Zinc, [1,2,3-propanetriolato(2-)-.kappa.O1,.kappa.O2]-, homopolymer, stereoisomer

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in bacteria but positive in a chromosome aberration study and positive in a mouse lymphoma assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Source: human blood was collected aseptically from healthy male donors.
- Type and identity of media: RPMI 1640 tissue culture medium (Sigma Chemical Company Ltd.)
- Preparation: Lymphocytes were separated by centrifugation
- Media after preparation: RPMI 1640 + 20% foetal calf serum (Imperial Laboratories) + phytohaemagglutinin (Wellcome) (approx. 175 µg/ml) to stimulate cell division
- Cell density: 1 x E+6 cells per ml.
- Incubation time: approx. 48 hours (37°C, 5% CO2)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from the livers of male 7-8 weeks old Sprague-Dawley derived rats, stimulated with Aroclor 1245 administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bw.

Test concentrations with justification for top dose:

Test article (first experiment): 1.0, 2.0, 3.9, 7.8, 15.6, 31.1, 62.5, 125, 250 and 500 µg/ml
Test article (second experiment): 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 62.5 and 100 µg/ml (without S9); 5, 7.5, 10, 15, 20, 30, 40, 50, 62.5 and 100 µg/ml (with S9)
Solvent control: 0.1 M HCl
Positive control: 500 and 750 µg/ml Ethyl methanesulphonate (without S9); 10 and 15 µg/ml Cyclophosphamide (with S9)

Vehicle / solvent:

- Solvent used: 0.1 M hydrochloric acid (HCl)
- Justification for choice of solvent: The test substance formed a dosable suspension in 0.1 M HCl on sonication at 50 mg/ml. This formed a fine dispersion in the culture medium when dosed at 1% v/v to give a final concentration of 500 µg/ml. This was used as the highest concentration for subsequent testing.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(0.1 M HCl)

Positive controls:

yes

Positive control substance:

other: Ethyl methanoesulphonate (without S9) and Cyclophosphamide (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period (+ phytohaemagglutinin): 48 h
- Expression time (cells in growth medium): 18 h (without S9-mix), 15 h (with S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h
- Treatment time: 18 h, no recovery period (without S9-mix); 3 h and 15 h recovery (with S9-mix)

SPINDLE INHIBITOR (cytogenetic assays): Colchicin (0.25 µg/ml), added 2 hours before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 experiments à 2 plates per dose

NUMBER OF CELLS EVALUATED: 100 per dose per experiment

CONCENTRATIONS OF THE TEST SUBSTANCE SELECTED FOR ANALYSIS:
- First test: 15.6, 7.8 and 3.9 µg/ml (without S9); 31.1, 15.6 and 7.8 µg/ml (with S9); posive cotrols: ehtyl methanesulphonate and cyclophosphamide: 500 and 15 µg/ml, respectively
- Second test: 20, 10 and 5 µg/ml (without S9); 40, 30, 20 and 10 µg/ml (with S9); posive cotrols: ehtyl methanesulphonate and cyclophosphamide: 500 and 10 µg/ml, respectively

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

SCORING OF SLIDE PREPARATION:
Metaphase cells were examined under a microscope for structural and numerical abnormalities. Structural abnormalities of chromosome were classified as gap (included chromosome type and chromatid type), chromatid break (ctb), chromosome break (csb), chromatid exchange (cte), chromosome exchange (cse), and other (fragmentation and so on). Observations of slides were carried out by the plural number of observers. The cell having at least one aberration was recorded as an aberrant cell.

Evaluation criteria:

Numbers of cells with chromosome aberration and polyploidy cells in the groups treated by the test substance are compared with those of the control groups for each dose. In principle, the test substance is judged to be clastogenic or aneugenic, if significant increase in aberration frequency, dose response and reproducibility of the result could be confirmed. For evaluation of the number of aberrant cells gaps were not included.

Statistics:

no data

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>15.6 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(30, 31.3 and 40 µg/ml)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 10 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: Following the exposure of the cells to the test substance, a sample of the supenatant medium from cultures treated with the solvent control and the two highest concentrations of test substance was removed and stored at -20°C. This was retained for osmolality measurement in the case of a positive response which is only detected at high dose levels and may be attributed to a hyperosmotic effect

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxicity data:

First experiment: In the absence of S9 -mix, there was a decrease in mitotic index to 47% of the solvent control value at 15.6 µg/ml. All higher concentrations were too toxic for analysis. In the presence of S9 -mix, the test substance caused a dose-related reduction in mitotic index leading to complete cell death at 125 µg/ml. The highest concentration suitable for analysis, 31.3 µg/ml, caused a decline in mitotic index to 45% of the control level.

Second experiment: In the absence of S9 -mix, there was a decrease in mitotic index to 53% of the solvent control value at 20 µg/ml. All higher concentrations were too toxic for analysis. In the presence of S9 -mix, the test substance caused a decrease in mitotic index to 51% of control levels at 40 µg/ml. The additional concentration of 30 µg/ml was included to assess the reproducibility of the response seen in the first test.

Metaphase data:

First experiment: In the absence of S9 -mix, the test substance caused no statistically significant increases in the proportion of metaphase figures with chromosome damage at any concentration. In the presence of S9 -mix, a statistically significant increase in the number of aberrant cells was observed at 31.3 µg/ml. This increase, to 10.5%, lies outside the historical control range.

Second experiment: In the absence of S9 -mix, the test substance caused no statistically significant increases in the number of aberrant cells at any concentration. In the presence of S9 -mix, a statistically significant increase in the number of aberrant cells was observed at 30 and 40 µg/ml. The increases, to 5.5 and 16%,respectively, lie outside the historical control range (0 -5.25%).

The test substance has shown evidence of clastogenic activity in human lymphocytes in the presence of S9 -mix, but not in its absence, in this in vitro cytogenetic test system.

First experiment, without metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	4.0
1.0	98	-
2.0	105	-
3.9	88	4.5
7.8	58	3.0
15.6	47	4.0
31.3	28	-
62.5	12	-
125	-	-
250	-	-
500	-	-
Etylmethanesulphonate	-	15.0

First experiment, with metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	2.25
1.0	96	-
2.0	88	-
3.9	96	-
7.8	63	3.0
15.6	73	3.0
31.3	45	10.5
62.5	6	-
125	-	-
250	-	-
500		-
Cyclophosphamide	-	21.5

Second experiment, without metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	1.5
2.5	89	-
5.0	83	1.5
7.5	69	-
10.0	61	1.5
15.0	56	-
20.0	53	2.5
30.0	8	-
40.0	11	-
50.0	11	-
62.5	28	-
100	-	-
Ethylmethanesulfonat	-	21.0

Second experiment, with metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	1.25
2.5	-	-
5.0	77	-
7.5	77	-
10.0	74	2.5
15.0	54	-
20.0	51	3.5
30.0	80	7.0
40.0	51	16.5
50.0	29	-
62.5	26	-
100	3	-
Cyclophosphamide	-	24.0

Conclusions:

The test item has shown evidence of clastogenic activity in the presence of S-9 mix, but not in its absence, in this in vitro cytogenetic test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1984)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

(1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (dutch modification) supplemented with 5 % heat inactivated donor horse serum (HIDHS), 2mM L-glutamine, 50 µg/ml gentamicin buffered with HEPES and bicarbonate.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Mixture of co-factors with S9 fraction of liver from rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Preliminary toxicity test: 5, 10, 15, 30, 60, 125, 250, 375, 500 µg/ml (with and without S9-mix)
Experiment I: 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 500 µg/ml (without S9-mix) and 5, 7.5, 10, 12.5, 15, 20, 25, 30, 500 µg/ml (with S9-mix)
Experiment II: 1, 2.5, 5, 10, 12, 13, 14, 15, 500 µg/ml (without S9-mix) and 1, 2.5, 5, 10, 15, 25, 27.5, 30, 500 µg/ml (with S9-mix)

Vehicle / solvent:

0.1 M HCl

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see details below

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix Migrated to IUCLID6: 500 µg/ml

Positive controls:

yes

Positive control substance:

other: 20-Methylcholanthrene (2.5 µg/ml)

Remarks:

with S9-mix

Details on test system and experimental conditions:

PRE-TEST:
A pre-test was performed in order to determine the concentration range of the main experiments.
6xE+5 cells were exposed to each concentration of the test item for 3 hours with and without S9-mix. Following treatment the cells were washed and finally re-suspended in 30 ml complete culture medium (supplemented with 10% HIDHS) for a 48 hour growth period. Growth was monitored by sampling at 24 and 48 hours. The cell density was adjusted to 2xE+5cells/ml at 24 hours after treatment. The relative suspension growth (RSG) of the treated cell cultures was calculated.

MAIN TEST:
Both experiments were performed in duplicate. The selection agent was Trifluorothymidine (TFT; 4µg/mL). Following treatment (3 h with and without S9-mix) the cells were washed and finally re-suspended in 60 ml complete culture medium (supplemented with 10% HIDHS) for a 48 hour growth period. Growth was monitored by sampling at 24 and 48 hours. The cell density was adjusted to 2xE+5cells/ml at 24 hours after treatment. After the 48 hour expression period the cells were assessed for viability and mutant frequency by plating in semi-solid agar. Viability was assessed by plating 200 cells in cloning medium in triplicate. Mutant frequency was assessed by plating 1E+6 cells in selective medium in triplicate. The plates were incubated at 37°C in an atmosphere of 5% CO2 in air for 12 days.
The results reported were based on colonies with a diameter of and greater than 100 µm. In addition the size (diameter) of all colonies was measured.

MUTANT STABILITY
To prove whether colonies grown on cloning medium arose from mutations or were due to epigenetic effects on the thymidine kinase activity, 8 large and 8 small colonies were picked from each of the top dose level of test substance and the solvent control. Each colony was allowed to grow for 7 days under non-selective conditions. An equal number of cells from each group were plated in the presence and absence of TFT in soft agar. The relative re-plating efficiency of picked off colonies was calculated as the plating efficiency on selective plates/plating efficiency on non-selective plates. If the relative re-plating efficiency was greater than 50%, the TFT resistance for that culture was deemed to be stable. The mutation was found to be stable.

Evaluation criteria:

A test was considered positive if a reproducible, at least two-fold and statistically significant increase in mutant frequency in treated cultures relative to the concurrent control with a dose relationship over two dose levels was seen. In addition, the mutant frequency must be above 150 mutants per 1E+6 survivors (historical control range).
Growth in suspension was calculated as follows: (24 hours post treatment cell count /amount of seeded cells) X (48 hours post treatment cell count/amount of seeded cells). If the cell count after 48 h treatment was below the density of seeded cells the cell count of the previous day was used.
Cell survival was estimated from the following equation: Survival = Suspension growth (% Control) x Viability in agar (% Control)/100
The mutant frequency per 1E+6 survivors (MF) was calculated as follows: (600/ Total no. of viable colonies) x (Total no. of mutant colonies/3)

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al. (UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of Mutagenicity Test Data. p.26. Cambridge University Press, Cambridge (1989)).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Viability of cultures treated with 5, 7.5, 10 and 12.5 µg/ml ranged from 84 to 23% of control (Experiment I) and of cultures treated with 10, 12, 13 and 15 µg/ml from 66 to 6% of control (Experiment II).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mean cell growths in the absence of S9-mix of cells treated with 1 - 20 µg/ml (Experiment I) ranged from 114 - 3% and from 107 - 17% when treated with 1 - 15 µg/ml (Experiment II). Viability and mutation frequency were investigated of cultures treated with 5, 7.5, 10 and 12.5 µg/ml and 10, 12, 13 and 15 µg/ml of Experiment I and II, respectively. The resulting cell survival levels ranged from 84 to 23 and from 66 to 6% of control in experiment I and II, respectively. The mutation frequency was significantly increased at 12.5 and 10, 12, 13 and 15 µg/ml in experiment I and II, respectively. Details are given in table 1.
Mean cell growths in the presence of S9-mix of cells treated with 5 - 30 µg/ml (Experiment I) ranged from 97 to 7 % and from 112 - 6% when treated with 1 - 30 µg/ml (Experiment II).
Viability and mutation frequency were investigated of cultures treated with 10, 12.5, 15, 20 and 25 µg/ml and 2.5, 5, 15 and 25 µg/ml of Experiment I and II, respectively. The resulting cell survival levels ranged from 74 to 12% and 101 to 8% in of control in experiment I and II, respectively. The mutation frequency was significantly increased at 15, 20, 25 and 15 and 25 µg/ml in experiment I and II, respectively. Details are given in table 1.
The mutation frequency of cells treated with the respective positive control was increased with and without S9-mix. Details are given in table 1.
A clearly dose related and pronounced decrease in colony size was observed with and without S9-mix. This shift towards smaller mutant colonies seen cultures was stated to be indicative of clastogenic effects. Details are given in table 1.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Summary on mutation frequency, survival and growth after treatment with test substance
Without S9-mix
	Mean Survival (%)		Mean Mutant Frequency		Mean colony diameter (mm)		Mean growth (%)
Experiment	I	II	I	II	I	II	I		II
0 µg/ml	100	100	76	111	0.8369	0.9631	100		100
5 µg/ml	75	--	68	--	0.8334	--	93		--
7.5 µg/ml	84	--	67	--	0.8400	--	110		--
10 µg/ml	56	66	108	185***	--	0.8022	79		78
12 µg/ml	--	37	--	382***	0.8058	0.7449	--		65
12.5 µg/ml	23	--	170**	--	0.7139	--	42		--
13 µg/ml	--	22	--	409***	--	0.7187	--		38
15 µg/ml	--	6	--	443***	--	0.6784	7#		17
EMS	38	36	596***	633***	0.6537	0.7026	41		60
With S9-mix
	Mean Survival (%)		Mean Mutant Frequency		Mean colony diameter (mm)		Mean growth (%)
Experiment	I	II	I	II	I	II	I	II
0 µg/ml	100	100	66	123	0.8132	0.8314	100	100
2.5 µg/ml	--	94	--	133	--	0.8587	--	92
5 µg/ml	--	101	--	150*	--	0.8199	--	103
10 µg/ml	74	--	64	--	0.8068	--	85	--
12.5 µg/ml	65	--	76	--	0.8509	--	82	--
15 µg/ml	55	57	163***	277***	0.7778	0.6845	68	71
20 µg/ml	36	--	284***	--	0.6674	--	52	--
25 µg/ml	12	8	353***	577***	0.6306	0.6212	24	18
20-MC	22	10	808***	1099***	0.6328	0.6069	52	29
*,**,***: p<0.05, 0.01, 0.001; #: culture discarded as superfluous to test requirements

Conclusions:

It was concluded that Zinc monoglycerolate did demonstrate mutagenic potential in this in vitro mammalian cell mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 16, 2008 to September 25, 2008

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:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The strain lacks an excision repair system. The sensitivity has been enhanced by Tris-EDTA treatment.

Metabolic activation:

with and without

Metabolic activation system:

Mixture of co-factors with S9 fraction of liver from Wistar rats induced with phenobarbital and beta-naphthoflavone.

Test concentrations with justification for top dose:

The results of a dose range-finding assay with 3, 10, 33, 100, 333, 1000, 3330 and 5000 microgram/plate in triplicates were reported as first experiment. Concentrations used in the second experiment were 10, 33, 100, 333 and 1000 microgram/plate (triplicates).The final concentration of S9-mix was 5% in the first and 10% in the second experiment.
The test item precipitated at dose levels of 1000 microgram/plate and upwards.

Vehicle / solvent:

- Vehicle used: Dimethyl sulfoxide (DMSO) (SeccoSolv, Merck, Darmstadt, Germany)
At concentrations of 0.03 mg/mL and higher, the test item was suspended in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Sodium azide (TA 1535) , 9-aminoacridine (TA1537), 2-nitrofluorene (TA98), methylmethansulfonate (TA100), 4-nitroquinoline N-oxide for WP2uvrA / With metabolic activation: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation
0.1 ml bacterial culture (1E+9 cells/ml), 0.1 ml of a dilution of the test substance in DMSO and either 0.5 ml S9-mix or 0.5 ml 0.1 M phosphate buffer were added to 3 ml molten top agar. After mixing the plates were incubated in the dark at 37.0 ± 10 °C for 48 h. After this period revertant colonies were counted. Each experiment was performed in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
Toxicity was assessed as clearing of the bacterial background lawn and/or as reduction of spontaneous revertants.

Evaluation criteria:

A test substance is considered positive in the test if: the total number of revertant in tester strain TA 100 is greater than 2 times the concurrent control or the total number of revertant in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater the 3 times the concurrent control and in case a positive response will be repeated, the positive repsonse should be reproducible in at least one independently repeated experiment.
The selected dose range exhibits limited solubility, as demonstrated by the preliminary toxicity range finding test.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test/Experiment I
Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards. Plates incubated with test article showed normal background growth in either concentration with and without metabolic activation. No biologically relevant increase in the number of revertants was observed.

Experiment II
Precipitation of the test item on the plates was observed at a concentration of 1000 µg/plate. Plates incubated with test article showed normal background growth in either concentration with and without metabolic activation.
In both mutation assays, there was no biologically relevant increase in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

SUMMARY OF RESULTS

Experiment 1:

	Mean revertants per plate
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
positive control	1073	354	378	425	1103	954	1048	1542	1347	374
solvent control	9	9	5	5	24	27	97	87	25	26
3	-	-	-	-	-	-	124	88	19	24
10	9	8	6	9	25	29	96	84	25	31
33	7	12	9	8	27	37	111	91	27	26
100	9	8	8	9	28	28	100	106	27	28
333	10	12	9	11	27	27	109	106	25	28
1000 SP	10	8	9	7	28	30	120	102	21	29
3330 SP	-	-	-	-	-	-	134	112	24	22
5000 SP	-	-	-	-	-	-	134	123	28	26

SP = slight precipitate

Experiment 2:

	Mean revertants per plate
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
positive control	994	206	590	477	1102	567	1121	987	1338	302
solvent control	11	7	4	5	23	26	108	66	28	34
10	9	7	6	6	20	33	112	63	20	21
33	13	6	7	8	22	31	119	79	21	26
100	9	8	6	10	24	26	114	71	22	28
333	8	10	5	8	24	30	118	83	21	28
1000 SP	6	9	8	7	24	26	138	101	24	25

SP = slight precipitate

Conclusions:

The test article was not genotoxic when tested in the Ames test with different strains of Salmonella Typhimurium and the Escherichia coli strain WP2 uvrA according to the OECD 471 (1997).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No mutagenic activity was found in an in vivo micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Bone marrow investigated in the course of this study was harvested from animals of a 90 d repeated dose toxicity feeding study conducted according to OECD guideline 408 (ESL FT930588, IUCLID Chapter 7.5.1). Animals of this study were taken to assess the potential of the test substance to induce genotoxicity in vivo by means of a micronucleous test. The conduct of the micronucleous test was in principle similar to OECD 474. It was stated in the report that the bone marrow micronucleus test, as described in this study, is an equally sensitive alternative to an (more common) acute micronucleus test. This was validated by MacGregor et al. (Fundamental and Applied Toxicology, 14,513-522, 1990) and Henderson, L. et al. (Mutation Research, 291, 79-85, 1993) for subacute 28 d studies. However, validity for a subchronic 90d study was not given. It was further stated that Wistar rats have been used in the course of this study, while the study report of the subchronic toxicity study mentioned above was performed with Sprague-Dawley rats.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 5-6 weeks, three females treated with the positive control were 21 weeks old.
- Weight at study initiation: 100 - 200 g
No further details are given in the study report. For further details see study FT930588 in IUCLID Chapter 7.5.1.

Route of administration:

oral: feed

Details on exposure:

The bone marrow investigated in the course of the study was harvested from male and female Wistar rats fed with the test substance at 0, 0.05, 0.2 and 1 % in purified diet over a 13 week period as part of study FT930588 (see IUCLID Chapter 7.5.1). The experimental diets in the mentioned study were based on the ESL Modified AIN-76A diet and were prepared weekly.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

The animals were given free access to the test diets at all times.

Post exposure period:

None

Dose / conc.:

500 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

10 000 ppm

No. of animals per sex per dose:

Treatment groups: 20 (10 animals per sex per dose were investigated in the course of this study)
Positive control group: 7
Bone marrow of 10 animals of each sex was investigated at 0 and 0.2% and the bone marrow of 5 animals of each sex was investigated at 0.05% and positive control.

Control animals:

yes

Positive control(s):

Cyclophosphamide was used as positive control. The dosing solution was prepared immediately before use by diluting in 0.9% physiological saline.
Seven male and seven female rats were dosed with cyclophosphamide during the final week of the live animal phase for this study (1994-02-01 to 1994-05-06). The positive control rats were dosed by oral gavage at 20mg/kg bw/d in an application volume of 10ml/kg bw. The animals were sacrificed 24 hours after treatment and bone marrow slides were prepared. The male rats and four of the female rats were spare animals from the feeding study (see study FT930588 in IUCLID Chapter 7.5.1). Three females were from the palatability study (see study ESL FP930559 in IUCLID Chapter 7.5.1) and were four weeks older at the time of dosing with the positive control (21 weeks).

Tissues and cell types examined:

The femoral bone marrow was examined for incidence of micronuclei in both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) together with the PCE to NCE ratio. The PCE/NCE ratio was recorded when 2000 of either PCEs or NCEs had been scored.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The dose selection was based on the results of a 14 day palatability study (ESL study FP930559).

TREATMENT AND SAMPLING TIMES
Bone marrow slides were prepared from 10 males and 10 females from each treatment group, and initially slides from 5 males and 5 females from each group were scored. Due to considerable variation in individual rat data at the 0.2% dose group further 5 males and 5 females were analysed from the 0 and 0.2 % dose. Rats of the top dose group were sacrificed during the study due to brittle bones and consequently were not assessed for micronuclei induction.

DETAILS OF SLIDE PREPARATION
The bone marrow was transferred from the femur to a clean glass slide by the use of a moistened paint brush and slides were prepared by carefully stroking the slide a few times with the brush. Two bone marrow smears were prepared from each femur. The smears were air dried, fixed in methanol and stained using a Feulgen staining procedure. All slides were dried and mounted with DPX.

METHOD OF ANALYSIS
One slide prepared from the femur of each test animal was examined by light microscopy. From each slide the incidence of micronuclei in both polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined together with the PCE to NCE ratio. The PCE/NCE ratio was recorded when either 2000 of either PCEs or NCEs had been scored.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Rats of the high dose group had to be sacrificed for humane reasons. The PCE/NCE-Ratio was, however, not affected by the treatment.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initially, the mean number of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of females treated with 0.2% test substance was doubled over the control value for the females (1.4 vs. 3.8, see table 2). Since, the individual rat data varied considerably at this dose level another five males and five females from the control group and the 0.2% dose group were analysed. The doubling of MPEC over the control value was not seen in the second assessment (combined results 2.4 vs. 3.6) and the doubling of MPECs seen in the first analysis was considered to be due to the low incidence of micronuclei in the first five control females. A summary of results is given in table 1.
The mean number of micronucleated PCEs in bone marrow of animals treated with the positive control was 25.4 and 18.6 for females and males, respectively.
The top dose assessed for micronuclei was limited due to systemic toxicity. Animals of the high dose group were sacrificed due to humane reasons (reported in ESLstudy FP930588). No deaths occurred in the lower dose groups.
The PCE/NCE-ratio indicated no evidence of cytotoxicity.

Table 1: Summary of Results
Dose	PCE/NCE (Mean)		MPCE incidence (%)		MNCE incidence (%)
	Male	Female	Male	Female	Male	Female
0 %	0.99	0.99	0.11	0.12	0.01	0.01
0.05 %	0.98	1.13	0.14	0.10	0.01	0.00
0.2 %	1.00	0.99	0.12	0.18	0.01	0.01
Positive Control	0.9	1.0	1.27	0.93	0.01	0.01

Table 2: Details on Analysis 1 and 2 of animals fed with 0 and 0.2% of test substance in the diet
	PCE/NCE-Ratio		MPCEs			N (NCE)
Analysis	I	II	I		II	I	II
0 % (5 Males)	0.84	1.07	4		3	2000	1875
	0.88	1.12	1		2	2000	1788
	1.05	0.93	0		2	1903	2000
	1.05	0.89	2		4	1898	2000
	1.01	1.10	2		2	1990	1812
Mean	0.97	1.02	1.8		2.6	1958.2	1895
Analysis	I	II	I		II	I	II
0 % (5 Females)	0.95	0.89	2		4	2000	2000
	1.28	0.87	2		3	1567	2000
	1.11	1.00	0		3	1802	1992
	1.17	0.83	1		4	1707	2000
	1.02	0.78	2		3	1960	2000
Mean	1.11	0.87	1.4		3.4	1807.2	1998.4
	PCE/NCE-Ratio		MPCEs			N (NCE)
Analysis	I	II	I	II		I	II
0.2 % (5 Males)	1.06	0.92	1	4		1889	2000
	0.78	0.97	3	2		2000	2000
	1.19	0.88	5	3		1675	2000
	1.12	1.08	1	2		1780	1846
	0.98	0.99	2	1		2000	2000
Mean	1.03	0.97	2.4	2.4		1868.8	1969.2
Analysis	I	II	I	II		I	II
0.2% (5 Females)	1.35	0.96	6	4		1481	2000
	0.83	1.04	4	3		2000	1928
	1.17	0.85	2	5		1709	2000
	0.88	0.89	1	3		2000	2000
	1.00	0.94	6	2		2000	2000
Mean	1.05	0.95	3.8	3.4		1838	1992.4

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

The test item was tested in an Ames test following OECD guideline 471 using Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100. Two independent tests were performed in the presence and absence of liver S9 fraction. The first test included a pre-incubation test procedure and the second test used plate incorporation. When diluted in 0.1 M HCl no toxicity was observed and the top dose chosen was the maximum recommended for this test,5000 µg/ml. No evidence of mutagenicity was seen in this study (Huntingdon, 1994).

In a second Ames study following OECD guideline 471 the test item was tested with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli strain WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). Based on the results of a dose range finding test, the test article dissolved in DMSO was tested in the first mutation assay at a concentration range of 10 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537and TA98. In an independent repeat of the assay with additional parameters, the test item was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test substance precipitated on the plates at the top dose of 1000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test substance did not induce a significant dose-related increase in the number of revertant colonies in each of the tester strains both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Notox, 2008).

Chromosome aberration

The test item was assessed for its ability to induce chromosome damage in cultured separated human lymphocytes. The test item was diluted in 0.l M HCl. Two independent tests were conducted in the presence and absence of liver S9 fraction. A single harvest time corresponding to approximalely 1.5 cell cycles was used. The top doses analysed were 15.6 µg/ml and 20 µg/ml in the absence of S9 in the first and second tests, respectively, and 31.3 µg/ml and 40 µg/ml in the presence of S9 in the first and second tests, respectively. These doses were chosen on the basis of toxicity, i.e. they induced approximately 50% reduction in mitotic indices. In the absence of S9 mix no evidence of clastogenicity was seen in either test. In the presence of S9 mix the test article showed evidence of clastogenicity, producing statistically significant increases in aberrant cells which lay outside the historical control range of the laboratory. It was concluded that the test item has clastogenic potential in mammalian cells (Huntingdon, 1994).

Mouse Lymphoma assay

The test item was tested for its ability to induce mutations at the thymidine kinase locus in mouse lymphoma L5178Y cells. Two independent tests were performed in the presence and absence of S9 fraction. The

top doses which produced acceptable toxicity for the assessment of mutagenic potential (i.e. approximately 10-20% relative survivaI) were 12.5 µg/ml (first test in absence of S9), 13 µg/ml (second test in absence of S9), and 25 µg/ml (first test and second test in presence of S9). Biologically and statistically significant increases in mutant frequency were seen in all tests. Sizing of the mutant colonies indicated an increased frequency of small colonies which may be indicative of claslogenicity. Zinc is involved in regulating the formation and enzymatie activity of thymidine kinase (Lieberman el al, 1963; Dreosti and Hurley, 1975; Jacobson and Turner, 1980). Due to the possibilily of zinc interfering with the uptake of the selective agent or the regulation of nucleotide metabolism the mutant colonies were picked off and assessed for phenotypic stabilily. All the colonies were found to be stable when reintroduced to the selective agent, TFT, and it was therefore concluded that the test item demonstrated mutagenic potential in this in vitro gene mutation assay and also the ability to induce phenotypically stable TFT-resistant colonies (Huntingdon, 1994).

In vivo micronucleus lest

A 13-week feeding study was performed on the test item. In view of the mutagenic potential demonstrated in the two mammalian cell in vitro tests described above, bone marrow from animals dosed in the 13-week study was analysed for micronuclei induction. When assessed at toxicity limits the assessment of micronuclei in bone marrow from sub-acute or sub-chronically dosed animals has been shown to be as sensitive as an acute micronucleus test (MacGregor el al. , 1990; Henderson el a1., 1993). Male and female Wistar rats were fed 0.05%,0.2% or 1% of the test item in purified diet. There were bone abnormalities at the high dose and as a result the animals were humanely killed during the course of the study. The incidence of micronuclei was analysed initially in smears taken from five animals of each sex from each of the remaining treatment and control groups. A positive control group treated with a single dose of cyclophosphamide, was also analysed. In order to clarify an equivocal response seen in the female group dosed with 0.2% the analysis was

extended to another five males and five females from this group and the negative control group. There was no increase in the number of micronucleated erythrocytes of the treated animals compared to the control animals (ESL, 1995).

Justification for classification or non-classification

There are conclusive but not sufficient data for classification of the test substance with regard to genotoxicity. The test substance is not classified for this endpoint in accordance to Regulation (EC) No 1272/2008.