regulation Annex VIII, section 8.4. Furthermore, the final testing program established by the Competent Authorities in July 2005 and September 2007 confirms this waiving (see letters attached in Section 13.2: French CA testing program July 2005 and French CA testing program Sep 2007).

Justification for classification or non-classification

Based on the classification criteria of UN/EU GHS, and considering the negative results in the five in vitro genetic toxicity tests using Iron oxide isostearate or similar substances, no classification for mutagenicity is required.