Neodecanoic acid, iron salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the test substance Neodecanoic acid, iron salt, data from a bacterial reverse gene mutation assay does not suggest any mutagenic potential of the substance.

In addition, studies performed with the suitable read-across partners Neodecanoic acid, and soluble iron salts (as iron trichloride hexahydrate and iron sulphate heptahydrate) were used in a weight-of-evidence approach for the assessment of the genotoxic potential of Neodecanoic acid, iron salt. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

In vitro gene mutation studies in mammalian cells performed with Iron trichloride hexahydrate did not suggest mutagenic effects. Iron sulphate heptahydrate induced mutation frequency with metabolic activation, but not without. Iron sulphate did not increase the rate of micronucleus formation in vivo. Overall, the iron part of Neodecanoic acid, iron salt, is not considered to be genotoxic.

Neodecanoic acid neither increased the number of mutations in an in vitro mammalian cell gene mutation assay nor induced chromosomal aberrations in vitro.

By assessing the information on the target substance itself and the information derived from suitable read-across partners in a weight-of-evidence approach, Neodecanoic acid, iron salt, can be considered as not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-08-31 to 2017-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline 471 (OECD, 1997).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number: E01133-065
Purity: 100%
Date received: 19 July 2017
Retest date: 31 July 2019
Storage conditions: 15-25°C, protected from light

Target gene:

The test item was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation using an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test item at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed except in strain TA1537 at a concentration of 5000 µg/plate in the absence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of the test item approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

Vehicle / solvent:

All the test item treatments in this study were performed using formulations prepared in dimethylformamide (DMF).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene (2NF), Sodium azide (NaN3), 9-aminoacridine (AAC), Mitomycin C (MMC), Benzo[a]pyrene (B[a]P), 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

The test item was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation using an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

Rationale for test conditions:

OECD guidline

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was =1.5-fold (in strain TA102), =2-fold (in strains TA98 or TA100) or =3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values

2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Key result

Species / strain:

other: TA98, TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: It was concluded that the test item did not induce mutation

Mutation Experiment 1:

Treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of the test item at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed, except for a marked reduction in revertant numbers noted in strain TA1537 at a concentration of 5000µg/plate.

Mutation Experiment 2:

treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 µg/plate, in order to examine more closely those concentrations of the test item approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments no evidence of toxicity was observed.

In Mutation Experiment 1, precipitation of the test article was observed on the test plates at concentrations of 5000 µg/plate in all strains in the absence and presence of S-9.

In Mutation Experiment 2, precipitation of the test article was observed on the test plates at concentrations of 2500 µg/plate and above in all strains in the absence of S-9 and at 1250 µg/plate and above in all strains in the presence of S-9.

The individual mutagenicity plate counts were averaged to give mean values. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges.The positive control chemicals all induced increases in revertant numbers of =1.5-fold (in strain TA102), =2-fold (in strains TA98 and TA100) or =3-fold (in strains TA1535 and TA1537) the concurrent vehicle controlsconfirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Following the test item treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were = 1.5-fold (in strain TA102), = 2-fold (in strains TA98 and TA100) or = 3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any test item mutagenic activity in this assay system.

Conclusions:

It was concluded that the test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study (OECD 471). These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

The test item was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation using an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. It was concluded that the test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9). Based on the results, the test item can be considered as not mutagenic in the bacterial reverse gene mutation assay.

Reference 2

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:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Toxicity Assay

A preliminary toxicity assay evaluated neodecanoic acid at concentrations of 3.4 to 1740 µg/mL using a 4-hour treatment with and without S9, and a 24 hour treatment without S9 (the maximum concentration tested approximated the 10 mM limit dose for this assay). Precipitate was observed at concentrations ≥435 µg/mL at the beginning of treatment only. The test article did not adversely impact the pH or osmolality of the cultures (454 and 446 mmol/kg for the solvent control and 218 µg/mL, the highest soluble concentration at the beginning of treatment, respectively). Relative suspension growth (RSG) was 52, 5 and 29% at concentrations of 435 µg/mL (4-hour treatment with S9), 870 µg/mL (4 hour treatment without S9), and 218 µg/mL (24-hour treatment without S9), respectively. RSG at all higher concentrations in all treatment groups was 0%.

 

Mutagenesis Assays

Cultures were treated in the mutagenicity assay at concentrations of 26.7, 53.4, 107, 142, 190, 253, 338, 450, 600 and 800 µg/mL (4-hour treatment with S9), 107, 142, 190, 253, 338, 450, 600 and 800 µg/mL (4-hour treatment without S9), and 26.7, 53.4, 107, 142, 190, 253, 338 and 450 µg/mL (24-hour treatment without S9) based on the preliminary toxicity assay results. No visible precipitate was observed during treatment. The test article did not adversely impact culture osmolality (444 and 433 mmol/kg for the solvent control and the highest treatment condition, respectively). Culture treatment concentrations of 107, 190, 338, 600 and 800 µg/mL (4-hour treatment without S9) produced RSGs of 11 to 96% and were cloned, while cultures treated at concentrations of 26.7, 53.4, 107, 190 and 253 µg/mL (24 hour treatment without S9) produced RSGs of 28 to 105% and were cloned. Cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available or due to excessive toxicity. One culture treated at 800 µg/mL for 4-hours without S9 was excluded from evaluation of mutagenicity due to excessive toxicity. The remaining cloned cultures had relative total growth (RTG) of 10 to 69% (4-hour treatment without S9) and 23 to 96% (24 hour treatment without S9). Induced mutant frequency (IMF) did not increase under either treatment condition. The average mutant frequency of the negative controls with S9 exceeded the acceptance criteria (not shown) and that portion of the assay was repeated.

A re-test was conducted under identical conditions using a 4-hour treatment with S9. No visible precipitate was observed during treatment. Culture treatment concentrations of 53.4, 142, 253, 450 and 600 µg/mL resulted in 44 to 93% RSG and were cloned (cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available or due to excessive toxicity). RTG of the cloned cultures ranged from 34 to 91%. No increases in IMF were observed.

The trifluorothymidine-resistant colonies for the positive and solvent control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. Colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Conclusions:

In conclusion, Neodecanoic acid is not mutagenic in the mouse lymphoma test.

Executive summary:

Neodecanoic acid was evaluated in vitro for potential to cause gene mutations in mammalian cells using the mouse lymphoma L5178Y assay conducted according to OECD Test Guideline 490. Rat liver S9 fraction was used for metabolic activation. Cells were exposed to concentrations from 0 to 800 micrograms/mL for 4 hours with and without metabolic activation and from 0 to 450 micrograms/mL for 24 hours with metabolic activation. Evidence for cytotoxicity (% relative growth less than 50%) was observed at concentrations of 218 micrograms/mL and higher. Positive controls (methylmethanesulfonate and 7,12-dimethylbenzanthracene) performed as expected, increasing induced mutant frequency ~5 to 20-fold in a concentration-related manner. These results indicate that neodecanoic acid was negative in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay under the conditions, and according to the criteria, of the test protocol.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

 

Reference 3

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:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Cytotoxicity was also markedly increased with metabolic activation. There was no increase in the number of small colonies, and the proportion of large and small colonies remained constant for all compounds. For detailed results please refer to box "Any other information on results incl. tables".

Table 1: Mutagenicity and cytotoxicity of iron sulphate heptahydrate in the L5178Y TK+/- Mouse Lymphoma Assay

	Dose [µg Fe/mL]	Rat S9 mix	Absolute cloning efficiency^a	Relative total growth [% of control]	Average no. TFT colonies	Mutant frequency/10^6 survivors^b
Solvent control (Distilled water)	0	-	0.90/0.98	100.0	26/20	25
	20.10	-	0.81/0.84	84.5	16/25	25
	50.25	-	0.74/0.83	72.5	19/26	29
	100.50	-	0.75/0.86	51.5	27/24	32
	150.75	-	0.72/0.71	27.5	28/30	46
	201.00	-	0.58/0.59	10.5	39/54	80
Positive control (EMS)		-	0.54	46.0	230	430
Solvent control (Distilled water)	0	+	0.92/0.91	100.0	28/21	27
	0.804	+	0.69/0.76	61.5	37/39	53
	1.005	+	0.68/0.70	54.5	43/49	67
	1.206	+	0.66/0.69	41.5	46/47	70
	1.508	+	0.49/0.58	5.0	47/43	88
Positive control (DMBA)		+	0.57	52.0	116	171

a: Based on the average of three petri dishes each plated with 200 cells.

b: 1 x 10⁸ cells in a measured volume were plated in each of three plates/culture in the presence of trifluorothymidine (3 µg/ml) after 2 days of expression.The values are for duplicate cultures (four cultures/solvent control).

The dose levels for the positive controls were: EMS 4.7 x 10^-6 M; DMBA 9.8 x 10^-6 M.

Conclusions:

Based on the results from an in vitro mouse lymphoma assay, iron sulphate heptahydrate was tested negative without metabolic activation and positive with metabolic acitvation.

Executive summary:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells cultured in vitro were exposed to iron sulphate heptahydrate, dissolved in distilled water at concentrations of 0.1, 50,25, 100.5, 150.75 and 201 µg Fe/mL in the absence and at concentrations of 0.804, 1.005, 1.206 and 1.508 µg Fe/mL in the presence of mammalian metabolic activation.

The induced mutation frequency without metabolic activation was not dose-dependently increased compared to the negative control. The induced mutation frequency with metabolic activation was dose-dependently increased in comparison to the negative control. The positive controls did induce the appropriate response.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 4

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:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: primary

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no statistical increases in the number of cells with chromosomal aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at the highest concentrations tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In conclusion, the test item Versatic 10 does not induce chromosome aberrations.

Executive summary:

The test substance Versatic 10 (neodecanoic acid) was examined for its potential to induce structural chromosome aberrations in cultured human lymphocytes in both the absence and presence of a metabolic activation system (S9 mix), in compliance with OECD guideline 473. Two independent chromosome aberration tests were conducted in both the absence and presence of S9. In the absence of S9, cells were exposed to the test substance continuously for 24 or 48 hours. In the presence of the S9, cells were exposed to the test substance for 3 hours and harvested at 24 or 48 hours later. The choice for the highest concentrations scored was based on toxicity. The test substance was dissolved in DMSO. In either chromosome aberration assay, Versatic 10 did not induce a statistically significant increase in the percentage of cells with structural chromosome aberrations at any of the concentrations and time points analyzed. The positive controls gave appropriate responses. It is concluded that Versatic 10 is not clastogenic under the conditions used in this study. 

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 5

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:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Four experiments were conducted, two in each activation condition. Precipitation was observed at 150 µg/mL. In no experiment was there a mutagenic response without precipitation of ferric chloride. A significant response was observed in one experiment without S9 mix at a concentration of 600 µg/mL, well above the saturation concentration. It was concluded that ferric chloride was not a mutagen. However, since a statistically significant response was observed after precipitation, in one experiment, longer exposure times may be required for a positive response to be demonstrated. For detailed results please refer to box "Any other information on results incl. tables".

Table 1: Trial 1_Without metabolic activation

Conc. [µg/mL]	CE	RTG	MC	MF	AVG MF
DMSO	98	97	151	51
0	102	108	164	54
	121	96	164	45
	95	95	225	79	61
37.5	91	118	126	46
	94	100	151	54	50
75	90	115	147	55
150	92	102	149	54
300	105	88	209	67
	100	95	188	63	65
600	95	51	290	102
	89	50	291	109	106
1200	Lethal	-	-	-
	Lethal	-	-	-
MMS [15 µg/mL]	33	23	418	424
	32	20	422	440	432

CE: cloning efficiency

RTG: relative total growth

MC: mutant colony count

MF: mutant fraction

AVE MF: group average mutant fraction

Statistically significant values are underlined

 

Table 2: Trial 2_Without metabolic activation

Conc. [µg/mL]	CE	RTG	MC	MF	AVG MF
DMSO	115	108	236	69
0	97	88	202	70
	80	95	153	64
	96	110	180	63	66
15	91	97	197	72
	112	106	218	65	69
30	104	98	211	68
	87	114	170	66	67
60	104	111	155	50
	84	108	165	66	58
120	89	86	160	60
	112	96	183	54	57
240	90	101	143	53
	103	100	150	49	51
MMS [15 µg/mL]	64	41	464	240
	45	36	348	257	249

 

Table 3: Trial 3_With metabolic activation

Conc. [µg/mL]	CE	RTG	MC	MF	AVG MF
DMSO	83	103	127	51
0	88	107	136	52
	81	90	173	71
	97	99	142	49	56
20	94	98	113	40
	92	92	131	47	44
50	91	85	121	44
	93	101	120	43	44
80	87	91	106	41
	101	97	152	50	45
110	93	102	120	43
	101	84	142	47	45
140	89	81	124	46
	96	78	134	47	47
MMS [15 µg/mL]	66	50	599	301
	51	46	581	381	341

 

Table 4: Trial 4_With metabolic activation

Conc. [µg/mL]	CE	RTG	MC	MF	AVG MF
DMSO	70	112	136	64
0	52	79	126	81
	87	95	134	51
	85	114	136	54	63
20	72	91	101	47
	72	85	135	62	55
50	80	96	123	52
	67	86	125	63	57
80	76	91	122	54
	83	102	122	49	52
110	81	91	157	65
	77	95	128	55	60
140	75	99	126	56
	74	102	141	64	60
MMS [15 µg/mL]	38	27	719	639
	35	32	684	645	642

 

Conclusions:

In this study under the given conditions, iron trichloride is considered to be non-mutagenic in the in vitro mouse lymphoma assay in the presence and absence of mammalian metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells were exposed to iron trichloride dissolved in DMSO at concentrations of 0 to 1200.0 µg/mL without and 0 to 140 µg/mL with metabolic activation in a total of 4 experiments. The positive controls did induce the appropriate responses. Precipitation was observed at 150 µg/mL. A significant mutagenic response was only observed at 600 µg/mL without S9 in the presence of precipitation, well above the saturation concentration. Based on these results, iron trichloride is considered to be non-mutagenic.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

To further support the the conclusion derived from the available in vitro data, in vivo data from the suitable read-across partner Iron sulphate was used. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Iron sulphate was tested negative in an in vivo micronucleus test. Based on the results from the suitable read-across partner, it is concluded that Neodecanoic acid does not cause cytogenetic damage in vivo.

Link to relevant study records

Reference

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:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

4 mortalities out of 6 at 180 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Table 1: Result of the micronucleus test using mouse bone marrow cells

No. of doses	Time between doses [h]	Sampling time [h]	Dose level [mg/kg bw]	MNPCE [%]	PCE [%]	Mortality	Trend Test*	Judgement
1	-	24	0	0.20±0.13	53.8±7.7	0/6	NS	negative
			25	0.23±0.23	48.9±16.8	0/6
			50	0.28±0.16	50.0±9.3	0/6
			100	0.40±0.17^a	54.2±12.7	0/6
			180	0.10±0.14	53.8±0.7	4/6
			MMC: 2.0	7.25±2.33^a	45.6±7.8	0/6		positive
1		24	0	0.27±0.20	71.8±9.2	0/6	NS	negative
			100	0.23±0.26	64.3±7.8	0/6
			150	0.30±0.10	65.4±17.7	0/6
4	24	24	50	0.23±0.18	43.2±6.7	0/6		negative

* dose-response relationships were tested using the Cochran-Armitage trend test. A positive dose-response was considered significant at p<0.05

MNPCE = micronucleated polychromatic erythrocytes

PCE = polychromatic erythrocytes

NS = not significant

^asignificantly different from historical control (p< 0.01)

Conclusions:

Under the reported experimental conditions iron sulphate did not induce structural and/or numerical chromosomal damage in the bone marrow cells of the mouse.

Executive summary:

In a ddY mouse bone marrow micronucleus test conducted similar to OECD guideline 474, six male mice per dose were treated intraperitoneally with iron sulphate (87.65% purity) at doses of 0, 25, 50, 100 and 180 mg/kg bw (experiment 1), in experiment 2 at doses of 0, 100 and 150 mg/kg bw and at 50 mg/kg bw in experiment 3. The animals were injected intraperitoneally with the test substance once (experiments 1 and 2) or four times at 24 h intervals (experiment 3) with an additional 24 hours period after the last dosing. The vehicle was water. Iron sulphate did not increase the level of micronuclei in comparison to the concurrent vehicle control. Thus, the substnace is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.

This information is used in a read-across approach in the assessment of the target substance.For details and justification of read-across please refer to the read-across report attached to IUCLID section 13

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the test substance Neodecanoic acid, iron salt, data from a bacterial reverse mutation assay was available. The test item, was assayed for mutation in a bacterial reverse mutation assay (according to OECD 471) using five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation using an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. Vehicle and positive control treatments were included for all strains in both experiments. It was concluded that the test item did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

In addition, studies performed with the suitable read-across partners neodecanoic acid, and soluble iron salts (iron trichloride hexahydrate and iron sulphate heptahydrate) were used in a weight-of-evidence approach for the assessment of the genotoxic potential of Neodecanoic acid, iron salt.

For the iron compound:

In a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells cultured in vitro were exposed to Iron sulphate heptahydrate, dissolved in distilled water at concentrations of 0.1, 50,25, 100.5, 150.75 and 201 µg Fe/mL in the absence and at concentrations of 0.804, 1.005, 1.206 and 1.508 µg Fe/mL in the presence of mammalian metabolic activation. The induced mutation frequency without metabolic activation was not dose-dependently increased compared to the negative control. The induced mutation frequency with metabolic activation was dose-dependently increased in comparison to the negative control. The positive controls did induce the appropriate response.

Furthermore, in a mammalian cell gene mutation assay conducted similar to OECD guideline 490, L5178Y mouse lymphoma cells were exposed to iron trichloride dissolved in DMSO at concentrations of 0 to 1200.0 µg/mL without and 0 to 140 µg/mL with metabolic activation in a total of 4 experiments. The positive controls did induce the appropriate responses. Precipitation was observed at 150 µg/mL. A significant mutagenic response was only observed at 600 µg/mL without S9 in the presence of precipitation, well above the saturation concentration. Based on these results, iron trichloride is considered to be non-mutagenic.

In a mouse bone marrow micronucleus test conducted similar to OECD guideline 474, six male mice per dose were treated intraperitoneally with iron sulphate (87.65% purity) at doses of 0, 25, 50, 100 and 180 mg/kg bw (experiment 1) and in experiment 2 at doses of 0, 100 and 150 mg/kg bw and at 50 mg/kg bw in experiment 3. The animals were injected intraperitoneally with the test substance once (experiments 1 and 2) or four times at 24 h intervals (experiment 3) with an additional 24 hours period after the last dosing. Iron sulphate did not increase the level of micronuclei in comparison to the concurrent vehicle control. Thus, the substance is considered not to cause cytogenetic damage according to the results of the in vivo micronucleus test.

For the neodecanoic acid compound: 

The read-across partner Neodecanoic acid was evaluated in vitro for potential to cause gene mutations in mammalian cells using the mouse lymphoma L5178Y assay according to OECD Test Guideline 490. Rat liver S9 fraction was used for metabolic activation. Cells were exposed to concentrations from 0 to 800 micrograms/mL for 4 hours with and without metabolic activation and from 0 to 450 micrograms/mL for 24 hours with metabolic activation. Evidence for cytotoxicity (% relative growth less than 50%) was observed at concentrations of 218 micrograms/mL and higher. Positive controls (methylmethanesulfonate and 7,12-dimethylbenzanthracene) performed as expected, increasing induced mutant frequency ~5 to 20-fold in a concentration-related manner.

These results indicate that neodecanoic acid was negative in the L5178Y/TK+/-Mouse Lymphoma Mutagenesis Assay under the conditions, and according to the criteria, of the test protocol.

Neodecanoic acid was additionally examined for its potential to induce structural chromosome aberrations in cultured human lymphocytes in both the absence and presence of a metabolic activation system (S9 mix), in compliance with OECD guideline 473.

Two independent chromosome aberration tests were conducted in both the absence and presence of S9. In the absence of S9, cells were exposed to the test substance continuously for 24 or 48 hours. In the presence of the S9, cells were exposed to the test substance for 3 hours and harvested at 24 or 48 hours later. In either chromosome aberration assay, Neodecanoic acid did not induce a statistically significant increase in the percentage of cells with structural chromosome aberrations at any of the concentrations and time points analyzed. The positive controls gave appropriate responses. It is concluded, that Neodecanoic acid is not clastogenic under the conditions used in this study. 

 

Justification for classification or non-classification

Based on the available data, Neodecanoic acid, iron salt is considered to be non-mutagenic and does not warrant classification for genotoxicity in accordance to CLP Regulation 1272/2008.