[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

865812-80-2

Cas Number:

865812-80-2

IUPAC Name:

865812-80-2

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.