Reaction products of sodium glucoheptonate with iron sulfate and ammonium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

To address the endpoint genetic toxicity, read-across on gluconates and derivatives and iron compounds was performed within the frame of a weight of evidence approach. The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

The overall conclusion on mutagenicity of iron glucoheptonate is based on several studies available for gluconates, its derivatives, and iron compounds. The registered substance is not considered to be genotoxic because all available genetic toxicity studies in vitro in bacterial cells and yeasts conducted with sodium ferric gluconate complex in sucrose injection, iron dichloride and sodium gluconate or glucono-delta-lactone were negative. The positive outcomes of the in vitro studies with sodium ferric gluconate complex in sucrose (Ferrlecit) and other iron compounds on mammalian cells are indicative of the potentiated formation of active oxygen species under overload condition. They are not indicative for a primary mutagenic effect and are not relevant to the genetic toxicity of the registered substance.

Data on sodium ferric gluconate complex in sucrose injection

Gene Mutation was evaluated in an Ames Test. The employed doses were 0.625 - 5.00 mg Fe/plate. The number of revertants per plate was counted. No positive increase in the number of revertants per plate of any of the tester strains with / without microsomal enzymes prepared from AroclorTM -induced rat liver (S9). The results of the tests show that Ferrlecit is negative for genetic mutagenicity sanofi-aventis, 2009).

Clastogenicity was evaluated in chinese hamster ovary cells. The employed doses were 225 - 1250 µg Fe/mL. The number of cells with chromosomal aberrations was counted.

No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed from the non-activation assay. In the assay with metabolic activation, however, significant increases in cells with chromosomal aberrations were observed at the 10-hour harvest in the cultures dosed with 1250 μg Fe/mL Ferrlecit® and at all dose levels (313 to 1250 μg Fe/mL Ferrlecit®) at the 20-hour harvest. The response was highly variable between replicate cultures and was not dose-related. These results were indicative of ferric ion potentiated formation of active oxygen species by the S9 system rather than any metabolic activation of the test article. Such a "test system-generated" response would make the utilization of S9 inappropriate for screening this test article for clastogenic activity. Taking this explanation into account, the results of the tests show that Ferrlecit is negative for genetic mutagenicity (sanofi-aventis, 2009).

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Data on iron dichloride

A bacterial reverse mutation test, according to OECD test guideline 471 with iron dichloride was performed in compliance with GLP. It was concluded that iron dichloride did not exhibit mutagenic activity to any test strains under the test conditions (SIDS 2004).

Data on iron gluconate

The mutagenicity iron gluconate was examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. Iron gluconate was negative in two tester strains (Fujita et al. 1994).

Brusick (1975) also evaluated iron gluconate (Compound 000299296) for genetic activity in a series of in vitro microbial assays with and without metabolic activation. In Salmonella typhimurium plate tests, ferrous gluconate was not mutagenic for the bacterial indicator organisms used in the direct and activation plate tests at a concentration of 0.079 %. In nonactivation suspension tests, the results were negative. However, in activation suspension tests, the compound was positive in activation tests with TA-1538 and primate liver tissue.

In nonactivation and activation tests with Saccharomyces cerevisiae, the results were negative (Brusick, 1975). In cotrast, iron gluconate, appeared to be mutagenic for indicator strain TA-1538 in activation tests with primate liver activation homogenate.

However, the overall result of iron gluconate is negative. This is based on the following arguments.

a) the "traditional" S- Typhimurium strains 1535 and 1537 were always negative

b) the "unusual" S. typhimurium strain 1538 was negative in tests with the "traditional" rats S9 metabolic activation system.

c) nonactivation and activation tests with saccharomyces cerevisiae were negative.

Therefore the single - even though repeatable - positive result obtained in Strain 1538 with monkey liver S9 fraction is overruled.

Data on several iron compounds and elemental iron

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds were evaluated for mutagenicity in L5178Y mouse lymphoma cells (Dunkel et al. 1999).

Ferric chloride (FeCl3) and ferric phosphate (FePO4), induced an increase in mutant frequency only with S9. With ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), on the other hand, positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9.

These divergent responses were related to the different oxidation states of iron in the different compounds. The oxidation state of iron is also influenced by liver S9 mix. NADP and reductase in the liver S9 mix lead to the reduction of ferric (Fe3+) to ferrous (Fe2+). The transferrin-independent transport system requires the reduced form of iron (Fe2 +). Thus, Fe2+ is absorbed by the cells more effectively, which is accompanied by an increased toxicity of Fe2 + when compared to Fe3 +.

The positive genotoxic response of mouse lymphoma cells to Fe2+ is most likely to be caused by the activation of the oxygen species H2O2 and superoxide (O2 -*), which may result in the formation of the hydroxyl radical (*OH), the most powerful DNA-damaging radical. Attack of *OH on DNA produces a variety of different products and all purine and pyrimidine bases can be modified (Dunkel et al. 1999).

Data on gluconates and derivatives

Sodium gluconate, glucono-delta-lactone and calcium gluconate were tested on Saccharomyces cerevisiae and Salmonella typhimurium with and without metabolic activation similar to OECD Guideline 471. The substances were tested on Saccharomyces cerevisiae (strain D4) and Salmonella typhimurium (3 strains) with and without metabolic activation. Only 3 concentrations were tested where OECD guideline recommends at least 5 concentrations. None of the test substances showed mutagenicity on the strains tested.

The genetic toxicity of monosaccharides, disaccharides, and related ingredients as used in cosmetics was determined. The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies and the results of these studies are negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Iron gluconate and iron glucoheptonate are structurally similar sugar-like substances with the same functional groups, whereby gluconate and glucoheptonate anions share similar chemical moiety. They differ only in one carbon segment (HCOH): glucoheptonate is longer (C7) than gluconate (C6). Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate, releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: sodium ferric gluconate complex in sucrose injection
The substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).

Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The calculation of a hazard value for iron glucoheptonate is based on 76 % content of iron glucoheptonate in the registered product. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconate and glucoheptonate anions share similar chemical moiety. Iron is also the same cation in both chemicals. They dissociate according the same scheme: 1:1 iron and gluconate or glucoheptonate are released. Therefore, genetic toxicity of both chemicals is expected to be very similar.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

No positive increase in the number of revertants per plate of any of the tester strains with / without microsomal enzymes prepared from AroclorTM -induced rat liver (S9).

Conclusions:

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Executive summary:

Gene Mutation was evaluated in an Ames Test (sanofi-aventis Canada Inc., 2009). The employed doses were 0.625 - 5.00 mg Fe/plate. The number of revertants per plate was counted. No positive increase in the number of revertants per plate of any of the tester strains with / without microsomal enzymes prepared from AroclorTM -induced rat liver (S9). The results of the tests show that Ferrlecit is negative for genetic mutagenicity.

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Reference 2

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:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Iron gluconate and iron glucoheptonate are structurally similar sugar-like substances with the same functional groups, whereby gluconate and glucoheptonate anions share similar chemical moiety. They differ only in one carbon segment (HCOH): glucoheptonate is longer (C7) than gluconate (C6). Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate, releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: sodium ferric gluconate complex in sucrose injection
The substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).

Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The calculation of a hazard value for iron glucoheptonate is based on 76 % content of iron glucoheptonate in the registered product. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconate and glucoheptonate anions share similar chemical moiety. Iron is also the same cation in both chemicals. They dissociate according the same scheme: 1:1 iron and gluconate or glucoheptonate are released. Therefore, genetic toxicity of both chemicals is expected to be very similar.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: all strains tested

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed from the non-activation assay. In the assay with metabolic activation, however, significant increases in cells with chromosomal aberrations were observed at the 10-hour harvest in the cultures dosed with 1250 μg Fe/mL Ferrlecit® and at all dose levels (313 to 1250 μg Fe/mL Ferrlecit®) at the 20-hour harvest. The response was highly variable between replicate cultures and was not dose-related. These results were indicative of ferric ion potentiated formation of active oxygen species by the S9 system rather than any metabolic activation of the test article. Such a "test system-generated" response would make the utilization of S9 inappropriate for screening this test article for clastogenic activity.

Conclusions:

The results of the tests are considered to show that Ferrlecit is negative for genetic mutagenocity.
These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Executive summary:

Clastogenicity was evaluated in chinese hamster ovary cells. The employed doses were 225 - 1250 µg Fe/mL. The number of cells with chromosomal aberrations was counted.

No significant increase in cells with chromosomal aberrations was observed in the cultures analyzed from the non-activation assay. In the assay with metabolic activation, however, significant increases in cells with chromosomal aberrations were observed at the 10-hour harvest in the cultures dosed with 1250 μg Fe/mL Ferrlecit® and at all dose levels (313 to 1250 μg Fe/mL Ferrlecit®) at the 20-hour harvest. The response was highly variable between replicate cultures and was not dose-related. These results were indicative of ferric ion potentiated formation of active oxygen species by the S9 system rather than any metabolic activation of the test article. Such a "test system-generated" response would make the utilization of S9 inappropriate for screening this test article for clastogenic activity. Taking this explanation into account, the results of the tests show that Ferrlecit is negative for genetic mutagenicity.

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Iron gluconate and iron glucoheptonate are structurally similar sugar-like substances with the same functional groups, whereby gluconate and glucoheptonate anions share similar chemical moiety. They differ only in one carbon segment (HCOH): glucoheptonate is longer (C7) than gluconate (C6). Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate, releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

There is limited information on purity of iron gluconate in this study. Purity of 100 % was therefore assumed.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconate and glucoheptonate anions share similar chemical moiety. Iron is also the same cation in both chemicals. They dissociate according the same scheme: 1:1 iron and gluconate or glucoheptonate are released. Therefore, genetic toxicity of both chemicals is expected to be very similar.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other:

Remarks:

with liver homogenate, lung homogentate or testes homogenate from mouse, rat or monkey

Compound 000299296, Ferrous Gluconate, was evaluated for genetic activity in a series of in vitro microbial assays with and without metabolic activation. The following results were obtained:

A. Salmonella typhimurium

1. Plate Tests - At a concentration of 0.079%, 000299296 was not mutagenic for the bacterial indicator organisms used in the direct and activation plate tests.

2. Nonactivation suspension tests - The results of these tests were negative.

3. Activation suspension tests - The results of these tests indicate that this compound is positive in activation tests with TA-1538 and primate liver tissue. The tests were repeated and the results appeared positive. No other tests were positive with this compound.

B. Saccharomyces cerevisiae

1. Nonactivation suspension tests - The results of these tests were negative.

2. Activation suspension tests - The results of these tests were negative.

C. Conclusions - Compound 000299296, Ferrous Gluconate, appeared to be mutagenic for indicator strain TA-1538 in activation tests with primate liver tissue. No other tests with this compound were positive.

Table 2 - Overall Summary Of Test Results
Plate Tests
A.	Name or code designation of test compound:					000299296
B.	Test date:	02/14/75
C.	Concentration of the test compound:				0.07%
Test		Species	Tissue	TA-1535		TA-1537		TA-1538
				1	2	1	2	1	2
1.   Non-activation
	Solvent Control	---	---	47	78	11	14	26	6
	Positive Control^a	---	---	>10^3	>10^3	209	301	147	140
	Test Compound	---	---	47	17	3	3	11	6
2. Activation
	Negative Control	---	---	40	90	14	22	14	4
	Solvent Control	---	---	27	89	10	33	17	18
	Reaction Mixture Control	---	---	47	31	12	34	20	22
	Positive Control^b	Mouse	Liver	>500	>500	83	75	>200	>200
	Positive Control		Lung	59	59	10	7	20	10
	Positive Control		Testes	42	60	11	10	11	10
	Positive Control	Rat	Liver	>300	>300	83	86	>10^2	>10^2
	Positive Control		Lung	63	77	10	7	19	9
	Positive Control		Testes	44	61	10	8	12	9
	Positive Control	Monkey	Liver	>100	>100	41	34	>100	93
	Positive Control		Lung	71	54	9	8	20	6
	Positive Control		Testes	47	61	10	9	13	10
	Test Compound	Mouse	Liver	16	23	7	3	2	2
	Test Compound		Lung	35	15	0	4	1	2
	Test Compound		Testes	35	25	2	0	2	3
	Test Compound	Rat	Liver	20	19	4	3	2	2
	Test Compound		Lung	33	17	0	3	2	2
	Test Compound		Testes	32	26	2	1	3	3
	Test Compound	Monkey	Liver	16	31	4	1	3	2
	Test Compound		Lung	35	23	0	6	1	3
	Test Compound		Testes	29	26	2	1	2	4
a	TA-1535	EMS	10	µL/plate	b	TA-1535	DMNA	50	µm/plate
	TA-1537	QM	20	µg/plate		TA-1537	AAF	100	µg/plate
	TA-1538	NF	100	µg/plate		TA-1538	AAF	100	µg/plate

Table 3 – Summary of Results of Non-activation experiments
Litton Bionetics Mutagenic Activity System
Report EXR34
Compound Frequency Summary Report 04/25/75
					Compound 000299296
		Non –activation experiments
		TA1535	TA1537	TA1538	0000D4	0000D4
		HIS	HIS	HIS	ADE	TRY
TEST	DRG	EX-8	EX-8	EX-8	EX-5	EX-5
NAN		1.60	5.71	4.89	2.89	46.67
NAP		1158.63	73.09	200.22	24.55	77.84
NA1		1.66	2.03	3.75	6.51	42.52
NA2		3.41	4.87	5.69	4. 74	36.50

Table 4 – Summary of Results of Activation experiments
Litton Bionetics Mutagenic Activity System
Report EXR34
Compound Frequency Summary Report 04/25/75
Species ICRFLO (Flow ICR Random Bred Mice)
					Compound 000299296
		TA1535	TA1537		TA1538	0000D4	0000D4
		HIS	HIS		HIS	ADE	TRY
TEST	DRG	EX-8	EX-8		EX-8	EX-5	EX-5
ACT	A + C	5.02	7.20		3.61	3. 84	19.18
ACT	A + T	16.14	2.38		4.54	6.72	49.80
ACT	A - C	3.28	7.02		8.13	2.60	32.95
ACT	PLI	538.06	17.26		25.91 '	6.95	106.58
ACT	PLU	5.71	8.54		4.83	6.50	72.42
ACT	PTE	14.00	4.13		3.04	5.84	81.70
ACT	LI1	1.93	8.16		5.77	7.82	50.95
ACT	LI2	3.30	4.88		1.69	8.58	62.88
ACT	LU1	3.11	9.29		2.86	6.36	23.93
ACT	LU2	3.46	8.11		4.58	8.53	42.23
ACT	TE1	3.49	2.13		2.13	6.56	26.00
ACT	TE2	3.09	8.44		5.01	5.94	37.03
Species SPRDAW (Sprague Dawley Rats)
					Compound 000299296
		TA1535	TA1537	TA1537	TA1538	0000D4	0000D4
		HIS	HIS	HIS	HIS	ADE	TRY
TEST	DRG	EX-8	EX-8	EX-8	EX-8	EX-5	EX-5
ACT	A + C	1.61	5.41		1.64	3.41	3.32
ACT	A + T		2.25		1.70	1.11	1.01
ACT	A - C	1.49	3,04	1 .97	1.65	2.45	3.15
ACT	PLI	19.21	11.93		13.62	6.67	10.95
ACT	PLU	2.07	1.90		1,49	1.75	3.06
ACT	PTE	3.56	2.84		1.74	2.17	2.96
ACT	LI1	1.94	3.86		2.83	2.69	2.98
ACT	LI2	3.07	161.11	2.03	3.68	3.03	3.55
ACT	LU1	2.42	242.11	2.07	2.54	1.67	3.13
ACT	LU2	1.02	62.50	3.00	2.63	2.55	1.67
ACT	TE1	7.58	35.94	2.78	2.26	4.18	1.83
ACT	TE2	4.66	2.25		2.84	5.08	4.95
Species RHESUS (Rhesus Monkey (Macaca mulatta))
					Compound 000299296
		TA1535	TA1537	TA1538	TA1538	0000D4	0000D4
		HIS	HIS	HIS	HIS	ADE	TRY
TEST	DRG	EX-8	EX-8	EX-8	EX-8	EX-5	EX-5
ACT	A + C	18.56	2.33	5.85		2.22	3.02
ACT	A + T	18.18	2.76	8.85		3.08	2.73
ACT	A - C	13.67	2.66	4.20	5.62	3.02	1.99
ACT	PLI	283.75	9.04	86.14		5.98	5.82
ACT	PLU	12.85	2.33	4.58		2.41	2.81
ACT	PTE	15.37	1.56	5.62		2.99	3.95
ACT	LI1	19.13	2.51	152.94	43.75	3.17	4.95
ACT	LI2	26.36	3.76	47.87	66.04	3.04	4.80
ACT	LU1	11.28	2.43	16.10		5.09	4.95
ACT	LU2	18.35	2.06	10.06		3.15	3.72
ACT	TE1	19.62	2.58	17.37		4.39	4.14
ACT	TE2	14.04	2.75	9.56		2.86	2.56

Data Table Terms and Abrreviations

COMPOUND = Client designated compound number appears in this column.

TEST CODES

NAN = Non Activation: Solvent Control

NAP = Non Activation: Positive Control

NAT = Non Activation: Test Compound Dose 1

NA2, etc. = Reflects the other dose level(s)

A+C = Negative Chemical Control

A-C = Activation: Solvent Control

ACP = Activation: Positive Control

ACT = Activation: Test Compound

A+T = Activation: Tissue Control

LI = Liver Tissue Activation Fraction

LU = Lung Tissue Activation Fraction

KI = Kidney Tissue Activation Fraction

TE = Testes Tissue Activation Fraction

1,2, etc. = Dose Levels

CONCENTRATION = All test compound dose levels are expressed as a whole number followed by an exponent (negative) identified by the appropriate units. Example: 0025-2PCT = 0.25 percent concentration

POPU = Total number of viable cells in the plating sample raised to some exponent printed directly below the abbreviation (i.e., EP + 6 = X 106).

MUT 1 = Total number of mutants or convertants obtained from the sample plated raised to some exponent printed directly below the abbreviation (i.e., EP + 0 = X 10°). For strain D4, MUT 1 represents the number of ADE+ convertants.

MUT 2 = Only used for strain D4 and represents the number of TRY+ convertants in the plated sample.

FREQ 1 = The calculated mutation or gene conversion frequency times the negative exponent written directly below. For strain D4, FREQ 1 represents the ADE+ value.

FREQ 2 = Only used for strain D4 and represents the TRY+ conversion frequency.

CONTAM = Presence of contamination on any plates.

AAF = 2-Acetylaminofluorene

DMSO = Dimethylsulfoxide

DMN = Dimethylnitrosamine

EMS = Ethyl Methanesulfonate

QM = Quinacrine Mustard

NF = Nitrofluorene

SPECIES = Animal Strains

SPRDAW = Sprague Dawley Rats

ICRFLO = Flow ICR Random Bred Mice

RHESUS = Rhesus Monkey (Macaca mulatta)

MIXEDB = Dog, Mixed Breed

NEWZEA = New Zealand White Rabbit

Conclusions:

The overall result of Ferrous gluconate is negative. This is based on the following arguments.
a) the "traditional" S- Typhimurium strains 1535 and 1537 were always negative
b) the "unusual" S. typhimurium strain 1538 was negative in tests with the "traditional" rats S9 metabolic activation system.
Therefore the single - even though repeatable - positive result obtained in Strain 1538 with monkey liver S9 fraction is overruled.
This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate,, which is therefore considered to be also "not mutagenic" according to 1272/2008/EC.

Executive summary:

Compound 000299296, Ferrous Gluconate, was evaluated for genetic activity in a series of in vitro microbial assays with and without metabolic activation (Brusick, 1975). In Salmonella typhimurium plate tests, 000299296 was not mutagenic for the bacterial indicator organisms used in the direct and activation plate tests at a concentration of 0.079 %. In nonactivation suspension tests, the results were negative. However, in activation suspension tests, the compound was positive in activation tests with TA-1538 and primate liver tissue. The tests were repeated and the results appeared positive. No other tests were positive with this compound.

In nonactivation and activation tests with saccharomyces cerevisiae, the results were negative.

Though, compound 000299296, Ferrous Gluconate, appeared to be mutagenic for indicator strain TA-1538 in activation tests with primate liver activation homogenate.

However, the overall result of Ferrous gluconate is negative. This is based on the following arguments.

a) the "traditional" S- Typhimurium strains 1535 and 1537 were always negative

b) the "unusual" S. typhimurium strain 1538 was negative in tests with the "traditional" rats S9 metabolic activation system.

c) nonactivation and activation tests with saccharomyces cerevisiae were negative.

Therefore the single - even though repeatable - positive result obtained in Strain 1538 with monkey liver S9 fraction is overruled.

These results are also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also not mutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: summary of available in vitro and in vivo genetic toxicity data

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate on releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects. So, information on genetic toxicity of gluconate anion allows to strengthen the read-across approach, excluding genetic toxicity potential of the organic part of the molecule.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconates are not mutagenic in bacterial strains. Due to high structural similarity and the fact that gluconates and glucoheptonates are carbohydrates used by cells in high amounts, no mutagenicity is expected for glucoheptonate anion.

4. DATA MATRIX
please refer to the detailled data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1535; TA 1537 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Cytotoxic concentration (50% survival) (μg/ml):

Sodium gluconate: 0.024 (bacteria), 50 (yeast);

Glucono-delta-lactone: 10 (bacteria), 50 (yeast);

Caclium gluconate: 50 (bacteria), 30 (yeast).

Conclusions:

The available in vitro mutagenicity data with glucono-delta-lactone, sodium or calcium gluconate were negative.
These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not mutagenic is important.

Executive summary:

Sodium gluconate, glucono-delta-lactone and calcium gluconate were tested on Saccharomyces cerevisiae and Salmonella typhimurium with and without metabolic activation. OECD Guideline 471 was deviated for the number of strains tested and the choice of positive controls. The substances were tested on Saccharomyces cerevisiae (strain D4) and Salmonella typhimurium (3 strains) with and without metabolic activation. Only 3 concentrations were tested where OECD guideline recommends at least 5 concentrations. None of the test substances showed mutagenicity on the strains tested.

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not mutagenic is important.

Reference 5

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: summary of mutagenicity data

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate on releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects. So, information on genetic toxicity of gluconate anion allows to strengthen the read-across approach, excluding genetic toxicity potential of the organic part of the molecule.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.
Gluconates used in genetic toxicity studies described in this record do not include impurities that are different from those contained in the target substance and which would influence genetic toxicity potential. Moreover, since the data originate from a review, no detailed information on purity and impurities could be extracted. However, the results on mutagenicity of gluconates summarised in this Cosmetic ingredient review are peer-reviewed and therefore any impurity that would influence mutagenicity result of the tested substances is assessed by the Panel and therefore could be excluded as not relevant for the present assessment.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconates are not mutagenic in bacterial strains. Due to high structural similarity and the fact that gluconates and glucoheptonates are carbohydrates used by cells in high amounts, no mutagenicity is expected for glucoheptonate anion.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

other: The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are overwhelmingly negative.

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

other: not applicable (summary result)

Untreated negative controls validity:

other: not applicable (summary result)

Positive controls validity:

other: not applicable (summary result)

Remarks on result:

other: all strains/cell types tested

Conclusions:

The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are overwhelmingly negative.
These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these mono- and disaccharides are not mutagenic is important.

Executive summary:

Summary of genetic toxicity study results conducted with monosaccharides, disaccharides, and related ingredients as used in cosmetics.

The genotoxicity of a number of the mono- and disaccharides has been evaluated in in vitro and in vivo studies. The results of these studies are overwhelmingly negative.

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these mono- and disaccharides are not mutagenic is important.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Iron gluconate and iron glucoheptonate are structurally similar sugar-like substances with the same functional groups, whereby gluconate and glucoheptonate anions share similar chemical moiety. They differ only in one carbon segment (HCOH): glucoheptonate is longer (C7) than gluconate (C6). Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate, releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

There is limited information on purity of iron gluconate in this study. Purity of 100 % was therefore assumed.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconate and glucoheptonate anions share similar chemical moiety. Iron is also the same cation in both chemicals. They dissociate according the same scheme: 1:1 iron and gluconate or glucoheptonate are released. Therefore, genetic toxicity of both chemicals is expected to be very similar.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 97

Remarks:

Iron gluconate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Remarks:

Iron gluconate

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1. Results of Mutation Test with iron (II) gluconate

Dose (mg/plate)	No of Revertants /plate
	TA97		TA102
	-S9	+S9	-S9	+S9
1	192	248	244	372
0.5	187	251	222	425
0.1	182	218	263	375
0.05	205	217	235	372
0.01	206	228	229	353
0	190	242	226	379
Positive control	1000 ± 205	3545 ± 396	3142 ± 309	1090 ± 195
Solvent: DW (not specified)

Conclusions:

Iron gluconate was negative in two tester strains Salmonella typhimurium TA97 and TA102. Thus, it is very likely that iron glucoheptonate is also negative in Ames test.

Executive summary:

The mutagenicity of 34 food additives including 10 seasonings, 6 bases for processing, 5 flavouring agents and 13 others were examined in Ames' tester strains, Salmonella typhimurium TA97 and TA102. The mutation test was carried out by the preincubation procedure described by Ames et al. The test chemicals were preincubated with S9 mix or phosphate buffer (pH7.4) for 20 min. Iron gluconate was negative in two tester strains.

This result is also relevant for iron glucoheptonate, as ferrous gluconate is highly similar to iron glucoheptonate, which is therefore considered to be also negative in Ames test.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate in organic fluids releasing their metals that can further interact with a variety of biomolecules, while no genetic toxicity can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, producing an adverse genotoxicity effect. Therefore, data on genetic toxicity potential of several organic and inorganic iron compounds have been taken into account to address genetic toxicity potential of iron cation which could be released from the iron glucoheptonate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Ammonium sulfate was not mutagenic in bacteria (Ames test) and yeasts with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures. Therefore, the iron ion is the only toxicological relevant component of the registered substance.

The purity of the iron compounds used in this study range between 98 and 100 %.

3. ANALOGUE APPROACH JUSTIFICATION
Since glucoheptonate anion is considered not mutagenic to bacterial strains, certain mutagenicity potential, if any, could be attributed to iron. Therefore, data on mutagenicity of inorganic iron compounds are used to address mutagenicity of iron.

4. DATA MATRIX
please refer to the detailled data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Electrolytic Fe (particle size 16 µm)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Carbonyl Fe (particle size 3 µm)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

FeSO4

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

ferrous fumarate

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

ferrous fumarate

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

FeCl3, FePO4

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

NaFeEDTA

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe3+) to ferrous (Fe2+). With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe2+ compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. NaFe EDTA is the most relevant source substance for the chelate iron glucoheptonate.
A possible explanation for the genotoxicity is the activation of the oxygen species, H2O2 and superoxide (O2 -*) by Fe, which may result in the formation of the hydroxyl radical (*OH), the most powerful DNA-damaging radical. Attack of *OH on DNA produces a variety of different products and all purine and pyrimidine bases can be modified.

Executive summary:

In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe3+) to ferrous (Fe2+). With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe2+ compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. NaFe EDTA is the most relevant source substance for the chelate iron glucoheptonate.

A possible explanation for the genotoxicity is the activation of the oxygen species, H2O2 and superoxide (O2 -*) by Fe, which may result in the formation of the hydroxyl radical (*OH), the most powerful DNA-damaging radical. Attack of *OH on DNA produces a variety of different products and all purine and pyrimidine bases can be modified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

To address the endpoint genetic toxicity read-across on gluconates and derivatives and iron compounds was performed within the frame of a weight-of-evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

The conclusion on mutagenicity of iron glucoheptonate is based on several studies available for gluconates, its derivatives, and different iron compounds. Overall, the registered substance iron glucoheptonate is not considered to be genotoxic. In vivo studies conducted with glucono-delta-lactone and sodium gluconate did not induce chromosomal aberrations in mice, showing that no genetic toxicity can be attributed to the glucoheptonate moiety. An in vivo study with iron sulfate (Comet and micronucleus assay) had positive results. However, the dose of the test substance was that high that the genotoxic effects can be attributed to oxidative stress due to overload conditions. They are not indicative for a primary mutagenic effect. Moreover, such high intracellular Fe concentrations cannot be reached with the registered substance, iron glucoheptonate complex, in comparable in vivo settings. Another in vivo micronucleus with sodium ferric gluconate had a negative outcome. Based on these data, iron glucoheptonate is considered to be not mutagenic.

Data on sodium ferric gluconate complex in sucrose injection

Clastogenicity was evaluated in an rat micronucleus test (Sanofi-Aventis, 2009). The employed doses were 26, 52.1; and 104 mg Fe/kg. The number of micronucleated polychromatic erythrocytes was counted.

No significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times, except in the 104 mg/kg males at the 72-hour harvest time. This is a statistical anomaly, since this was not significant compared with the female vehicle control animals and not very different from the female 104 mg/kg group at the 24-hour harvest time.

In conclusion, sodium ferric gluconate complex in sucrose injection has been shown to be negative for clastogenicity in the rat micronucleus test (sanofi-aventis, 2009).

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Data on iron sulfate

Mice were gavaged for six consecutive days with either water or 33.2 mg/kg body weight iron. On the 7th day, the bone marrow micronucleus test and the comet assay were conducted. The results show significant genotoxicity for iron at the high doses tested. Iron induced damage to the chromosomes or the mitotic apparatus of erythroblasts and double strand breaks of DNA (Pra et al. 2008). However, it has to be considered that the test dose was very high (33.2 mg/kg body weight/day). The recommended daily iron intake for adult human is between 10 and 15 mg/day (Deutsche Gesellschaft für Ernährung). This corresponds to about 0.2 mg/kg body weight/day. It can be assumed that the need in iron is not significantly higher for mice than for human. Therefore, the mice in this study were exposed to an enormous iron overload. This might have induced cellular stress leading to the formation of reactive oxygen species, which can cause DNA damage. Considering the fact that iron glucoheptonate has an iron content of only 12 %, an even higher dose of iron glucoheptonate would be required to induce the same effect.

Data on gluconates and derivatives

Sodium gluconate and glucono-delta-lactone were tested for induction of chromosomal aberrations in mouse bone marrow cells after an oral single and a 4 days repeated dose administration. At least 200 metaphase cells per mouse were scored (C57BL male mice) and were examined for the presence or absence of chromosomal aberrations (gaps, breaks, translocation, fragments, ring chromosomes and minutes chromosomes). The frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administration and 4-d repeat administration. In conclusion, the test item is considered to be negative for genetic mutagenicity (SIDS 2004, CIR 2014).

Moreover, single dose administration tests of sodium gluconate at doses of 2.5, 5 and 10 g/kg were performed.

Induction of chromosomal aberration by sodium gluconate was not detected after in vivo single and repeated dose treatment. Thus, sodium gluconate is considered negative for mutagenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate on releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects. So, information on genetic toxicity of gluconate anion allows to strengthen the read-across approach, excluding genetic toxicity potential of the organic part of the molecule.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Based on the negative results from in vitro studies and the negative results in the micronucleus test in vivo with ammonium chloride a mutagenic activity of ammonium sulfate in vivo is unlikely (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.
Gluconates used in genetic toxicity studies described in this record do not include impurities that are different from those contained in the target substance and which would influence genetic toxicity potential. Moreover, since the data originate from a review, no detailed information on purity and impurities could be extracted. However, the results on mutagenicity of gluconates summarised in this Cosmetic ingredient review are peer-reviewed and therefore any impurity that would influence mutagenicity result of the tested substances is assessed by the Panel and therefore could be excluded as not relevant for the present assessment.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconates are not mutagenic in bacterial strains. Due to high structural similarity and the fact that gluconates and glucoheptonates are carbohydrates used by cells in high amounts, no mutagenicity is expected for glucoheptonate anion.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Remarks:

in both experiments: single administration and 4-d repeated dose administration.

Toxicity:

yes

Remarks:

At 8 g/kg, all mice died (single dose administration experiment)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Single dose administration:

At 8 g/kg, all mice died.

MMC induced chromosomal aberrations in at least 20% of bone marrow cells.

GDL induced chromosomal aberrations in the cells at a frequency of about 0.5% comparable to the control.

4-day repeated dose administration:

MMC induced chromosomal aberrations at about 30% cells.

The frequency of cells with chromosomal aberrations was 1 % or less in the test groups which is comparable to the control group. Induction of chromosomal aberration by GDL was not detected after in vivo single and repeated dose treatment.

Conclusions:

The frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administartion and 4-d repeat administration. The test item is considered to be negative for genetic mutagenicity.
These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not mutagenic is important.

Executive summary:

Sodium gluconate and glucono-delta-lactone were tested for induction of chromosomal aberrations in mouse bone marrow cells after an oral single and a 4 days repeated dose administration. At least 200 metaphase cells per mouse were scored (C57BL male mice) and were examined for the presence or absence of chromosomal aberrations (gaps, breaks, translocation, fragments, ring chromosomes and minutes chromosomes).

After single dose administration of glucono-delta-lactone: at 2 and 4 g/kg the number of chromosomal aberration was unchanged compared to control (both at a frequency of about 0.5 %). At 8 g/kg all mice died and the presence or absence of chromosomal aberration could not be determined. The positive control Mitomycin C induced chromosomal aberration in at least 20 % of bone marrow cells.

GDL induced chromosomal aberrations in the cells at a frequency of about 0.5% comparable to the control.

After a 4-day repeated dose administration of glucono-delta-lactone the positive control mitomycin C induced chromosomal aberrations at about 30% cells. However, the frequency of cells with chromosomal aberrations in the test groups (2 g/kg and 4 g/kg) was comparable to the control group.

In conclusion, the frequency of cells with chromosomal aberrations in the test groups was comparable to the control group in both experiments: single administration and 4-d repeat administration. The test item is considered to be negative for genetic mutagenicity.

These results are also relevant for iron glucoheptonate, as the glucoheptonate-residue is also a derivative of gluconic acid. Therefore, the fact that these gluconates are not mutagenic is important.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate in organic fluids releasing their metals that can further interact with a variety of biomolecules, while no genetic toxicity can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, producing an adverse genotoxicity effects. Therefore, data on genetic toxicity potential of several organic and inorganic iron compounds have been taken into account to address genetic toxicity potential of iron cation which could be released from the iron glucoheptonate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source Chemical: sodium ferric gluconate complex in sucrose injection
Each vial of 5 mL of FERRLECIT contains 12.5 mg/mL (62.5 mg/5 mL vial) of elemental iron as the sodium salt of a ferric ion gluconate complex in alkaline aqueous solution with approximately 20% sucrose w/v (195 mg/mL) in Water for Injection (pH 7.7 - 9.7). The solution contains 0.9% w/v (9 mg/mL) benzyl alcohol as preservative.
It is used to replete and maintain the total body content of iron.
The substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

Target Chemical: Sodium Iron glucoheptonate complex (HGA:Fe-1:1), CAS 1821694-04-5,
SMILES [Na+].[H]C([H])(O)C([H])(O)C1([H])O[Fe]2OC([H])(C([O-])=O)C([H])(O2)C1([H])O,
MW 354.8 g/mol (trihydrated from) or 300.8g/mol (anhydrous form)
The molecular formula is C7FeH10NaO8 *3H2O (trihydrated form).

Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The calculation of a hazard value for iron glucoheptonate is based on 76 % content of iron glucoheptonate in the registered product. Another component is Na2SO4. Sodium is a macroelement occurring in surface waters and in living organisms in considerable amounts. Sulfur species are also found in living organisms. Thus, these cations and anions are considered not to impact the toxicity of iron glucoheptonate.


3. ANALOGUE APPROACH JUSTIFICATION
Gluconate and glucoheptonate anions share similar chemical moiety. Iron is also the same cation in both chemicals. They dissociate according the same scheme: 1:1 iron and gluconate or glucoheptonate are released. Therefore, genetic toxicity of both chemicals is expected to be very similar.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

No significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times, except in the 104 mg/kg males at the 72-hour harvest time. This is a statistical anomaly, since this was not significant compared with the female vehicle control animals and not very different from the female 104 mg/kg group at the 24-hour harvest time. Due to toxicity, the PCE/NCE (poly- to normochromatic erythrocyte) ratio of the 26.0, 52.1, and 104 mg/kg dose groups at the 48-hour harvest time, and the positive control males and females were significantly lower than the vehicle control group. The positive control, cyclophosphamide, induced significant increases in micronucleated PCEs in both sexes as compared to the vehicle control, with means and standard errors of 3.44%"0.66% and 2.02%"0.36% for the males and females, respectively. The results indicate that Ferrlecit is not mutagenic in the rat micronucleus test.

Conclusions:

The results indicate that Ferrlecit is not mutagenic in the rat micronucleus test.
These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Executive summary:

Clastogenicity was evaluated in an rat micronucleus test. The employed doses were 26, 52.1; and 104 mg Fe/kg. The number of micronucleated polychromatic erythrocytes was counted.

No significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times, except in the 104 mg/kg males at the 72-hour harvest time. This is a statistical anomaly, since this was not significant compared with the female vehicle control animals and not very different from the female 104 mg/kg group at the 24-hour harvest time. Due to toxicity, the PCE/NCE (poly- to normochromatic erythrocyte) ratio of the 26.0, 52.1, and 104 mg/kg dose groups at the 48 -hour harvest time, and the positive control males and females were significantly lower than the vehicle control group. The positive control, cyclophosphamide, induced significant increases in micronucleated PCEs in both sexes as compared to the vehicle control, with means and standard errors of 3.44%"0.66% and 2.02%"0.36% for the males and females, respectively.

The results indicate that Ferrlecit is not mutagenic in the rat micronucleus test.

These results are of high relevance for the substance iron glucoheptonate, since sodium ferric gluconate complex in sucrose injection is a highly recommended read across substance, due to its similar chemical behaviour and its similar uses.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate on releasing their metals that can further interact with a variety of cellular proteins, while no genetic toxicity potential can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, leading to adverse mutagenicity and/or clastogenicity effects. So, information on genetic toxicity of gluconate anion allows to strengthen the read-across approach, excluding genetic toxicity potential of the organic part of the molecule.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Based on the negative results from in vitro studies and the negative results in the micronucleus test in vivo with ammonium chloride a mutagenic activity of ammonium sulfate in vivo is unlikely (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.
Gluconates used in genetic toxicity studies described in this record do not include impurities that are different from those contained in the target substance and which would influence genetic toxicity potential. Moreover, since the data originate from a review, no detailed information on purity and impurities could be extracted. However, the results on mutagenicity of gluconates summarised in this Cosmetic ingredient review are peer-reviewed and therefore any impurity that would influence mutagenicity result of the tested substances is assessed by the Panel and therefore could be excluded as not relevant for the present assessment.

3. ANALOGUE APPROACH JUSTIFICATION
Gluconates are not mutagenic in bacterial strains. Due to high structural similarity and the fact that gluconates and glucoheptonates are carbohydrates used by cells in high amounts, no mutagenicity is expected for glucoheptonate anion.

4. DATA MATRIX
data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Remarks:

in both experiments: single dose administration and 4-d repeated dose administration.

Toxicity:

yes

Remarks:

At 10 and 5 g/kg, all mice died (single dose administration); at 1.25 and 2.5 g/kg, one mouse died in each group (4-day repeated dose administration).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Single dose administration:

At 10 and 5 g/kg, all mice died.

At 2.5 g/kg, observation could be made only on 2 animals (preparation of the chromosome specimen failed).

MMC induced chromosomal aberrations in at least 20% of bone marrow cells. Sodium gluconate induced chromosomal aberrations in the cells at a frequency of about 0.5% is comparable to the control. (1 gap and 1 minute chromosome for 283 cells).

4-day repeated dose administration:

At 1.25 and 2.5 g/kg, one mouse died in each group.

MMC induced chromosomal aberrations at about 30% cells. The frequency of cells with chromosomal aberrations was 0.5% in the test groups which is comparable to the control group.

Conclusions:

Interpretation of results (migrated information): negative
Induction of chromosomal aberration by sodium gluconate was not detected after in vivo single and repeated dose treatment. The test item is considered negative for mutagenicity.

Executive summary:

Single dose administration tests of sodium gluconate at doses of 2.5, 5 and 10 g/kg were performed. At 10 and 5 g/kg, all mice died. At 2.5 g/kg, observations could be made only on 2 animals (preparation of the chromosome specimen failed). The positive control mitomycin C induced chromosomal aberrations in at least 20% of bone marrow cells. Sodium gluconate induced chromosomal aberrations in the cells at a frequency of about 0.5% is comparable to the control. (1 gap and 1 minute chromosome for 283 cells).

After 4-day repeated dose administration of sodium gluconate, one mouse of 2 or 3 animals, respectively, died in each group after doses of 1.25 and 2.5 g/kg. The positive control mitomycin C induced chromosomal aberrations at about 30% cells. The frequency of cells with chromosomal aberrations was 0.5% in the test groups which is comparable to the control group.

In conclusion, induction of chromosomal aberration by sodium gluconate was not detected after in vivo single and repeated dose treatment. The test item is considered negative for mutagenicity.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate in organic fluids releasing their metals that can further interact with a variety of biomolecules, while no genetic toxicity can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, producing an adverse genotoxicity effect. Therefore, data on genetic toxicity potential of several organic and inorganic copper compounds have been taken into account to address genetic toxicity potential of iron cation which could be released from the iron glucoheptonate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Based on the negative results from in vitro studies and the negative results in the micronucleus test in vivo with ammonium chloride a mutagenic activity of ammonium sulfate in vivo is unlikely (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

The purity of the iron compounds used in this study range between 98 and 100 %.

3. ANALOGUE APPROACH JUSTIFICATION
Since glucoheptonate anion is considered not mutagenic to bacterial strains, certain mutagenicity potential, if any, could be attributed to iron. Therefore, data on mutagenicity of inorganic iron compounds are used to address mutagenicity of iron.

4. DATA MATRIX
please refer to the detailled data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Dose / conc.:

33.23 mg/kg bw/day

Remarks:

elemental iron

Sex:

male/female

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The current results show significant genotoxicity for iron at the high doses tested. Iron induces damage to the chromosomes or the mitotic apparatus of erythroblasts. Due to the fact that the chelated iron of the target substance iron glucoheptonate is released in the body, these results are of high relevance.

Executive summary:

Mice were gavaged for six consecutive days with either water or 33.2 mg/kg body weight iron. On the 7th day, the bone marrow micronucleus test was used as genotoxicity endpoint. The results show significant genotoxicity for iron at the high doses tested. Iron induces damage to the chromosomes or the mitotic apparatus of erythroblasts.

Due to the fact that the chelated iron of the target substance iron glucoheptonate is released in the body, these results are of high relevance.

Reference 5

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

comet assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to read-across statement attached under section 13 of this IUCLID file.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Genetic toxicity potential of gluconates and glucoheptonates is believed to be driven by metal cation bonded to the organic part of the molecule and not by the organic part itself. Gluconates and glucoheptonates can dissociate in organic fluids releasing their metals that can further interact with a variety of biomolecules, while no genetic toxicity can be attributed to the organic part of the molecule. This is because gluconates and glucoheptonates are structurally similar sugar-like substances with the same functional groups (sugar backbone), which are not known to bind to DNA, producing an adverse genotoxicity effect. Therefore, data on genetic toxicity potential of several organic and inorganic iron compounds have been taken into account to address genetic toxicity potential of iron cation which could be released from the iron glucoheptonate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Information on purity of the registered substance is provided in the target record under "Test material" as confidential. The iron glucoheptonate content in the registered product is 76 %. Other components are ammonium sulfate and sodium sulfate. Ammonium sulfate and sodium sulfate are considered not to impact the genetic toxicity of the target substance to a significant degree. Based on the negative results from in vitro studies and the negative results in the micronucleus test in vivo with ammonium chloride a mutagenic activity of ammonium sulfate in vivo is unlikely (OECD SIDS, 2004). Therefore, the iron ion is the only toxicological relevant component of the registered substance.

The purity of the iron compounds used in this study range between 98 and 100 %.

3. ANALOGUE APPROACH JUSTIFICATION
Since glucoheptonate anion is considered not mutagenic to bacterial strains, certain mutagenicity potential, if any, could be attributed to iron. Therefore, data on mutagenicity of inorganic iron compounds are used to address mutagenicity of iron.

4. DATA MATRIX
please refer to the detailled data matrix attached in section 13.

Reason / purpose for cross-reference:

read-across source

Dose / conc.:

33.23 mg/kg bw/day

Remarks:

elemental iron

No. of animals per sex per dose:

5 males and 5 females

Sex:

male/female

Genotoxicity:

positive

Remarks:

iron sulfate

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The current results show significant genotoxicity for iron at the high doses tested. Iron seems to induce double strand breaks, single strand breaks and alkali labile sites at a considerable level. Continuous treatment with iron probably led to an accumulation of oxidative lesions in DNA that act as alkali labile sites or excision repair sites. Due to the fact that the chelated iron of the target substance iron glucoheptonate is released in the body, these results are of high relevance. It indicates that iron glucoheptonate might be genotoxic.

Executive summary:

Mice were gavaged for six consecutive days with either water or 33.2 mg/kg body weight iron. On the 7th day, the neutral and alkaline comet assays in whole blood was used as genotoxicity endpoint. The results show significant genotoxicity for iron at the high doses tested. Iron seems to induce double strand breaks, single strand breaks and alkali labile sites at a considerable level. Continuous treatment with iron probably led to an accumulation of oxidative lesions in DNA that act as alkali labile sites or excision repair sites. Due to the fact that the chelated iron of the target substance iron glucoheptonate is released in the body, these results are of high relevance. It indicates that iron glucoheptonate might be genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on all available information, the substance not classified as mutagenic and therefore does not have to be labelled as mutagenic substance according to European Regulation (EC) No 1272/2008.