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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
- genetic toxicity in vitro: • FeCl3: no classification, negative for bacterial gene mutation (several studies; key study: Dunkel 1999a), negative (with and without metabolic activation) for in vitro mammalian gene mutation ( 3 studies; key study:Dunkel 1999b), negative for in vitro chromosomal aberration (1 study, key study: Schulz 2009) and weak positive response at cytotoxic concentration with metabolic activation in one in vitro mammalian gene mutation study (Dunkel 1999b) • Fe2(SO4)3: no classification, no studies available, accordingly read across is used from FeCl3 • FeCl2: no classification, negative for bacterial gene mutation (1 study; key study: Kim 2004), data for in vitro mammalian gene mutation (1 study; disregarded study: Shires 1982), read-across to FeCl3 for in vitro chromosomal aberration (1 study, key study: Schulz 2009) • FeSO4: no classification, weak positive response at dose-related cytotoxic concentration with and without metabolic activation in one in vitro mammalian gene mutation study (Dunkel, 1999c) • FeClSO4: no classification, no studies available, accordingly read across is used from FeCl3 - genetic toxicity in vivo: • FeCl3: no classification, no data for in vivo mammalian gene mutation, negative for in vivo mammalian chromosome aberration (several studies; key studies: Bianchini 1988b and Ogawa 1992), • Fe2(SO4)3: no classification, no studies available, accordingly read across is used from FeCl3 • FeCl2: no classification, no reliable data for in vivo mammalian gene mutation (1 study; disregarded study: Shires 1982), negative for in vivo chromosomal aberration (1 study, key study: Ji Yoon 2004) • FeSO4: no classification, no reliable data for in vivo mammalian gene mutation (1 study; supporting study: Lee 1983), negative for in vivo chromosomal aberration (2 studies, key study: Bianchini 1988b) • FeClSO4: no classification, no studies available, accordingly read across is used from FeCl3
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date 11 June 2014, Experimental Completion Date 03 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Valid and conclusive guideline study under GLP; Relevant and adequate for this endpoint
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) number 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
(including METI, MHLW and MAFF guidances)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Statement of compliance in accordance with Directive 2004/9/EC, Department of Health of the Government of the U.K., 12 September 2014, inspection date 12 to 14 March 2014
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella typhimurium, Tryptophan for Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4, protein content adjusted to 20 mg/mL
Test concentrations with justification for top dose:
Formulated concentrations were adjusted to allow for the stated water/impurity content (3 %) of the test item.
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 μg/plate.
Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide)
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (dimethyl sulphoxide)
True negative controls:
no
Positive controls:
yes
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9-Aminoacridine (9AA): 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation (-S9)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (dimethyl sulphoxide)
True negative controls:
no
Positive controls:
yes
Remarks:
2-Aminoanthracene (2AA): 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA Benzo(a)pyrene (BP): 5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With metabolic activation (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In suspension
Experiment I: In agar (plate incorporation; 0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer or S9 mix. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
Experiment II: Preincubation: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer or S9 and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37±3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.

DURATION
- Preincubation period (Experiment 2): 20 minutes
- Exposure duration: Approximately 48 hours

NUMBER OF REPLICATIONS
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHER EXAMINATIONS:
- Other: All of the plates were scored for the presence of revertant colonies using an automated colony counting system.

OTHER:
Incubation temperature 37±3 °C
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can
be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviations
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was considered not initially soluble and thus tested in suspension.
- Precipitation: A test item precipitate (greasy/particulate in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES
Experiment 1 – Plate Incorporation Method

COMPARISON WITH HISTORICAL CONTROL DATA
The controls were found in line with the historical control data.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:
Interpretation of results (migrated information):
negative

Hydronium Jarosite was considered to be non-mutagenic under the conditions of this test.
Executive summary:
The capability of the test item Hydronium Jarosite to induce bacterial reverse mutation as a sign of gene mutation indicating genetic toxicity was measured in a GLP-compliant study using the “Bacterial Reverse Mutation Assay” compliant with EU Method B.13/14 (Commission Regulation (EC) No. 440/2008), OECD TG 471 (1997), US EPA OCSPP (former OPPTS) 870.5100 (1998) and the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals protocols. The validity criteria were met and the experiment can be considered relevant and adequate for the endpoint. Therefore it is deemed conclusive and was rated „reliable without restrictions“, i.e. “Klimisch 1” according to the scale of Klimisch et al. (1997). Five endobacterial strains from the species Salmonella (Salmonella typhimurium), TA 1535, TA 1537, TA 98 and TA 100, and coliform bacteria (Escherichia coli), WP2 uvr A, were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 μg/plate. The control system comprised untreated negative, solvent and positive controls using substances known to give a positive response in also absence (N-ethyl-N'-nitro-N-nitrosoguanidine, 9-Aminoacridine and 4-Nitroquinoline-1-oxide) or only in presence (2-Aminoanthracene, Benzo(a)pyrene) of metabolic activation (S9). The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test (pre-incubation method). A test item precipitate (greasy/particulate in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). In conclusion the test item was considered to be non-mutagenic under the conditions of this test.

  • Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxiclogical and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

This endpoint is covered by the category approach for dissociating, inorganic and non-toxic iron compounds (please see the section on toxicokinetics, metabolism and distribution for the category justification/report format).

-in vitro

bacterial gene mutation:

• FeCl3 is deemed negative for bacterial gene mutation. One reliable study (Dunkel 1999a) is available, conducted according to OECD 471 using the strains Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535, TA1537 and TA1538 with and without activation in a plate incorporation assay. The test was performed with FeCl3 x 6H2O with concentrations up to 10’000 µg/plate which is equivalent to 6001 µg/plate anhydrous FeCl3.

• Fe2(SO4)3 is deemed negative for bacterial gene mutation. No studies are available for this substance accordingly a read across from FeCl3 is employed.

• FeCl2 is deemed negative for bacterial gene mutation. One reliable study (Kim 2004) is available, conducted according to OECD 471 and GLP using the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A and concentrations up to 5000 µg/plate in a plate incorporation assay.

• FeSO4 is deemed negative for bacterial gene mutation. No studies are available for this substance accordingly a read across from FeCl2 and FeCl3 is employed.

 • FeClSO4: no classification, no studies available, accordingly read across is used from FeCl3.

 

mammalian gene mutation:

 • FeCl3 is negative for in vitro mammalian gene mutation. Two reliable studies (Dunkel 1999b; McGregor 1988) are available, conducted according to OECD 476 (Dunkel 1999b only) in mouse lymphoma L5178Y cells with cytotoxic concentrations or precipitation as the upper boundaries. In Dunkel,1999b the test was positive with metabolic activation only at cytotoxic concentration and otherwise negative, whereas in the McGregor study the test was negative. This was supported by the study Ohno,1982.

 • FeCl2 is deemed negative for in vitro mammalian gene mutation. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 • FeSO4 is deemed negative for in vitro mammalian gene mutation. One reliable study with mouse lymphoma L5178Y cells

is available (Dunkel 1999c) presenting a negative result in non-cytotoxic concentrations.

 • FeClSO4 is deemed negative for in vitro mammalian gene mutation. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 

mammalian chromosome aberration:

 • FeCl3 is deemed negative for in vitro mammalian chromosome aberration. One reliable OECD 487, GLP study (Schulz 2009) is available, with Chinese hamster lung fibroblasts (V79) up to 1650 µg/mL reporting a negative result.

 • FeCl2 is deemed negative for in vitro mammalian chromosomal aberration. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 • FeSO4 is deemed negative for in vitro mammalian chromosomal aberration. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 • FeClSO4 is deemed negative for in vitro mammalian chromosomal aberration. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 

-in vivo

mammalian gene mutation:

No studies available for any of the members of this iron salt category.

 

mammalian chromosome aberration:

• FeCl3 is deemed negative for in vivo mammalian chromosome aberration. One reliable study (Bianchini 1988a) is available, conducted according to Wargovich et al, J Natl Cancer Inst 71 125-131 1983 analysing micronuclei induction in the GI tract and reporting a negative result. This result is supported by a negative Drosophila wing spot test (Ogawa 1994). All other studies of low reliability except two (Liao, 1988; BASF, 1992) also show negative results. Overall weight of evidence suggests that the substance is negative in chromosomal aberration test.

 • FeCl2 is deemed negative for in vivo mammalian chromosome aberration. One reliable study (Ji Yoon 2004) according to OECD 474/GLP with ICR mice and doses of 2, 5, 10, 20, 50, 100 and 200 mg/ml in the dose range-finder and 1.25, 2.5 and 5 mg/ml in the micronucleus experiment was negative. No further data are available for FeCl2.

 • FeSO4 is deemed negative for in vivo mammalian chromosome aberration. Two reliable studies are available (Bianchini 1988b and Hayashi 1988). Bianchini 1988b analysed micronuclei induction in the GI tract and reported a negative result. Hayashi 1988 a Mammalian Erythrocyte Micronucleus Test according to OECD Guideline 474 using ddy mice treated intraperitoneal with 25, 50, 100, 180 mg/kg bw. The test result was negative. This result is supported by the Drosophila sex linked lethal test (Lee, 1983)

 • FeClSO4 is deemed negative in vivo mammalian chromosome aberration. No studies are available for this substance accordingly a read across from FeCl3 is employed.

 

General:

Human data is not available for genetic toxicity. Both, gene mutation and cytogenicity are cover by a plethora of studies. All relevant studies express that the iron compounds are non-genotoxic. Two short abstracts (Liao 1988 and BASF AG 1992) indicate that FeCl3 might have shown positive results in mammalian chromosomal aberration studies. Nevertheless, these data are considered to be unreliable since insufficiently reported.

In summary, the overwhelming majority of data support the conclusion that iron salts of this category are non-genotoxic. Accordingly, classification for this category is not appropriate or for any of the individual category members.


Justification for selection of genetic toxicity endpoint
Reliable study conducted according to OECD TG 487 and GLP in Chinese hamster lung fibroblasts (V79) using FeCl3

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential all members of this iron salt category are deemed non-genotoxic and accordingly do not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (5th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.