1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-nortall-oil alkyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Five in vitro genetic toxicity studies have been conducted on the test material as follows:

A. Bowles (2011) REVERSE MUTATION ASSAY “AMES TEST” USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI
San & Wagner (1990) Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay (Ames Test)
In both Ames studies the test material was considered to be non-mutagenic.

A. Bowles (2012) Chromosome Aberration Test in Human Lymphocytes in vitro
Putman & Morris (1990) Chromosome Aberrations in Chinese Hamster Ovary (CHO) Cells
In both Chromosome Aberration studies the test material was considered to be non-clastogenic.

Morris (2012) CHO HPRT FORWARD MUTATION ASSAY
The test material was considered to be non-mutagenic.

Endpoint Conclusion:No adverse effect observed (negative)

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 29 June 2011 and 12 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 not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

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.

Deviations:

no

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/plate
Experiment one: 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
Experiment two: All Salmonella strains without S9-mix: 0.15, 0.5, 1.5, 5, 15, 50, 150, µg/plate.
All Salmonella strains (with S9-mix) and WP2uvrA (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test item was immiscible 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: 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: 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: 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: 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 mix 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 mix 4-Nitroquinoline-1-oxide: 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 mix 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 mix 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation (20 minutes at 37 deg C) for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

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, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 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 material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following 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.

Statistics:

Standard deviation
Dunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Tested up to the toxic limit of the test item.

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:

Tested up to the toxic limit of the test item.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was immiscible 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.
- Precipitation: A test item precipitate (globular in appearance) was observed at 5000 µg/plate. However, due to excessive test item toxicity, this
observation was only noted in the preliminary assay.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test item initially exhibited toxicity at and above 50 and 150 µg/plate to TA100 and WP2uvrA respectively. The test item formulation and S9-mixused 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). These data are not given in the report. 
A history profile of vehicle and positive control values is presented in attached background material.

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 below in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

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: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains beginning at 50 and 150 µg/plate in the absence and presence of S9-mix respectively.
The test item was tested up to the toxic limit.

RESULTS

Preliminary ToxicityTest

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	109	104	109	113	105	99	78*	0*	0*	0*	0P*
+	TA100	88	96	89	96	92	81	91	34*	0*	0*	0P*
-	WP2uvrA	34	36	31	28	29	35	24	22*	0*	0*	0P*
+	WP2uvrA	26	37	34	33	33	27	34	26*	0*	0*	0P*

*       Partial or complete absence of bacterial bckground lawns

P       Precipitate

MutationTest

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test item, vehicle and positive controls both with and without metabolic activation, are presented in attached background material

The results are also expressed graphically in attached backgroung material.

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.

Table1               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
103		13		19		10		7
120	(101)	12	(15)	28	(24)	19	(16)	6	(6)
79		19		24		20		4

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
116		23		12		14		10
95	(105)	27	(24)	15	(16)	24	(18)	11	(11)
104		21		22		15		13

 

Conclusions:

The test item, 1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline, 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,1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline, 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 0.5 to 500 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to 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.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains beginning at 50 and 150 µg/plate in the absence and presence of S9-mix respectively. The test item was tested up to the toxic limit. A test item precipitate (globular in appearance) was observed at 5000 µg/plate. However, due to excessive test item toxicity, this observation was only noted in the preliminary assay.

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,1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline, 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 30 August 2011 and 13 December 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 not 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)

Deviations:

no

Qualifier:

according 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: yes

Cell 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:

S9 was prepared in house from the livers of male rats weighing approximately 250g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

The dose range of test item used in the preliminary toxicity test was to 0.5 to 48 µg/ml.
The dose range of test item selected for the mutagenicity test was based on the results of the preliminary toxicity test and were as follows:
Exposure Group Final concentration of test item (µg/ml)
4-hour without S9 0.25, 0.5, 1, 2, 4, 8, 12
4-hour with S9 (2%) 2, 4, 8, 16, 32, 40, 48
24-hour without S9 0.25, 0.5, 1, 2, 3, 4, 8
4-hour with S9 (1%) 1, 2, 4, 8, 16, 32, 40

Vehicle / solvent:

Culture media (Hams F12) was used as the solvent as the test item dissolved in it at the required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA)

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hams F12

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test:
A preliminary cytotoxicity test was performed on cell cultures plated approximately 24 hours before dosing at 3 x 10E6 cells/75 cm2 flask for the 4
hour exposure groups and at 1.5 x 10E6 cells/75 cm2 flask for the 24 hour exposure group. On dosing, the growth media was removed and replaced with serum free media (Ham's F12) for the 4 hour exposure groups and with Hams F12 with 1% serum for the 24 hour exposure group. One flask per dose level was treated for 4 hours with and without S9 metabolic activation and for 24-hours without metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The test item was seen to be excessively toxic in the partner chromosome aberration study (Harlan
Laboratories Ltd Project Number 41102291) performed on the same test item and therefore the dose range of test item used was limited to 0.5 to
48 µg/ml. At the end of the exposure period the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in Hams F12 with 5% serum; a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 ml of Hams F12 with 5% serum and incubated for 6 to 7 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies.
Results from the preliminary cytotoxicity test were used to select the test item dose levels for the mutagenicity experiments.

Mutagenicity Test:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient
cells for use in the test. Cells were seeded at 3 x 10E6/75 cm2 flask for the 4 hour exposure groups of Experiment 1 and allowed to attach overnight
before being exposed to the test or control items. In Experiment 2 the cells were seeded at 1.5 x 10E6 cells/75 cm2 flask for the 4-hour exposure
group and at 1.0 x 10E6 cells/75 cm2 flask for the 24 hour exposure group approximately 48 hours before exposure to the test or control items.
Duplicate cultures were set up, both in the presence and absence of metabolic activation, with a minimum of six dose levels of test item, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Hams F12) or for 24 hours in Hams F12 with 1% serum at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test item selected for the mutagenicity test was based on the results of the preliminary toxicity test and were as follows:

Exposure Group Final concentration of test item (µg/ml)
4-hour without S9 0.25, 0.5, 1, 2, 4, 8, 12
4-hour with S9 (2%) 2, 4, 8, 16, 32, 40, 48
24-hour without S9 0.25, 0.5, 1, 2, 3, 4, 8
4-hour with S9 (1%) 1, 2, 4, 8, 16, 32, 40

At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in Hams F12 with 5% serum. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10E6 cells/flask in a 225 cm2 flask to allow
growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in
Hams F12 with 5% serum and incubated at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to
estimate cytotoxicity.
During the 7 Day expression period the cultures were subcultured and maintained at 2 x 10E6 cells/225 cm2 flask on days 3 to 4 to maintain
logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and
plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 ml of Hams F12 with 5% serum to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/75 cm2 flask (5 replicates per group) in Hams F12 with 5% serum, supplemented with 10 µg/ml 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with
methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.

The percentage of viability and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An 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 section on test system and conditions

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to the attached results tables

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 0.5 to 48 µg/ml was used in the preliminary cytotoxicity test. The dose range was limited due to the excessive toxicity seen in the
partner chromosome aberration study on the same test item, Harlan Laboratories Ltd Project Number 41102291. The results of the individual flask
counts and their analysis are presented in the attached Table 1. It can be seen that there was very marked toxicity in all three exposure groups. A
reduction in cloning efficiency of 90% was achieved at 8 µg/ml in the 4-hour exposure group in the absence of S9. In the 4-hour exposure group in
the presence of S9 the toxicity was less severe, probably due to the protective effects of the S9, and reductions in cloning efficiency of 75% and 97%
were seen at 32 and 48 µg/ml respectively when compared to the vehicle control group. The 24-hour exposure group demonstrated a reduction in
cloning efficiency of 94% at 4 µg/ml when compared to the vehicle control group.

Precipitate was observed at the end of the exposure period at and above 8 µg/ml in the 4-hour and 24-hour exposure groups in the absence of S9. In the 4-hour exposure group in the presence of S9 precipitate was seen at and above 16 µg/ml at the end of the exposure period.

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 S9 0*, 0.25*,0.5*, 1*, 2*, 4*, 8, 12, , EMS 500* and 750*
4-hour with S9 (2%) 0*, 2*, 4*, 8*, 16*, 32*, 40, 48, DMBA 0.5* and 1*

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. The Day 0 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 35% toxicity at 4 µg/ml at Day 0 when compared to the vehicle control group and the dose levels of 8 and 12 µg/ml were considered too toxic for plating with toxicity of greater than 90%. In the 4-hour exposure group in the
presence of S9 the toxicity was too great for plating at 40 and 48 µg/ml at Day 0 with no surviving cells at these dose levels. The dose level of 32 µg/ml demonstrated a reduction in cloning efficiency of 88% when compared to the vehicle control group.

The Day 7 viability and mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3. In both the 4-hour exposure groups in the absence and presence of S9 there was no increase in the mutation frequency per survivor which exceeded the vehicle control value by 20 x 10-6 at any dose level of the test item. It should also be noted that no marked toxicity was observed with the viability data in both the absence and presence of S9.

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.

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 S9 0*, 0.25*, 0.5*, 1*, 2*, 3*, 4*, 8, EMS 200* and 300*
4-hour with S9 (1%) 0*,1*, 2*, 4*, 8*, 16*, 32, 40, DMBA 0.5* and 1*
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. The Day 0 cloning efficiencies for the vehicle controls of both exposure groups did not achieve 70% but were considered acceptable as they did achieve the 50% minimum. The toxicity seen in the 4-hour exposure group in the presence of S9 was greater than that seen in Experiment 1, this is considered to be due to the reduction in the S9 concentration and not unexpected. The maximum dose plated for mutation frequency in the 4-hour exposure group in the presence of S9 was 16 µg/ml with an increase in toxicity of 56% when compared to the vehicle control group. The dose levels of 32 and 40 µg/ml were not plated due to there being no viable cells at the end of the exposure period. The 24-hour exposure group demonstrated a steep toxicity curve with 30% toxicity at 4 µg/ml when compared to the vehicle control group and the higher dose level of 8 µg/ml being too toxic for plating.

The Day 7 viability and mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in the attached Tables 4 and 5. 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 should also be noted that no marked toxicity was observed with the viability data in both the absence and presence of S9.

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.

*= Dose levels plated for mutant frequency
EMS= Ethylmethanesulphonate
DMBA= Dimethyl benzanthracene

Please refer to the attached tables for results:

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

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

Conclusions:

The 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	0.25, 0.5, 1, 2, 4, 8, 12
4-hour with S9 (2%)	2, 4, 8, 16, 32, 40, 48
24-hour without S9	0.25, 0.5, 1, 2, 3, 4, 8
4-hour with S9 (1%)	1, 2, 4, 8, 16, 32, 40

 

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 18 July 2011 and 04 January 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:

equivalent or similar to guideline

Guideline:

other: is acceptable to the Japanese New Substance Law (METI)

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection: 19-21 July 2011, Certificate signed 31 August 2011

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Test Concentrations:

Cell Growth Inhibition Test:
4(20)-hour without S9: 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 µg/ml.
4(20)-hour with S9: 0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 40.0, 48.0 µg/ml.
20-hour without S9: 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 µg/ml.

Experiment 1
4(20)-hour without S9: 0*,2, 4, 8*, 12*, 16*, 24 µg/ml
4(20)-hour with S9: 0*, 4, 8, 12, 16*, 24*, 32*, 40 µg/ml

Experiment 2
24-hour without S9: 0*, 1, 2*, 4*, 8*, 12, 16 µg/ml
4(20)-hour with S9: 0*, 4*, 8*, 12*, 16*, 20, 24, µg/ml

* Dose levels selected for metaphase analysis

Vehicle / solvent:

Vehicle: MEM (Eagle's minimal essential medium with HEPES buffer)

Justification for choice of solvent/vehicle:
The test item was accurately weighed, dissolved in Eagle's minimal essential medium with HEPES buffer (MEM) and serial dilutions prepared. The test item was regarded as a complex mixture, therefore the maximum dose level was 5000 µg/ml, the maximum recommended dose level. No correction for purity was made.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Details on test system and experimental conditions:

METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

SELECTION AGENT (MUTATION ASSAYS):
No selection agent selected

SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When slides were dry they were stained in 5% giemsa for 5 minutes, 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 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 were approximately 30 to 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 (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

OTHER:
none

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-response 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 polyploidy cells were 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:

RESULTS

Preliminary Toxicity Test (Cell Growth Inhibition Test)
The mitotic index data are presented in Appendix 1 (5) and (6). The test item exhibited clear evidence of dose-related toxicity in all three exposure groups. No test item precipitate was observed in the parallel blood-free cultures at the end of the exposure period.
Microscopic assessment of the slides prepared ffrom the treatment cultures showed that metaphase cells were present up to 8 and 16 µg/mL in the abscence of metabolic activation (continuous and 4(20)-hour expsures, respectively). Metaphase cells were present up to 32 µg/mL in the presence of metabolic activation (4(20)-hour exposure).

Therefore, the dose selection for Experiments 1 and 2 was based on toxicity in accordance with the OECD 473 test guideline.

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of 1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline (µg/ml)
4(20)-hour without S9: 0*, 2, 4, 8*, 12*, 16*, 24, MMC 0.4*
4(20)-hour with S9: 0*, 4, 8, 16*, 24*, 32*, 40, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide

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 32 µg/ml in the presence of metabolic activation (S9). In the absence of metabolic activation (S9) the maximum test item dose level with metaphases suitable for scoring was 16 µg/ml.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 1, Appendix 2. These data show an approximate 50% growth inhibition (60%) was achieved at 16 µg/ml in the absence of S9 and at 32 µg/ml in the presence of S9 (growth inhibition of 54%).
The selection of the maximum dose level for metaphase analysis was based on toxicity, and was 16 and 32 µg/ml respectively in the absence and presence of S9 where optimum levels of toxicity had been achieved.
The chromosome aberration data are given in Form 1, Appendix 2. All of the vehicle control groups had frequencies of cells with chromosome aberrations which were generally considered to be within the expected range for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
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. 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:

Group Final concentration of 1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline (µg/ml)
24-hour without S9 0*, 1, 2*, 4*, 8*, 12, 16, MMC 0.2*
4(20)-hour with S9 0*, 4*, 8*, 12*, 16*, 20, 24, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 12 µg/ml in the absence of S9 and 24 µg/ml in the presence of S9.
The results of the mitotic indices (MI) from the cultures after their respective treatments are presented in Form 2, Appendix 2. These data show an approximate 50% growth inhibition (47%) was achieved at 16 µg/ml in the presence of S9 and at 8 µg/ml in the absence of S9 (growth inhibition 55%). The selection of the maximum dose level for metaphase analysis was similar to Experiment 1, and was based on toxicity.
The chromosome aberration data are given in Form 2, Appendix 2. All of the vehicle control cultures had frequencies of cells with chromosome aberrations which were generally considered to be within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
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. 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.

see 'Overall Remarks and Attachments'

Conclusions:

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 approximately 50% mitotic inhibition.
The test item was 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 Experiments 1 and 2 were selected using data from the preliminary toxicity test and were as follows:

Experiment	Group	Final concentration of test item (µg/mL)
1	4(20)-hour without S9	2, 4, 8, 12, 16 and 24
	4(20)-hour with S9 (2%)	4, 8, 16, 24, 32 and 40
2	24-hour without S9	1, 2, 4, 8, 12 and 16
	4(20)-hour with S9 (1%)	4, 8, 12, 16, 20 and 24

 

Results.All of the vehicle (solvent) control groups had frequencies of cells with chromosome aberrations which were generally considered to be within the expected rangefor normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

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 approximately 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

Bowles A & Thompson P W (2011)

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,1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline, 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 0.5 to 500 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to 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.

The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains beginning at 50 and 150 µg/plate in the absence and presence of S9-mix respectively. The test item was tested up to the toxic limit. A test item precipitate (globular in appearance) was observed at 5000 µg/plate. However, due to excessive test item toxicity, this observation was only noted in the preliminary assay.

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,1-(2-Hydroxyethyl)-2-Tall Oil-2-Imidazoline, was considered to be non-mutagenic under the conditions of this test.

San R H C & Wagner V O (1990)

The test material was submitted for testing in the Salmonella/ Mammalian-Microsome Plate Incorporation Mutagenicity Assay. This assay evaluates the mutagenic potential of the test article (or its metabolites) for its ability to induce back mutations at selected loci of several strains of Salmonella typhimurium in the presence and absence of microsomal enzymes derived from Aroclor-induced rat liver. The tester strains used in the study were TA98, TA100, TA1535, TA1537 and TA1538.

The results of the Salmonella/ Mammalian-Microsome Plate Incorporation Mutagenicity Assay indicate that under the conditions of the study, the test article did not cause a positive response with any of the tester strains in the presence and absence of microsomal enzymes prepared from Aroclor-induced rat liver.

Chromosome Aberration : (A Bowles (2012))

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 (Scottet 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 Experiments 1 and 2 were selected using data from the preliminary toxicity test and were as follows:

Experiment	Group	Final concentration of test item (µg/mL)
1	4(20)-hour without S9	2, 4, 8, 12, 16 and 24
	4(20)-hour with S9 (2%)	4, 8, 16, 24, 32 and 40
2	24-hour without S9	1, 2, 4, 8, 12 and 16
	4(20)-hour with S9 (1%)	4, 8, 12, 16, 20 and 24

 

Results.

All of the vehicle (solvent) control groups had frequencies of cells with chromosome aberrations which were generally considered to be within the expected rangefor normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

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 approximately 50% mitotic inhibition.

 

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

Chromosome Aberration: Putman D L & Morris M J (1990)

The test article was tested in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.0013, 0.0025, 0.005, 0.01 and 0.02 µL/mL for the non-activated study and 0.0065, 0.013, 0.025, 0.05 and 0.1 µL/mL for the S-9 activated study. Due to a significant delay in cell cycle kinetics observed in both the non-activated and S-9 activated test systems, metaphase cells were collected at 20 hours after treatment for microscopic evaluation in order to assure that first division metaphase cells were evaluated for chromosome aberrations. Toxicity, as measured by a reduction in mitotic index, was a limiting factor in the analysis of test concentrations in both the non-activated and S-9 activated studies. No increase in chromosome aberrations was observed in either the non-activated or S-9 activated test system. The test material was concluded to be negative in the CHO cytogenetics assay.

Chinese Hamster Ovary Cells: Morris A (2012)

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 methof used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 'In vitro Mammaloan 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 (6 -TG) 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 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 cytotoxcity test and were as follows:-

Exposure Group	Final concentration of test item (µg/mL
4-hour without S9	0.25, 0.5, 1, 2, 4, 8, 12
4-hour with S9 (2%)	2, 4, 8, 16, 32, 40, 48
24-hour without S9	0.25, 0.5, 1, 2, 3, 4, 8
4-hour with S9 (1%)	1, 2, 4, 8, 16, 32, 40

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 CHOcells at the HPRT locus under the conditions of the test.

Justification for classification or non-classification

Five separate in vitro genetic toxicity studies have been conducted on the test material, two Ames studies, two Chromosome Aberration studies and a Chinese Hamster Ovary cell study.

Of the five studies, all were carried out to the required standards however the latest studies (A. Bowles (2011) REVERSE MUTATION ASSAY “AMES TEST” USING SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI, A. Bowles (2012) Chromosome Aberration Test in Human Lymphocytes in vitro and Morris (2012) CHO HPRT FORWARD MUTATION ASSAY) can be considered to be the most reliable.

All studies showed the test material to have no significant effects for genetic toxicity and as such the test material can be considered to be non-classified.