Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not genotoxic during in vitro exposure of bacteria, mouse lymphoma cells and human lymphocytes

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:

Between 04 November 2011 and 05 December 2011 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) number 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(including METI, MHLW and MAFF guidances)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011; no analysis carried out to determine the homogeneity, concentration or stability of the test item formulation

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimurium, Tryptophan for Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: Genotypes: TA1535 = his G 46, rfa-, uvr B-; TA1537 = his C 3076, rfa-, uvr B-; TA 98 = his D 3052, rfa-, uvr B-, R-factor; TA 100 = his G 46, rfa-, uvr B-, R-factor; WP2 = trp-, uvr A-

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and ß-Naphthoflavone 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/plate
Mutation Test - Experiments 1 and 2 (Range-finding Test and Main Test): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide (DMSO) at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system. Acetone was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of all strains used

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

4-Nitroquinoline-N-oxide (CAS 56-57-5) at 0.2 µg/plate for TA 98; 9-Aminoacridine (CAS 90-45-9) at 80 µg/plate for TA 1537; N-ethyl-N'-nitro-N-nitrosoguanidine (CAS 4245-77-6) at 2, 3 and 5 µg/plate for WP2, TA 100 and TA 1535, respectively

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation (-S9)

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of all strains used

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

Benzo(a)pyrene (CAS 50-32-8) at 5 µg/plate for TA98 or 2-Aminoanthracene (CAS 613-13-8) at 1, 2, 2 and 10 µg/plate for TA 100, TA 1535, TA 1537 and WP2, respectively

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With metabolic activation (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10 h
- Exposure duration: 48 - 72 h

NUMBER 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 tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 0.9 to 9 • 10^9 bacteria per mL.
- Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test item dose levels.
- There should not be an excessive loss of plates due to contamination.

EVALUTION CRITERIA
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive 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 a definitive judgement about the test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation and Dunnett's linear regression analysis

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

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
- Effects of pH: There was no marked change in pH when the test item was dosed into media; pH 7.33 without test item, range from 9.77 to 2500 µg test item/mL was pH 7.29 to 7.32 with no visible trend, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media; mOsm 383 without test item; range from 9.77 to 2500 µg test item/mL was 387 to 307 decreasing with increasing test item concentration, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Evaporation from medium: Unlikely due to the low vapour pressure of the test item (0.00043 Pa at 25 °C, Tremain & Atwal 2011, Harlan Report no. 41103264)
- Water solubility: The test item can be considered water insoluble (water solubility < 0.1 mg/L at 20 °C, Fox & White 2012, Harlan Report no. 41103263), therefore it was dissolved in acetone.
- Precipitation: A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: The test item was non-toxic to the strains of bacteria used (TA100 and WP2). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
All master strains were found to be satisfactory when were checked for characteristics, viability and spontaneous reversion rate prior to use. The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. There was also no evidence of excessive contamination. The culture density for each bacterial strain used in each experiment was also checked and considered 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

The following table shows the colony counts and precipitation observations.

Table 1: Numbers of revertant colonies in the preliminary toxicity test and observation of precipitation

Strain	With (+) or without (-) S9-mix	Dose [µg/plate]
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
TA 100	-	113	107	96	113	78	75	94	86	113	99	83P
	+	98	81	94	74	95	79	93	86	81	81	99P
WP2	-	25	24	26	22	20	24	22	19	21	21	22P
	+	39	25	40	26	28	38	22	37	35	33	33P

P: Precipitate observed

Mutation Test

The results for the negative controls (spontaneous mutation rates) are presented below in Table 2 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment	Strain		Number of revertants [colonies per plate]
			Replicate			Mean
			1	2	3
Range-finding Test	Base pair substitution type	TA 100	75	73	79	76
		TA 1535	14	13	9	12
		WP2	20	19	13	17
	Frameshift substitution type	TA 98	30	22	22	25
		TA 1537	14	10	13	12
Main Test	Base pair substitution type	TA 100	87	97	98	94
		TA 1535	15	19	21	18
		WP2	25	35	18	26
	Frameshift substitution type	TA 98	24	27	32	28
		TA 1537	14	12	11	12

The mean number (from 3 replicates) of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 below.

Table 3: Number of revertants in the treatments and the positive control and observation of precipitation

Experiment	Strain	Mean Number of revertants± Standard Deviation
		Dose [µg/plate]						Positive control (as indicated above)
		0 (Negative control)	50	150	500	1500	5000
-S9 Range-finding Test	TA 100	70±8.4	72±10.8	63±2.0	70±8.5	66±2.5	71P±11.0	539±40.0
	TA 1535	16±1.5	11±0.6	15±3.1	16±1.5	9±1.2	12P±0.6	218±60.9
	WP2	23±6.1	26±0.6	19±3.1	14±5.8	19±2.1	19P±4.7	960±31.5
	TA 98	14±2.1	16±2.5	15±2.3	16±3.8	9±0.6	9P±1.0	120±21.7
	TA 1537	14±2.6	13±4.4	10±4.2	9±3.2	6±1.5	6P±1.2	1014±144.4
+S9 Range-finding Test	TA 100	76±10.5	78±8.1	80±1.0	73±3.2	80±3.5	70P±10.6	623±37.6
	TA 1535	13±3.2	11±1.2	10±1.5	13±3.8	16±5.0	12P±1.7	394±43.7
	WP2	28±3.2	28±3.5	21±4.0	30±4.0	25±4.5	32P±4.7	121±15.1
	TA 98	14±4.7	11±0.6	11±1.7	12±2.1	11±1.2	13P±3.6	311±8.7
	TA 1537	15±2.9	11±3.2	13±4.9	12±3.0	20±0.6	14P±1.5	335±25.1
-S9 Main Test	TA 100	85±7.2	91±2.9	85±6.9	84±9.0	91±4.9	94±6.1	532±3.2
	TA 1535	18±3.2	14±4.4	16±2.3	19±5.5	14±1.5	13±5.0	170±4.0
	WP2	28±4.4	25±5.5	23±4.6	24±5.0	26±5.9	30±8.7	769±54.8
	TA 98	26±6.1	33±5.0	31±7.2	22±6.8	19±5.5	9±2.3	127±20.6
	TA 1537	9±3.5	10±3.6	6±1.2	6±1.7	6±0.6	5±1.2	1319±210.8
+S9 Main Test	TA 100	91±10.1	84±3.5	90±12.9	77±13.1	75±9.2	76±14.5	1354±153.5
	TA 1535	11±2.5	13±4.7	9±1.0	12±1.5	9±2.6	11±2.5	357±6.1
	WP2	33±4.5	28±7.0	27±6.0	29±4.6	31±2.5	25±4.6	147±5.3
	TA 98	13±4.4	13±5.0	10±1.7	12±3.8	10±2.6	11±2.6	286±1.0
	TA 1537	11±4.6	9±1.5	12±3.5	7±2.6	7±2.5	12±1.7	295±1.7

P: Precipitate observed

A graphical presentation of the data is given in the illustration below.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests. 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.

Conclusions:

Interpretation of results (migrated information):
negative (no gene mutagenic effect in bacteria with and without metabolic activation)

Non-mutagenic to bacteria (absence of reverse mutation)

Executive summary:

The genetic toxicity (in vitro) of the test item was investigated in a GLP-compliant study by testing for bacterial reverse mutation (Ames test) using the strains TA 1535, TA 1537, TA 98, TA 100 (Salmonella typhimurium) and WP2 (Escherichia coli) according to the EU B.13/14 (2008), OECD TG 471 (1997) and OPPTS 870.5100 (1998) protocols and compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

The five bacterial strains were treated with the test item, using the Ames plate incorporation method at five 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 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (acetone) 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.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests.

In conclusion the test item was found non-mutagenic (in vitro) to bacteria in that it did not cause reverse mutation under the conditions of this test.

Reference 2

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:

Between 18 October 2011 and 14 February 2012 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) number 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(acceptable to the Japanese New Chemical Substance Law METI)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011; no analysis carried out to determine the homogeneity, concentration or stability of the test item formulation

Type of assay:

in vitro mammalian chromosome aberration test

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.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and ß-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

0, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL in the main Experiments 1 and 2 (6 concentrations, +S9 at 2 and 1 % in Experiments 1 and 2, respectively)
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL in the Preliminary Toxicity Test (9 concentrations, +S9 at 2 and 1 % in Experiments 1 and 2, respectively)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Acetone was selected as the solvent because the test item was readily soluble in it at the required concentrations. Due to the use of Acetone as the solvent which was dosed at a 0.5 % concentration to minimise its effect on the cells the maximum achievable dose was 2500 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, 0.5 % of the culture volume

True negative controls:

no

Positive controls:

yes

Remarks:

Mitomycin C (CAS 50-07-7) at 04 and 0.2 µg/mL in experiments 1 and 2, respectively

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9)

Migrated to IUCLID6: MMC

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, 0.5 % of the culture volume

True negative controls:

no

Positive controls:

yes

Remarks:

Cyclophosphamide (CAS 50-18-0) at 5 µg/mL in experiments 1 and 2

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9)

Migrated to IUCLID6: CP

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 h
- Exposure duration: Experiment 1 to 4 h with and without S9. Experiment 2 to 24 h without S9, 4 h with S9
- Expression time (cells in growth medium): 20 h for 4 h exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR: Demecolcine (CAS 477-30-5)

STAIN: When the slides were dry they were stained in 5 % Giemsa for 5 min, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100/culture

DETERMINATION 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 30 % 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.
-Reference: Savage JRK (1976). Annotation: Classification and relationships of induced chromosomal structural changes. J Med Genet 13:103-22.

OTHER EXAMINATIONS
- Determination of polyploidy: 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 - effect 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:

In 24-h exposure treatments only, from 78.13 and 625 µg/mL on in the Preliminary Toxicity Test and Experiment 2, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media; pH 7.33 without test item, range from 9.77 to 2500 µg test item/mL was pH 7.29 to 7.32 with no visible trend, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media; mOsm 383 without test item; range from 9.77 to 2500 µg test item/mL was 387 to 307 decreasing with increasing test item concentration, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Evaporation from medium: Unlikely due to the low vapour pressure of the test item (0.00043 Pa at 25 °C, Tremain & Atwal 2011, Harlan Report no. 41103264)
- Water solubility: The test item can be considered water insoluble (water solubility < 0.1 mg/L at 20 °C, Fox & White 2012, Harlan Report no. 41103263), therefore it was dissolved in acetone.
- Precipitation
Preliminary toxicity test: A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, at and above 39.06 µg/mL, in the 4(20)-h exposure groups. The precipitate increased in intensity as the dose concentration increased and became greasy/oily precipitate at and above 625 and 156.25 µg/mL in the absence and presence of S9 respectively. A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period in the 24-h exposure group at and above 39.06 µg/mL which became greasy/oily precipitate at and above 312.5 µg/mL.
Experiment 1: A cloudy precipitate of the test item was observed at the end of the treatment period at and above 78.13 µg/mL and a greasy/oily precipitate was observed at and above 312.5 µg/mL in the 4(20)-h exposure group in the absence of S9. In the 4(20)-h exposure in the presence of S9 cloudy precipitate and precipitate was observed at and above 78.13 µg/mL with a greasy/oily precipitate being noted at and above 625 µg/mL at the end of the treatment period.
Experiment 2: A precipitate greasy/oily precipitate of the test item was observed at the end of the treatment period at and above 312.5 µg/mL in the presence of S9. In the 24-h exposure group a cloudy precipitate was observed at and above 78.13 µg/mL and a greasy/oily precipitate at and above 156.25 µg/mL in the presence of S9 at the end of the treatment period.

RANGE-FINDING/SCREENING STUDIES: In the preliminary Toxicity Test (Cell Growth Inhibition Test) the test item showed no evidence of toxicity in any of the 4(20)-h exposure groups but demonstrated marked toxicity in the 24-h exposure group with 60 % mitotic inhibition at 2500 µg/mL. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 2500 µg/mL in all three exposure groups. With evidence of marked toxicity in the 24-h exposure group, the maximum dose level selected in Experiment 1 and Experiment 2 was the maximum achievable dose of 2500 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item was shown on the basis of reduced Mitotic Index (MI) to be toxic to human lymphocyte cells (in vitro) in the 24-h exposure groups only (no cytotoxicity in the 4-h treatments). Accordingly in Experiment 1, were only 4-h exposure was included, the results of the mitotic indices (MI) from the cultures after their respective treatments showed no marked toxicity in either the presence or absence of S9. In the 24-h exposure group of Experiment 2 (in the absence of S9) there was a plateau in toxicity between 625 and 2500 µg/mL with 29 % mitotic inhibition being achieved at 2500 µg/mL. This toxicity was not as marked as that seen in the Preliminary Toxicity Test although a similar plateau of toxicity was seen in the upper dose range. The data are visualized in the illustration below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Chromosome Aberration Test – Experiment 1

The culture showed no marked cytotoxicity after treatment as indicated by the mitotic indices (MI) shown in Table 1, below.

The maximum dose level selected for metaphase analysis was the maximum dose level tested of 2500 µg/mL which was the maximum achievable dose level and was selected based on the toxicity seen in the 24-h exposure group in the Preliminary Toxicity Test. A dose level before the onset of greasy/oily precipitate was also included in the dose range for metaphase analysis in both exposure groups

The chromosome aberration data are given in Table 2, below. 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 polyploid cell frequency data are given in Table 3, below. 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 results of the mitotic indices (MI) from the cultures after their respective treatments show no marked growth inhibition at 2500 µg/mL in the presence of S9, while in the absence of S9 toxicity from 625 on is visible with 29 % mitotic inhibition being achieved at 2500 µg/mL (Table 1, below).

The chromosome aberration data are given in Table 2, below. 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 either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 3, below. 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.

Table 1: Mean Mitotic Indices (cytotoxicity)

Treatment			Mitotic Indices (MI) [%]
Experiment	Exposure duration [h]	Metabolic Activation	at Test item concentration [µg/mL]
			0	9.77	19.53	39.06	78.13	156.25	312.5	625	1250	2500
Preliminary	4	-S9	100	102	99	93	99	98	108	107	95	96
	4	+S9	100	108	102	134	157	136	189	125	121	149
	24	-S9	100	115	99	113	85	67	74	64	39	40
1	4	-S9	100	-	-	-	-	104	98	-	90	91
	4	+S9	100	-	-	-	-	-	92	85	84	73
2	4	+S9	100	-	-	-	-	-	74	62	76	97
	24	-S9	100	-	-	-	-	-	110	75	78	71

Table 2: Structural Chromosome Aberration Data

Treatment			Mean Percentage of Cells showing any Structural Chromosome Aberration (or Gaps) [%]
Experiment	Exposure duration [h]	Metabolic Activation	at Test item concentration [µg/mL]
			0	156.25	312.5	625	1250	2500
1	4	-S9	1.5 (0.0)	0.0 (0.0)	2.0 (0.0)	-	0.5 (0.0)	1.0 (0.0)
	4	+S9	0.0 (0.0)	-	0.5 (0.5)	0.0 (0.5)	1.0 (0.5)	0.0 (0.0)
2	4	+S9	0.0 (0.0)	-	0.0 (0.5)	0.5 (0.0)	0.0 (0.0)	0.0 (0.0)
	24	-S9	0.0 (0.0)	-	0.0 (0.0)	1.0 (1.0)	0.0 (0.0)	0.0 (0.0)

Table 3: Polyploid cell frequency

Treatment			Mean Percentage of Cells showing numerical Chromosome Aberration [%]
Experiment	Exposure duration [h]	Metabolic Activation	at Test item concentration [µg/mL]
			0	156.25	312.5	625	1250	2500
1	4	-S9	0.0	0.0	0.0	-	0.0	0.5
	4	+S9	0.0	-	0.0	0.0	0.0	0.0
2	4	+S9	0.0	-	0.0	0.0	0.0	0.0
	24	-S9	0.0	-	0.0	0.0	0.0	0.0

Conclusions:

Interpretation of results (migrated information):
negative (no cytogenic effect/chromosome aberration in mammalian cells with and without metabolic activation)

Non-clastogenic to mammalian cells (human lymphocytes), no cytogenicity (induction of chromosome aberration)

Executive summary:

The genetic toxicity (in vitro) of the test item was investigated in a GLP-compliant study by testing for cytogenicity (chromosome aberration) in mammalian cells using human lymphocytes according to the EU B.10 (2008) and OECD TG 473 (1997) protocols and acceptable to the Japanese new chemical substance law (METI). The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. in Experiment 1, a 4-h exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2 % final concentration with cell harvest after a 20-h expression period and a 4-h exposure in the absence of metabolic activation (S9) with a 20-h expression period. In Experiment 2, the 4-h 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 h. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were in the range from 78.13 to 2500 µg/mL.

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 the maximum achievable dose level.

In conclusion the test item was considered to be non-clastogenic (in vitro) to mammalian cells (human lymphocytes) as it did not cause cytogenicity (induction of chromosome aberration) under the conditions of this test.

Reference 3

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:

Between 17 October 2011 and 13 December 2011 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) number 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011; no analysis carried out to determine the homogeneity, concentration or stability of the test item formulation

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/- (heterozygous), locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Clone used: L5178Y TK +/- 3.7.2c mouse lymphoma cells (generation time ca. 12 h)
- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % carbon dioxide in air. In the treatment Experiments 1 and 2 the volume was doubled during the Exposure using R0 so that eventually R5 was achieved. In the expression period R20 was used.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes (master cell stocks)
- Periodically checked for karyotype stability: No
- Periodically “cleansed” against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

S9 (phenobarbital and ß-naphthoflavone induced rat liver) used in concentrations of 2 and 1 % in the Experiments 1 and 2, respectively

Test concentrations with justification for top dose:

Experiment 1: 0, 156.25, 312.5, 625, 1250, 1875 and 2500 µg test item/mL (6 concentrations, +S9 at 2 %)
Experiment 2: 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 1875 and 2500 µg test item/mL (8 concentrations, +S9 at 1 %)
Preliminary Toxicity Test 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg test item/mL

Vehicle / solvent:

Vehicle used: Acetone, concentration 0.5 % of the culture volume, was used in the main study (Experiments 1 and 2), while DMSO was used in the Preliminary Toxicity Test
Justification for choice of vehicle: Miscible at the required concentration; acetone is toxic to L5178Y at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 2500 µg/mL.

Untreated negative controls:

yes

Remarks:

Solvent / vehicle only

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, 0.5 % of the culture volume

True negative controls:

no

Positive controls:

yes

Remarks:

Ethylmethanesulphonate (CAS 62-50-0) at 400 and 150 µg /mL in Experiments 1 and 2, respectively.

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation (-S9)

Untreated negative controls:

yes

Remarks:

Solvent / vehicle only

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, 0.5 % of the culture volume

True negative controls:

no

Positive controls:

yes

Remarks:

Cyclophosphamide (CAS 50-18-0) at 2 µg/mL in Experiments 1 and 2

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period (prior to dosing):
75 min in Experiment 1 and Experiment 2 (-S9) without metabolic activation;
65 min in Experiment 2 (+S9) with metabolic activation;
55 min in the Preliminary Toxicity test
- Exposure duration:
4 h in Experiment 1 (-S9 and +S9), in Experiment 2 (+S9) with metabolic activation and in the Preliminary Toxicity Test (-S9 and +S9);
24 h in Experiment 2 and the Preliminary Toxicity Test, both (-S9) without metabolic activation
- Expression time (cells in growth medium): 2 d (48 h)
- Selection time: 10 to 14 d

SELECTION AGENT: Trifluorothymidine (TFT, CAS 70-00-8) at 4 µg/mL

NUMBER OF REPLICATIONS: Duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: Relative Total Growth (RTG); The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

OTHER EXAMINATIONS
- Cell Colony types: Two distinct types of mutant colonies were recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

OTHER
-No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within 2 h of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
- The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood.
- Measurement of Survival, Viability and Mutant Frequency: At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 ∙ 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % carbon dioxide in air and subcultured every 24 h for the expression period of 2 d, by counting and dilution to 2 ∙ 10^5 cells/mL. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
- Plate Scoring: Microtitre plates were scored using a magnifying mirror box after 10 to 14 d incubation at 37 °C with 5 % carbon dioxide in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two h. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Normal test laboratory range for Mutant Frequency (MF) per survivor: 50-200 ∙ 10^-6 for the TK+/- locus in L5i178Y; vehicle control values should not be markedly greater than 250 ∙ 10^-6 MF
Positive control chemicals should induce at least three to five fold increases in MF greater than the corresponding vehicle control.
Cytotoxicity: Should be ca. 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity)
%RSG: Values less than 10 % to be excluded from any statistical analysis
Mutagenic response: Test item must produce a statistically significant increase in the Induced Mutant Frequency (IMF) over the concurrent vehicle MF value. According to Moore et al (2003) IMF must exceed the Global Evaluation Factor (GEF) value, set at 126 ∙ 10^-6 for the microwell method (Moore et al 2006). Finally a positive linear significantly dose-related trend is necessary for a clearly positive consideration.
- Moore, M.M., Honma M, Clements J, Bolcsfoldi G, Cifone M, Delongchamp R, Fellows M, Gollapudi B, Jenkinson P, Kirby P, Kirchner S, Muster W, Myhr B, O’Donovan M, Ouldelhkim M-C, Pant K, Preston R, Riach C, San R, Stankowski LF, Thakur A, Wakuri S, Yoshimura I (2003). Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: International Workshop on Genotoxicity Tests Workgroup Report – Plymouth U.K. 2002. Mut Res 540:127-140.
- Moore MM, Honma M, Clements J, Bolcsfoldi G, Burlinson B, Cifone M, Clarke J, Delongchamp R, Durward R, Fellows M, Gollapudi B, Hou S, Jenkinson P, Lloyd M, Majeska J, Myhr B, O’Donovan M, Omori T, Riach C, San R, Stankowski LF, Thakur AK, Van Goethem F, Wakuri S, Yoshimura I (2006). Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: International Workshop on Genotoxicity Testing - Aberdeen, Scotland, 2003 - Assay acceptance criteria, positive controls, and data evaluation. Environ Mol Mutagen 47(1):1–5.

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson et al 1989).
- Robinson WD et al (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS subcommittee on guidelines for mutagenicity testing (Kirkland DJ ed),Cambridge University Press Report part III, p 102-40.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Optimum levels of toxicity achieved in the 24 h exposure group in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media; pH 7.33 without test item, range from 9.77 to 2500 µg test item/mL was pH 7.29 to 7.32 with no visible trend (see Table 1)
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media; mOsm 383 without test item; range from 9.77 to 2500 µg test item/mL was 387 to 307 decreasing with increasing test item concentration (see Table 1)
- Evaporation from medium: Unlikely due to the low vapour pressure of the test item (0.00043 Pa at 25 °C, Tremain & Atwal 2011, Harlan Report no. 41103264)
- Water solubility: The test item can be considered water insoluble (water solubility < 0.1 mg/L at 20 °C, Fox & White 2012, Harlan Report no. 41103263), therefore it was dissolved in acetone.
- Precipitation: At and above 156.25 and 78.13 in Experiment 1 and 2, respectively; in the Preliminary Toxicity Test at and above 39.06 µg/mL in the 4 h exposure group in the absence of metabolic activation, and at and above 78.13 µg/mL in the 4 h exposure group in the presence of metabolic activation and the 24 h exposure group in the absence of metabolic activation. The precipitate generally increased in intensity with increase in dose concentration.

RANGE-FINDING/SCREENING STUDIES
The dose range of the test item used in the Preliminary Toxicity Test was 9.77 to 2500 µg/mL (0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg test item/mL). The experiments comprised three treatments: A 4 h exposure group without metabolic activation (-S9), a 4 h exposure group in the presence of metabolic activation (+S9) and a 24 h exposure group in the absence of metabolic activation (-S9). Precipitate of the test item was observed at lower concentrations than in the main studies (see above). With evidence of marked toxicity in the 24 h exposure group, the maximum dose level in the subsequent mutagenicity test was the maximum achievable dose of 2500 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical control data demonstrate that no deleterious or mutagenic effects were induced by the chosen solvent.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item-induced toxicity of the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).
In the Preliminary Toxicity Test was in the 4 h exposure groups evidence of only very modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. However, much greater reductions were observed in the 24 h exposure group.
In Experiment 1 the levels of toxicity observed, as indicated by the %RSG and RTG values, in both the absence and presence of metabolic activation were greater than those of the preliminary toxicity test and near optimum levels of toxicity were approached in the absence of metabolic activation. The difference in toxicity between Experiment 1 and the Preliminary Toxicity Test was considered to be due to a combination of inter-experimental variation, the use of acetone as the vehicle, and the presence of test item precipitate. However, the levels of toxicity observed were taken to confirm that exposure of the test item to the cells had been achieved. There was also evidence of modest dose related reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. The slightly increased viability (%V) observed at 2500 µg/mL was considered to be due to the presence of precipitate effectively reducing exposure of the test item to the cells. Acceptable levels of toxicity were seen with both positive control substances.
In Experiment 2 the levels of toxicity observed in both the absence and presence of metabolic activation were once again greater than those of the preliminary toxicity test and optimum levels of toxicity were achieved in the absence of metabolic activation, and near optimum levels of toxicity were approached in the presence of metabolic activation. The levels of toxicity were more consistent with those of Experiment 1 and were once again taken to confirm that exposure of the test item to the cells had been achieved. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation.
- Moore MM, Honma M, Clements J, Harrington-Brock K, Awogi T, Bolcsfoldi G, Cifone M, Collard D, Fellows M, Flanders K, Gollapudi B, Jenkinson P, Kirby P, Kirchner S, Kraycer J, Muster W, Myhr B, O’Donovan M, Oliver J, Ouldelhkim M-C, Pant K, Preston R, Riach C, San R, Shimada H, Stankowski L (2002). Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: Follow-up international workshop on genotoxicity Test procedures. Environ Mol Mutagen 40:292-9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Vehicle control

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus (50 to 200 ∙ 10^-6 viable cells).

Positive control

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Osmolality and pH

The following Table 1 demonstrates the absence of any relevant effects. There was no change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm, which is suitable according to Scott et al (1991).

Table 1: Effects of the test item on pH and osmolality

Dose [µg/mL]	0	9.77	19.53	39.06	78.13	156.25	312.5	625	1250	2500
pH	7.33	7.31	7.30	7.32	7.30	7.31	7.29	7.29	7.30	7.30
mOsm	383	387	384	373	378	381	372	366	349	307

• Scott D, Galloway SM, Marshall RR, Ishidate M, Brusick D, Ashby J, Myhr BC (1991). Genotoxicity under Extreme Culture Conditions. A report from ICPEMC task Group 9. Mutation Res 257:147-204.

Test item effect data

The 24-h exposure in Experiment 2 without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1 % in this second experiment resulted in greater levels of toxicity being observed when compared to 4-h exposure groups in the presence of 2 % metabolic activation in the Preliminary Toxicity Test and Experiment 1.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency ∙ 10^-6 per viable cell in the absence of metabolic activation. A very modest dose related (linear trend) increase in mutant frequency was observed in the presence of metabolic activation. However, none of the individual dose levels were of statistical significance, there was no evidence of a marked increase in the absolute number of mutant colonies, and the GEF was not exceeded at any of the dose levels. A slight increase in mutant frequency was observed at 2500 µg/mL; however, the mutant frequency observed only marginally exceeded the current acceptable upper limit for vehicle controls. The response observed was, therefore, considered to be spurious and of no toxicological significance.

The numbers of small and large colonies were normally distributed and showed no marked dose dependency.

Table 2: Summary of results

Experiment 1
Treatment [µg/L]	4 h -S9			Treatment [µg/L]	4 h (+S9)
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	125.53	0	100	1.00	119.06
156.25	96	0.90	116.25	156.25	100	0.97	110.32
312.5	90	0.90	125.20	312.5	94	0.95	106.17
625	80	0.81	111.55	625	85	0.91	116.35
1250	60	0.53	126.20	1250	77	0.75	113.73
1875	43	0.32	144.08	1875	65	0.73	94.95
2500	42	0.36	138.53	2500	55	0.60	134.14
400 EMS	86	0.57	944.15	2 CP	66	0.35	978.34
Experiment 2
Treatment [µg/L]	24 h -S9			Treatment [µg/L]	4 h (+S9)
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	148.63	0	100	1.00	156.12
39.06	86	1.03	155.58	39.06 ø	103
78.13	72	0.87	172.98	78.13 ø	86
156.25	51	0.69	153 99	156.25	99	1.12	163.97
312.5	23	0.38	151.03	312.5	100	1.10	167.86
625	17	0.24	127.66	625	68	0.76	185.09
1250 ø	6			1250	49	0.54	198.98
1875 ø	4			1875	45	0.43	187 81
2500 ø	3			2500	35	0.37	229.10
150 EMS	51			2 CP	69	0.37	1319.11

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/10^6 viable cells 2 d after treatment

ø = Not plated due to toxicity or surplus to requirements

Conclusions:

Interpretation of results (migrated information):
negative (no gene mutagenic effect in mammalian cells with and without metabolic activation)

Non-mutagenic in mammalian (mouse lymphoma) cells

Executive summary:

The genetic toxicity (in vitro) of the test item was investigated in a GLP-compliant study by testing for gene mutation in mammalian cells using mouse lymphoma L5178Y cells according to the EU B.17 (2008), OECD TG 476 (1997) and OPPTS 870.5300 (1998) protocols and acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4 h exposure group in the presence of metabolic activation (1 % S9) and a 24 h exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test, and in Experiment 1 was 156.25 to 2500 µg/mL in both the absence and presence of metabolic activation. In Experiment 2 the dose range was 39.06 to 2500 µg/mL in both the absence and presence of metabolic activation. The highest concentration was considered the highest achievable dose avoiding solvent effects.

Precipitate of test item was observed at and above 156.25 and 78.13 in Experiment 1 and 2, respectively. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment using a dose range that included the maximum achievable dose level.

In conclusion the test item was considered to be non-mutagenic (in vitro) to L5178Y cells under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Testing is not required.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

The generic toxicity was tested in accordance with the recommendation of ECHA (2008, Guidance on information requirements and chemical safety assessment. Chapter R.7a: Endpoint specific guidance. Guidance for the implementation of REACH, Figure R.7.7-1 Flow chart of the mutagenicity testing strategy, p 395):

First the genetic toxicity (in vitro) was investigated in a GLP-compliant study (Thompson 2012, Harlan Report no. 41103489) by testing for bacterial reverse mutation (Ames test) as required in Annex VII, 8.4.1. (REACH) using the strains TA 1535, TA 1537, TA 98, TA 100 (S. typhimurium) and WP2 (E. coli) according to the EU B.13/14 (2008), OECD TG 471 (1997) and OPPTS 870.5100 (1998) protocols and compatible with the Japanese guidelines of METI, MHLW and MAFF. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions. The submission item gave a negative, non-mutagenic response in absence and in presence of metabolic activation.

Next the genetic toxicity (in vitro) was investigated in a GLP-compliant study (Morris 2012, Harlan Report no. 41103490) by testing for cytogenicity in mammalian cells as required in Annex VIII, 8.4.2. (REACH) using human lymphocytes according to the EU B.10 (2008) and OECD TG 473 (1997) protocols and acceptable to Japanese METI. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions. The submission item responded negative, non-cytogenic in absence and in presence of metabolic activation.

Finally the genetic toxicity (in vitro) was investigated in a GLP-compliant study (Flanders 2012, Harlan Report no. 41103491) by testing for gene mutation in mammalian cells as required in Annex VIII, 8.4.3. (REACH) using mouse lymphoma L5178Y cells according to the EU B.17 (2008), OECD TG 476 (1997) and OPPTS 870.5300 (1998) protocols and acceptable to the Japanese METI/MHLW guidelines. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions. The submission item responded in absence and in presence of metabolic activation negative indicating the absence of gene mutagenic effects in mammalian cells. In conclusion the submission item can be considered not genotoxic.

Justification for selection of genetic toxicity endpoint
According to the Integrated Testing Strategy (ITS) foreseen in the REACH legislation ECHA (2008, Guidance on information requirements and chemical safety assessment. Chapter R.7a: Endpoint specific guidance. Guidance for the implementation of REACH, Figure R.7.7-1 Flow chart of the mutagenicity testing strategy, p 395) considers the gene mutation test in bacteria (Ames test) as a first tier (available from Thompson 2012, Harlan Report no. 41103489). In absence of mutagenicity an in vitro micronucleus test or an in vitro cytogenicity (chromosome aberration) study in mammalian cells (available from Morris 2012, Harlan Report no. 41103490) should be considered. As the result is negative the next tier is an in vitro gene mutation study in mammalian cells (available from Flanders 2012, Harlan Report no. 41103491), which is selected as it represents the highest tier experiment.

Justification for classification or non-classification

Based on the available data, the submission item is not classified.