Phosphoric acid, 2-ethylhexyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test = non-mutagenic. Thompson P.W., 2011. OECD 471 (Bacterial Reverse mutation assay).

Chromosomal abberation = non-clastogenic. Lacey F.E. & Durward R., 2011. OECD 473 (In vitro mamalian chromosome abberation test.

CHO HPRT forward mutation assay = non-mutagenic. Morris A., 2012. OECD 476 (In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 25 May 2011 and 21 July 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.Tryptophan for E.coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plateExperiment one: 5, 15, 50, 150, 500, 1500 and 5000 µg/plateExperiment two: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide- Justification for choice of solvent/vehicle: The test item formed an emulsion in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA): 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mixMigrated to IUCLID6: (BP): 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mixMigrated to IUCLID6: (4NQO): 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mixMigrated to IUCLID6: (9AA): 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mixMigrated to IUCLID6: (ENNG): 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mixMigrated to IUCLID6: (ENNG): 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mixMigrated to IUCLID6: (ENNG): 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation (Experiment 1) and pre-incubation (Experiment 2))DURATION- Preincubation period for bacterial strains: 10h- Exposure duration: Plates were incubatede for approximately 48 hours- Expression time (cells in growth medium): Not applicable- Selection time (if incubation with a selection agent): Not applicableNUMBER OF REPLICATIONS: Triplicate plating.DETERMINATION OF CYTOTOXICITY - Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:The reverse mutation assay may be considered valid if the following criteria are met:All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic dose levels.There should be no evidence of excessive contamination.Evaluation criteria:There are several criteria for determining a positive result. Any, one, or all of thefollowing can be used to determine the overall result of the study:1. A dose-related increase in mutant frequency over the dose range tested 2. A reproducible increase at one or more concentrations.3. Biological relevance against in-house historical control ranges.4. Statistical analysis of data as determined by UKEMS.5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviationDunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic but tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

cytotoxicity

Remarks:

Toxic but tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Solubility: The test item was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.RANGE-FINDING/SCREENING STUDIES: Preliminary Toxicity Test:The test item initially exhibited toxicity at and above 1500 µg/plate to TA100 and 5000 µg/plate to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.COMPARISON WITH HISTORICAL CONTROL DATA: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix beginning at 1500 µg/plate (TA1537) and at 5000 µg/plate for all of the remaining strains. In the main test (pre-incubation method) weakened bacterial background lawns or a reduction in revertant frequency were initially noted at 500 µg/plate (absence of S9-mix) and from 1500 µg/plate (presence of S9-mix). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS

Preliminary ToxicityTest

The test item initially exhibited toxicity at and above 1500 µg/plate to TA100 and 5000 µg/plate to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	79	77	70	82	68	82	69	77	59	60	32*
+	TA100	69	67	62	63	65	60	67	64	64	29*	20*
-	WP2uvrA^-	31	41	26	29	42	33	34	27	27	27	7*
+	WP2uvrA^-	42	33	33	44	35	31	31	29	29	30	15*

*: Partial absence of bacterial background lawn

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 (see attached background material) with the results also expressed graphically in Figure 1 to Figure 4 (see attached background material)

A history profile of vehicle and positive control values is presented in attached background material (Appendix 3 historic controls).

In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix beginning at 1500 µg/plate (TA1537) and at 5000 µg/plate for all of the remaining strains. In the main test (pre-incubation method) weakened bacterial background lawns or a reduction in revertant frequency were initially noted at 500 µg/plate (absence of S9-mix) and from 1500 µg/plate (presence of S9-mix). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
111		30		31		21		14
103	(115)	18	(24)	39	(37)	19	(19)	15	(12)
131		24		42		18		6

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
102		26		32		22		10
108	(103)	22	(24)	33	(34)	23	(22)	11	(11)
99		24		37		22		13

Data Tables and graphs for Experiments 1 and 2 can be found in "Attached background material"

Conclusions:

Interpretation of results (migrated information):negativeNo significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Results.

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

In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix beginning at 1500 µg/plate (TA1537) and at 5000 µg/plate for all of the remaining strains. In the main test (pre-incubation method) weakened bacterial background lawns or a reduction in revertant frequency were initially noted at 500 µg/plate (absence of S9-mix) and from 1500 µg/plate (presence of S9-mix). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion.

The test item was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 22 June 2011 and 05 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for Mycoplasma contamination:yes- Periodically checked for karyotype stability: no- Periodically "cleansed" against high spontaneous background: yesCell Line :The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.Cell Culture:The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Hams F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.Cell Cleansing:Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

The test item was considered to be a mixture, therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level. The dose range of test item used in the preliminary cytotoxicity test was 19.53 to 5000 µg/ml. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-Exposure GroupFinal concentration of test item (µg/ml)4-hour without S92.5, 5, 10, 20, 30, 404-hour with S9 (2%)5, 10, 20, 40, 60, 8024-hour without S91.25, 2.5, 5, 10, 20, 30, 404-hour with S9 (1%)5, 10, 20, 30, 40, 50, 60

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)- Justification for choice of solvent/vehicle: The test material formed a solution with the solvent suitable for dosing at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

Dimethyl benzanthracene (DMBA) at 0.5 and1.0 µg/ml was used as the positive controls in cultures with S9. All positive controls were dissolved in dimethyl sulphoxide and dosed at 1%.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethyl methane sulphonate (EMS) was used at 500 and 750 µg/ml as the positive control in the 4-hour cultures without S9 and at 200 and 300 µg/ml for the 24-hour cultures without S9.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.DURATION- Exposure duration: 4 hours (with and without S9), 24 hours (without S9)- Expression time (cells in growth medium): 7 daysSELECTION AGENT (mutation assays): 6-thioguanine (6-TG)NUMBER OF REPLICATIONS: Duplicate culturesDETERMINATION OF CYTOTOXICITY- Method: Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.ASSAY ACCEPTANCE CRITERIAAn assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25 x 10-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 10-6 will not be used for the evaluation of a test item.iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.iv) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/ml) or twice the solubility limit of the test item in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.vi) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Evaluation criteria:

Please see 'Assay Acceptance criteria', in details on test system and conditions section.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST:A dose range of 19.53 to 5000 µg/ml was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in the attached Table 1 (attached background material). It can be seen that there was very marked toxicity at and above 19.53 µg/ml in both the 4 hour exposure group in the absence of S9 and the 24 hour exposure group with no surviving cells above this dose level. In the 4 hour exposure group in the presence of S9 the toxicity was slightly less severe with approximately 70% survival at 39.06 µg/ml when compared to the negative control and a few cells surviving up to 78.13 µg/ml. A precipitate of the test item was noted in all three exposure groups at the end of exposure at and above 1250 µg/ml.MUTAGENICITY TEST - EXPERIMENT 1:The dose levels of the controls and the test item are given in the table below:Group Final concentration of test item (µg/ml)4-hour without S90*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 500* and 750*4-hour with S9 (2%)0*, 5*, 10*, 20*, 40*, 60*, 80, DMBA 0.5* and 1** Dose levels plated for mutant frequency.No precipitate of the test item was seen at the end of exposure in either exposure group. The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3 (attached background material). The Day 0 and Day 7 cloning efficiencies for the vehicle control groups in both the with and without S9 exposure groups did not achieve 70% cloning efficiency but all achieved at least 50% cloning efficiency and were therefore considered to be acceptable. The test item demonstrated a steep toxicity curve in both exposure groups consistent with that seen in the preliminary toxicity test. In the absence of S9 the test item achieved 58% toxicity at 30 µg/ml at Day 0 when compared to the vehicle control group. The dose level of 40 µg/ml had no surviving cells and was too toxic for plating. In the 4 hour exposure group in the presence of S9 the toxicity was too great for plating at 80 µg/ml at Day 0 with no surviving cells at this dose level. The test item achieved a 21% increase in toxicity when compared to the vehicle control group at 40 µg/ml. The dose level of 60 µg/ml was plated although it exceeded 90% toxicity as it provided an intermediate dose in a steep toxicity curve. The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3 (attached background material). In the 4 hour exposure group in the absence of S9 there was an increase in the mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 at 2.5 µg/ml, however since this increase was not dose related and the mutant frequency for this exposure group was generally high this was considered to be a random fluctuation. There were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 in the presence of S9 with the exception of the 60 µg/ml dose level which can be excluded on the basis of excessive toxicity.MUTAGENICITY TEST - EXPERIMENT 2:The dose levels of the controls and the test item are given in the table below:Group Final concentration of test item (µg/ml)24-hour without S90*, 1.25*, 2.5*, 5*, 10*, 20*, 30*, 40, EMS 200* and 300*4-hour with S9 (1%)0* ,5*, 10*, 20*, 30*, 40*, 50*, 60 DMBA 0.5* and 1** Dose levels plated for mutant frequency.No precipitate of the test item was seen at the end of exposure in either exposure group The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation are presented in the attached Tables 4 and 5 attached background material). The Day 0 cloning efficiencies for the vehicle control groups in both exposure groups did not achieve 70% but were considered acceptable as they did achieve the 50% minimum. It can be seen that the toxicity is similar to that seen in Experiment 1. The maximum dose plated for mutation frequency in the 4 hour exposure group in the presence of S9 was 50 µg/ml with an increase in toxicity of 66% when compared to the vehicle control group. The dose level of 60 µg/ml was not plated due to toxicity greater than 90%. The 24 hour exposure group demonstrated an increase in toxicity of 51% when compared to the vehicle control at 30 µg/ml. The dose level of 40 µg/ml was too toxic for plating with no surviving cells. The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in the attached Table 4 and 5 (attached background material). In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6.It can be seen that the vehicle control values were all within the maximum upper limit of 25 x 10-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. In the positive control groups dosed with DMBA at 1 µg/ml and EMS at 300 µg/ml there were insufficient cells for plating 5 mutant flasks due to the toxicity of these positive control items and the mutant frequency was therefore calculated from the counts of the available flasks and adjusted according to the number of flasks. The positive response was clearly demonstrated and therefore the reduction in mutant flasks was considered to be acceptable.

Remarks on result:

other: strain/cell type: GHO

Remarks:

Migrated from field 'Test system'.

See attached background material for:

Table 1: Preliminary Cytotoxicity Results

Table 2: Experiment 1 - 4 Hour Exposure Without Metabolic Activation (S9)

Table 3: Experiment 1 - 4 Hour Exposure With Metabolic Activation (S9)

Table 4: Experiment 2 - 24 Hour Exposure Without Metabolic Activation (S9)

Table 5: Experiment 2 - 4 Hour Exposure WIth Metabolic Activation (S9)

Conclusions:

Interpretation of results (migrated information):negative Non-mutagenicThe test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476' In Vitro Mammalian Cell Gene Mutation Tests', Method B17 of Commission Regulation (EC) No 440/2008, the United Kingdom Environmental Mutagen Society (Cole et al, 1990) and the US EPA OPPTS 870.5300 Guideline. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods

Chinese hamster ovary (CHO) cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration of test item (µg/ml)
4-hour without S9	2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%)	5, 10, 20, 40, 60, 80
24-hour without S9	1.25, 2.5, 5, 10, 20, 30, 40
4-hour with S9 (1%)	5, 10, 20, 30, 40, 50, 60

 

Results

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The Experimental phases of the study were performed between 19 April 2011 and 18 August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions. Cell Culture:Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal, bovine serum, at 37°C with 50/0 C02 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test (Cell Growth Inhibition Test):The dose range of test item used was 19.53 to 5000 µg/mlExperiment 1: 4(20)-hour without S9 (µg/ml)0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*4(20)-hour with S9 (µg/ml)0*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*Experiment 224-hour without S9 (µg/ml)0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*4(20)-hour with S9 (µg/ml)0*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, CP 5**Dose levels selected for metaphase analysisMMC: Mitomycin CCP: Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test item was readily miscible in it.Preparation of Test Item and Control Items:The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The test item was considered to be a mixture, therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level. There was a modest decrease in pH of less than 1 pH unit when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated. The decrease in pH was within acceptable limits.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9 at 5 µg/ml.Migrated to IUCLID6: (CP)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9 at 0.4 and 0.2 µg/ml for 4(20)-hour and 24-hour culture respectively.Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 48 hrs- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.- Selection time (if incubation with a selection agent): Not applicable.- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid 0.1 µg/ml)STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.NUMBER OF REPLICATIONS: Duplicate culturesNUMBER OF CELLS EVALUATED: 100/cultureDETERMINATION OF CYTOTOXICITY - Method: mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.OTHER EXAMINATIONS: - Determination of polyploidy: In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(Refer to information on results and attached tables.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTSPRELIMINARY TOXICITY TEST (CELL GROWTH INHIBITION TEST)The mitotic index data are presented in Appendix 1 (5) and (6) (see attached background material - Appendix 1). It can be seen that the test item showed clear evidence of dose-related toxicity in all three exposure groups. A greasy oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 2500 µg/ml, in all three of the exposure groups.Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 156.25 µg/ml, in all three of the exposure groups. Dose selection for Experiments 1 and 2 was based on test item induced toxicity.CHROMOSOME ABERRATION TEST - EXPERIMENT 1:The dose levels of the controls and the test item are given in the table below:GroupFinal concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (µg/ml)4(20)-hour without S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.4*4(20)-hour with S90*, 9.77, 19.53, 39.06*, 78.13*, 156.25*, 312.5, CP 5*The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 156.25 µg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 µg/ml in either exposure group.The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2 (see attached background material - Appendix 2). These data show that 59% growth inhibition was achieved at 156.25 µg/ml in the absence of S9 and 40% growth inhibition was achieved at 156.25 µg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either exposure group.The maximum dose level selected for metaphase analysis was based on the maximum surviving dose level and toxicity, and was 156.25 µg/ml in both of the exposure groups.The chromosome aberration data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation (S9).The polyploid cell frequency data are given in Form 1, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.CHROMOSOME ABERRATION TEST - EXPERIMENT 2:The dose levels of the controls and the test item are given in the table below:GroupFinal concentration of Reaction mass of bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate (µg/ml)24-hour without S90*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, MMC 0.2*4(20)-hour with S90*, 9.77, 19.53*, 39.06*, 78.13*, 156.25*, 312.5, CP 5*The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the test item dose level of 156.25 µg/ml in both the absence and presence of metabolic activation (S9). No metaphases suitable for scoring were observed at 312.5 µg/ml in either of the exposure groups.The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2 (see attached background material - Appendix 2). These data show that 75% growth inhibition was achieved at 156.25 µg/ml in the absence of S9 and 50% growth inhibition was achieved at 156.25 µg/ml in the presence of S9.No precipitate of the test item was observed at the end of the treatment period in either of the exposure groups.The maximum dose level selected for metaphase analysis was the same as Experiment 1, and was based on toxicity at 156.25 µg/ml, for both exposure groups. In the absence of S9 the toxicity observed at 156.25 µg/ml was high at 75%, however the dose level was selected for metaphase analysis because the next dose level down demonstrated no toxicity.The chromosome aberration data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.The polyploid cell frequency data are given in Form 2, Appendix 2 (see attached background material - Appendix 2). The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

See attached background material for:

Appendix 1: Report of Results of Chromosomal Aberration Test in Cultured Mammalian Cells

Appendix 2: Results of Chromosome Aberration Test

Appendix 3: Dose Response Curves

Conclusions:

Interpretation of results (migrated information):negativeThe test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, UKDoH Guidelines for the Testing of Chemicals for Mutagenicity as detailed in the UKEMS Recommended Procedures for Basic Mutagenicity Test (1990), US EPA OPPTS 870.5375 Guideline and is acceptable to the Japanese New Chemical Substance Law (METI).

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration ofTest Item(µg/ml)
4(20)-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (2%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
24-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (1%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5

 

 

Results.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded the optimum 50% mitotic inhibition.

Conclusion. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

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

In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix beginning at 1500 µg/plate (TA1537) and at 5000 µg/plate for all of the remaining strains. In the main test (pre-incubation method) weakened bacterial background lawns or a reduction in revertant frequency were initially noted at 500 µg/plate (absence of S9-mix) and from 1500 µg/plate (presence of S9-mix). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

The test item was considered to be non-mutagenic under the conditions of this test.

Chromosome aberration test in human lymphocytes

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of Test Item(µg/ml)
4(20)-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (2%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
24-hour without S9	9.77, 19.53, 39.06, 78.13, 156.25, 312.5
4(20)-hour with S9 (1%)	9.77, 19.53, 39.06, 78.13, 156.25, 312.5

 

 

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced or exceeded the optimum 50% mitotic inhibition.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

CHO HPRT forward mutation assay

Chinese hamster ovary (CHO) cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration of test item(µg/ml)
4-hour without S9	2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%)	5, 10, 20, 40, 60, 80
24-hour without S9	1.25, 2.5, 5, 10, 20, 30, 40
4-hour with S9 (1%)	5, 10, 20, 30, 40, 50, 60

 

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Justification for selection of genetic toxicity endpoint
A full suite of in-vitro genetic toxicity studies were conducted, and all studies generated negative results. The substance is therefore concluded to be negative for genetic toxicity.

Short description of key information:
Genetic Toxicity = Negative; Thompson, P.W. (2011); OECD 471 (Bacterial Reverse Mutation Assay)
Genetic Toxicity = Negative; Lacey, F.E. & Durward, R. (2011); OECD 473 (In Vitro Mammalian Chromosome Aberration Test)
Genetic Toxicity = Negative; Morris, A (2012); OECD 476 (In Vitro Mammalian Cell Gene Mutation Test)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in the three following in-vitro studies, the substance is not classified for mutagencity.

 

- Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli:

The test item was considered to be non-mutagenic under the conditions of this test.

- Chromosome aberration test in human lymphocytes:

The test item is considered to be non-clastogenic in this chromosome aberration test.

- CHO HPRT forward mutation assay:

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.