2-ethylhexyl 4-(dimethylamino)benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several in vitro studies are available on 2-ethylhexyl 4-(dimethylamino)benzoate. The Ames test, in vitro gene mutation test and the Chromosome aberration test showed negative results (no mutagenic or clastogenic activity). 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 24 May 2021
Experimental Completion Date: 29 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Labcorp were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37°C. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S-9 post-mitochondrial fraction from rat liver induced with ß-Naphthoflavone/Phenobarbital

Test concentrations with justification for top dose:

Range-Finder Experiment- 3 Hour Treatments in the Absence and Presence of S-9:
0, 0.7813, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 µg/mL.
(limited by solubility in the culture medium)

Mutation Experiment - (3 hour treatment)
In the absence of S-9: 0, 10, 20, 30, 40, 45, 50, 55, 60, 80,100 µg/mL
In the presence of S-9: 0, 20, 40, 80, 100, 120, 130, 140, 150, 175, 200 µg/mL

Vehicle / solvent:

(DMSO) diluted 100 fold in the treatment medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 50 mL RPMI 10.
All cultures were placed in an incubator set to 37°C for 1 day, counted and where possible, diluted to 2 x 10^5 cells/mL. Cultures were incubated for a further day and counted.

Mutation Experiment
Treatment of Cell Cultures
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.

After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 50 mL RPMI 10 medium.

Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment

Expression Period
Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of retaining an appropriate number of cells/flask.

In the Range-Finder Experiment, at the end of the expression period, toxicity was assessed by measuring suspension growth (and hence relative suspension growth (RSG)), compared to the concurrent vehicle control values. Cultures were not plated for viability assessment.

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^4 cells/mL in readiness for plating for TFT resistance. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator set to 37°C, gassed with 5% v/v CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL) was diluted approximately 100 fold into these suspensions to give a final concentration of 3 µg/mL. Using an eight channel pipette, 0.2 mL of each suspension was placed into each well of four 96 well microlitre plates (384 wells at 2 x 10^3 cells/well). Plates were placed in a humidified incubator set to 37°C, gassed with 5% v/v CO2 in air until scoreable (12 days). Wells containing viable clones were identified by eye using background illumination and counted.

In addition, the number of wells containing large colonies and the number containing small colonies were scored for the vehicle and positive controls.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF
2. The linear trend test was statistically significant.
The test article was considered as positive in this assay if both of the above criteria were met.
The test article was considered as negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Toxicity
In the cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S-9 ranging from 0.7813 to 200 µg/mL (limited by solubility in the culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested in the absence and presence of S-9 (25 to 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the absence of S-9 only (200 µg/mL). The highest concentrations to provide >10% RSG were 50 µg/mL in the absence of S 9 and 100 µg/mL in the presence of S-9, which gave 14% and 32% RSG, respectively (see table 1 in Any other information on results incl. tables).

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations analysed (50, 100 and 200 µg/mL) as compared to the concurrent vehicle controls (measured data not reported).
In the Mutation Experiment, ten concentrations were tested, ranging from 10 to 100 µg/mL in the absence of S-9 and from 20 to 200 µg/mL in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest nine concentrations tested in the absence of S-9 (20 to 100 µg/mL) and at all concentrations tested in the presence of S-9 (20 to 200 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration in the presence of S-9 only (200 µg/mL). Two days after treatment, the highest four concentrations in the absence of S-9 (55 to 100 µg/mL) and the highest three concentrations in the presence of S-9 (150 to 200 µg/mL) were considered too toxic for selection to determine viability and TFT resistance. All other concentrations were selected in the absence and presence of S-9. However, the highest two concentrations selected in the absence of S-9 (45 and 50 µg/mL) and in the presence of S-9 (130 and 140 µg/mL) were later rejected from analysis due to extreme toxicity (<10% RTG). The highest concentrations analysed were 40 µg/mL in the absence of S-9 and 120 µg/mL in the presence of S-9, which gave 16% and 6% RTG, respectively.

Although 120 µg/mL in the presence of S-9 gave less than 10% RTG, the second highest concentration (100 µg/mL) gave 24% RTG and therefore both concentrations were analysed (see table 2 in Any other information incl. tables)

Mutation.
The acceptance criteria were met and the study was accepted as valid.
No increases in mutant frequency (MF), which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 10^6 viable cells (compared to concurrent controls), were observed in any treated cultures and no significant linear trends were observed in either the absence or presence of S-9. The maximum concentration analysed in the presence of S-9 gave <10% RTG, however, no increases in MF were observed at this concentration and therefore it was considered to be a suitable maximum dose.

In addition, for the vehicle and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated. For the vehicle controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 24% to 27%. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P.

Table 1: RSG Values - Range-Finder Experiment - 3 Hour Treatments in the Absence and Presence of S-9

Concentration	-S-9	+S-9
µg/mL	%RSG	%RSG
0	100	100
0.7813	96	102
1.563	101	106
3.125	99	103
6.25	106	104
12.5	112	111
25 P	99	96
50 P	14	90
100 P	2	32
200 P	1 PP	4

%RSG                  Percent relative suspension growth

P                           Precipitation noted at time of treatment
PP                        Precipitation noted following treatment incubation period

 

 

Table 2: Summary of Mutation Data - 3 Hour Treatments in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration	%RTG	MF §	Concentration	%RTG	MF §
µg /mL			µg/mL
0	100	81.69	0	100	60.37
10	106	81.27	20 P	78	114.24
20 P	103	87.93	40 P	80	91.51
30 P	65	90.54	80 P	56	102.17
40 P	16	85.56	100 P	24	90.33
			120 P	6	83.86
MMS 15	62	345.29	B[a]P 4	49	615.08
MMS 20	39	521.15	B[a]P 6	39	713.97

-S-9 Linear trend test on mutant frequency: Not Significant
+S-9 Linear trend test on mutant frequency: Not Significant (negative slope)
§                           5-TFT -resistant mutants/10⁶ viable cells 2 days after treatment
%RTG                 Percent Relative Total Growth
P                           Precipitation noted at time of treatment

Conclusions:

It is concluded that SpeedCure EHA did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested in the absence and presence of a rat liver metabolic activation system (S-9). The highest concentration analysed was limited by toxicity in both the absence and presence of S-9.

Executive summary:

Summary

Introduction

SpeedCure EHA was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.

Method

A 3 hour treatment incubation period was used for each experiment.
In the cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S-9 ranging from 0.7813 to 200 μg/mL (limited by expected solubility in the culture medium). The highest concentrations to provide >10% relative suspension growth (RSG) were 50 μg/mL in the absence of S-9 and
100 μg/mL in the presence of S-9, which gave 14% and 32% RSG, respectively.
In the Mutation Experiment, ten concentrations were tested, ranging from 10 to
100 μg/mL, in the absence of S-9 and ranging from 20 to 200 μg/mL in the presence of S-9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 40 μg/mL in the absence of S-9 and 120 μg/mL in the presence of S-9, which gave 16% and 6% RTG, respectively. Although 120 μg/mL in the presence of S-9 gave less than 10% RTG, the second highest concentration (100 μg/mL) gave 24% RTG and therefore both concentrations were analysed.
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.

Results

No increases in mutant frequency (MF), which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 10⁶ viable cells (compared to concurrent controls), were observed in any treated cultures and no significant linear trends were observed in either the absence or presence of S-9. The maximum concentration analysed in the presence of S-9 gave <10% RTG, however, no increases in MF were observed at this concentration and therefore it was considered to be a suitable maximum concentration.

Conclusion

It is concluded that SpeedCure EHA did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested in the absence and presence of a rat liver metabolic activation system (S-9). The highest concentration analysed was limited by toxicity in both the absence and presence of S-9.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2016 to 16 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon)
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media: Overnight subcultures of stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours.
- Properly maintained: Yes, each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates. Prior to use, the master strain was checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). All of the strains were stored at approximately -196 °C in a iquid nitrogen freezer.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Experiment 1 (with and without metabolic activation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate)
- Experiment 2 (with and without metabolic activation): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1; pre-incubation for Experiment 2

EXPERIMENT 1: PLATE INCORPORATION METHOD
0.1 mL of the appropriate concentration of test material, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and either 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively). These were then mixed and overlayed onto a Vogel-Bonner minimal agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 µg/plate because of test material precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

EXPERIMENT 2: PRE-INCUBATION METHOD
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of S9-mix or phosphate buffer (for assays with and without metabolic activation, respectively) and 0.1 mL of the test material formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner minimal agar plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method.
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 µg/plate because of test material precipitation.

NUMBER OF REPLICATIONS: Triplicate assays were performed

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the growth of the bacterial background lawn and increases in the frequency of revertant colonies were recorded to determine cytotoxicity. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

ACCEPTABILITY CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows: TA1535: 7 to 40; TA100: 60 to 200; TA1537: 2 to 30; TA98: 8 to 60; WP2uvrA: 10 to 60.
- All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.

EVALUATION CRITERIA
There are several criteria for determining a positive result. Any, one, or all of 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 (Mahon et al., 1989).
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 material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean number of revertant colonies and the standard deviations for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.
The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test material formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 µg/plate; this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.2 times the concurrent vehicle control.
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.

Table 1: Number of revertants (mean) ± SD in Experiment 1- without metabolic activation

Dose Level per plate (µg)	TA100		TA1535		WP2uvrA		TA98		TA1537
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Solvent control	65	1.2	11	4.6	21	4.5	19	1.7	13	3.6
1.5	63	2.3	10	2.3	21	5.5	20	1.5	18	6.8
5	64	0.6	9	1.7	26	9.7	14	6.0	16	4.6
15	67	0.6	15	4.6	24	8.2	18	4.7	15	4.5
50	65	1.5	10	0.6	19	6.8	19	1.5	11	3.2
150	68	4.9	9	2.0	23	8.1	21	4.0	11	2.9
500	68	5.3	11	4.6	19	10.7	18	6.4	8	1.5
1500	62P	1.0	10P	1.5	18P	2.3	20P	0.6	13P	0.0
5000	62P	1.0	11P	2.6	13P	5.5	17P	5.0	9P	3.2
Positive Controls (µg)	ENNG		ENNG		ENNG		4NQO		9AA
	3		5		2		0.2		80
	536	28.3	591	69.7	856	52.1	267	1.7	313	25.1

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

P = Precipitate

Table 2: Number of revertants (mean) ± SD in Experiment 1- with metabolic activation

Dose Level per plate (µg)	TA100		TA1535		WP2uvrA		TA98		TA1537
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Solvent control	75	11.5	12	2.3	18	4.7	30	8.7	18	5.5
1.5	69	13.3	13	4.4	24	5.8	28	3.1	18	4.5
5	79	6.1	9	0.0	19	3.2	28	4.4	13	5.9
15	71	9.6	11	1.0	23	0.6	34	11.2	10	1.7
50	70	12.4	11	0.6	23	3.5	27	8.7	12	2.1
150	76	4.4	15	5.0	18	2.1	33	6.7	9	1.0
500	81	8.0	10	1.0	20	3.8	35	1.7	11	7.8
1500	70P	3.2	10P	1.2	21P	2.3	26P	0.6	13P	1.2
5000	66P	3.8	8P	0.6	19P	2.5	24P	3.8	11P	2.3
Positive Controls (µg)	2AA		2AA		2AA		BP		2AA
	1		2		10		5		2
	1119	64.1	257	9.0	460	70.7	128	5.5	261	12.1

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

 

Table 3: Number of revertants (mean) ± SD in Experiment 2- without metabolic activation

Dose Level per plate (µg)	TA100		TA1535		WP2uvrA		TA98		TA1537
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Solvent control	91	6.1	17	7.6	33	7.2	26	6.2	15	6.9
15	76	5.8	18	6.1	27	1.0	25	5.0	11	1.2
50	95	4.0	18	8.7	30	12.0	27	6.1	14	1.5
150	108*	12.1	21	10.7	28	12.0	19	4.0	16	3.1
500	99	1.5	13	1.7	27	6.1	24	5.2	20	2.5
1500	89P	3.1	14P	4.0	29P	0.6	18P	4.0	21P	1.2
5000	77P	4.2	11P	3.0	20P	3.2	19P	0.6	9P	1.7
Positive Controls (µg)	ENNG		ENNG		ENNG		4NQO		9AA
	3		5		2		0.2		80
	969	346.8	1862	173.4	1025	65.0	283	7.1	301	126.7

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

*p=0.05

P = Precipitate

Table 4: Number of revertants (mean) ± SD in Experiment 2- with metabolic activation

Dose Level per plate (µg)	TA100		TA1535		WP2uvrA		TA98		TA1537
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Solvent control	91	1.2	17	4.2	31	6.0	24	2.1	17	8.5
15	87	13.5	18	5.1	28	5.0	26	3.8	22	4.6
50	108	13.5	13	2.9	31	6.4	32	4.7	19	1.0
150	101	12.1	13	2.6	33	2.9	26	7.9	20	4.2
500	96	9.9	10	2.6	21	10.7	30	5.1	17	4.0
1500	103P	6.1	12P	2.1	26P	8.1	29P	9.5	22P	4.0
5000	73P	5.9	10P	1.5	22P	7.0	26P	4.6	11P	2.0
Positive Controls (µg)	2AA		2AA		2AA		BP		2AA
	1		2		10		5		2
	1173	25.2	181	12.9	195	16.7	108	14.4	129	3.5

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

P = Precipitate

Conclusions:

Under the conditions of this study the test material was considered to be non-mutagenic.

Executive summary:

A bacterial reverse mutation assay was carried out in accordance with the standardised guidelines OECD 471, EU Method B13/14, US EPA OCSPP 870.5100 and the testing published by the Japanese METI, MHLW and JMAFF under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in DMSO using both the Ames plate incorporation and pre-incubation methods at up to eight 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 Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Bacteria were also exposed to vehicle controls and appropriate positive controls.

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

In Experiment 1 there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 µg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain.

Under the conditions of this study the test material was considered to be non-mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 1991 to 30 April 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n/a

Species / strain / cell type:

lymphocytes: human whole blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: A single human donor. Venous blood was drawn into sterile, heparinised "Vacutainers".
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Cell culture: Cultures were initiated by adding 0.3 mL of blood to 4.7 mL of culture medium in the range-finding assays and 0.6 mL of whole blood to 9.4 mL of culture medium in the aberrations assays in 15 mL centrifuge tubes. The cells were incubated at about 37 °C on a slope, with loose caps, in an atmosphere of about 5 % CO2 in air.

MEDIA USED
- Type and identity of media: RPMI 1640, supplemented with 15 % foetal calf serum (FCS), 1 % phytohaemagglutinin (PHA-M), 1 % penicillin and streptomycin and 1 % L-glutamine.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

- Range-finding test (without metabolic activation): 167, 501, 1670 and 5010 µg/mL
- Range-finding test (with metabolic activation): 50.1, 167, 501, 1670 and 5010 µg/mL
- Trial 1 (with and without metabolic activation): 315, 630, 1260, 2510 and 5010 µg/mL
- Trial 2 (with and without metabolic activation): 313, 625, 1250, 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent: According to the sponsor the test material was insoluble in water. Solubility was evaluated in DMSO and clear colourless solutions were obtained at concentrations of 201, 301, 401 and 501 mg/mL. These were sham dosed using 1 % in RPMI-1640 medium, but the resulting solutions had visible precipitation and oily globules. Solubility was then evaluated in ethanol, using the same dilutions; at a final concentration of 5020 µg/mL, a clear solution with very fine oily droplets and no precipitate that adhered to the sides of the dilution tube (pH 7.0) was obtained. Ethanol was therefore chosen as the solvent. The test material was dissolved at a stock concentration of 501 mg/mL in ethanol before diluting.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: Cultures were initiated approximately 48 hours prior to treatment by adding 0.6 mL of whole blood to 9.4 mL of culture medium in 15 mL centrifuge tubes
- Exposure duration: 21 or 45 hours (non-activation assay) and 2 hours (activation assay)
- Expression time (cells in growth medium): 2 hours (non-activation assay) and 21 or 45 hours with Colcemid only present for the last 2 hours (activation assay)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL

PROCEDURE
- Non-activation assays: Following treatment with the test material, the cultures were then washed with buffered saline and refed with complete RPMI 1640 medium containing Colcemid. Approximately two hours later the cells were harvested and slides were made.
- Activation assays: The cultures were incubated at 37 °C for two hours in the presence of the test material and the S9 reaction mixture in RPMI culture medium without FCS. After the two hour exposure period, the cells were washed twice with buffered saline and the cells were refed with complete RPMI 1640 medium. The cells were incubated with Colcemid then the cells were harvested and prepared for cytogenetic analysis.

STAIN (for cytogenetic assays): 5 % Giemsa solution

REPLICATES: 2

HARVESTING
The cell suspension was centrifuged, supernatant discarded and the cells treated with hypotonic KCl (0.075 M) for 6 to 10 minutes. This treatment helps to swell the cells and thus disperse the chromosomes. After centrifugation and removal of the KCl, the cells were fixed three times with freshly prepared fixative (absolute methanol: glacial acetic acid 3:1 v:v).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared by dropping the harvested cultures on clean slides. The slides were stained and all slides were then air-dried and coverslipped using Depex mounting medium.

NUMBER OF CELLS EVALUATED: One hundred cells, if possible, from each replicate culture at four dose levels of the test material and from each of the negative and solvent control cultures were analysed for the different types of chromosomal aberrations. At least 25 cells were analysed for chromosomal aberrations from one of the positive control cultures.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index was first analysed from one of the replicate cultures from the first set of assays to select doses for analysis. In the second set of experiments, the top four dose levels were analysed. Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 46 ± 2 (range 44 to 48) were analysed.

Rationale for test conditions:

The dosing concentrations were determined as a result of a range finding test performed with and without metabolic activation in which no reduction in mitotic index was observed for any of the tested concentrations in the range 167 to 5010 µg/mL without activation and 50.1 to 5010 µg/mL with activation.

Evaluation criteria:

The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequency
2. The percentage of cells with any aberrations
3. The percentage of cells with more than one aberration
4. Any evidence for increasing amounts of damage with increasing dose, i.e. a positive dose response.

Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test material to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity.

Statistics:

Statistical analysis employed the Fisher’s Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test material significance was established where p<0.01. All factors as stated previously were taken into account and the final evaluation of the test material was based upon scientific judgement.

Key result

Species / strain:

lymphocytes: human whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
Mitotic index was evaluated by analysing the number of metaphases in the first 1000 cells. The analysis was performed on the highest dose levels with metaphases (four from the non-activation assay and five from the activation assay). No reduction in the mitotic index was evident in the cultures analysed either with or without activation.

CHROMOSOMAL ABERRATIONS ASSAY WITHOUT METABOLIC ACTIVATION
In the first 24 hour trial, oily droplets were observed after dosing and an oily precipitate was observed on the sides of the culture tubes prior to wash in the cultures dosed with 5010 µg/mL. A slight amount of precipitate was observed on the sides of the culture tubes prior to wash in the cultures dosed with 2510 µg/mL. No reduction in mitotic index was observed in the test cultures. Results were analysed from the cultures dosed with 630, 1260, 2510 and 5010 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 24 hour repeat trial, oily droplets were observed after dosing and a precipitate was observed on the sides of the culture tubes prior to washing in the cultures dosed with 5000 µg/mL. A slight amount of precipitate was observed on the sides of the culture tubes in the cultures dosed with 2510 µg/mL. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 48 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Slight lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
The sensitivity of the cell culture for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test material was considered negative for inducing chromosomal aberrations under non-activation conditions.

CHROMOSOMAL ABERRATIONS ASSAY WITH METABOLIC ACTIVATION
In the first 24 hour trial, oily droplets were observed after dosing the cultures with 5010 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. No reduction in mitotic index was observed in the test cultures. Results were analysed from the cultures dosed with 630, 1260, 2510 and 5010 µg/mL. Due to toxicity sparse numbers of metaphases were available on the slides prepared from the cultures dosed with 1260 µg/mL and only 95 metaphases were available for analysis from one of the replicate cultures dosed with 5010 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 24 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. Due to toxicity, probably arising from the lysis of erythrocytes, only 73 and 76 cells were available for analysis from one of the replicate cultures dosed with 625 and 1250 µg/mL, respectively. The remainder of metaphases for a total number of 200 cells for those dose levels was analysed from the other culture. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
In the 48 hour repeat trial, oily droplets were observed after dosing the cultures with 5000 µg/mL. Lysis of red blood cells was observed prior to washing in all test cultures. Results were analysed from the cultures dosed with 625, 1250, 2500 and 5000 µg/mL. . Due to toxicity, probably arising from the lysis of erythrocytes, 100 metaphases were not available for analysis from one of the replicate cultures dosed with 625, 1250 and 2500 µg/mL. No significant increase in cells with chromosomal aberrations was observed at the concentrations analysed.
The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test material was considered negative for inducing chromosomal aberrations under conditions of metabolic activation.

Conclusions:

Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

Executive summary:

A chromosomal aberration assay using human lymphocytes was carried out using methodology equivalent to the standardised guideline OECD 473 under GLP conditions.

Whole blood lymphocytes from a single human donor were used for the study. The test material was dissolved in ethanol which was then diluted in the medium for exposure to the cells.

A range finding test was performed to determine the concentrations to be used for testing, both with and without metabolic activation. Without metabolic activation, mitotic index was evaluated in cultures dosed with 167, 501, 1670, and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the non-activation aberrations assay. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500 and 5000 µg/mL with 24 and 48 hour harvests.

With metabolic activation, mitotic index was evaluated in cultures dosed with 50.1, 167, 501, 1670 and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the aberrations assay with metabolic activation. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500, and 5000 µg/mL with 24 and 48 hour harvests.

Each concentration was tested in duplicate both with and without metabolic activation, in the form of S-9 mix. Negative (medium), solvent (ethanol) and positive controls (mitomycin C or cyclophosphamide) were also tested concurrently.

Following exposure the cells were harvested, fixed and stained with 5 % Giemsa solution. 100 cells, if possible, from each replicate culture at four test material dose levels and controls were analysed for chromosome aberrations. 25 cells were evaluated for the positive control.

No significant increase in the number of cells with chromosomal aberrations was observed at the concentrations analysed either with or without metabolic activation. Positive and negative controls were deemed to be valid.

Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo mammalian erythrocyte micronucleus test is available on 2-ethylhexyl 4-(dimethylamino)benzoate. Under the conditions of this study, the test material is not considered to be a clastogenic agent.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 December 1986 to 09 February 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Mice have historically been used in the Micronucleus test and have been shown to exhibit micronuclei indicative of chromosome breakage or lagging chromosomes. Intraperitoneal administration was chosen to maximise bioavailability of the test material.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 weeks old
- Weight at study initiation: Males: 35 to 44 g; Females: 30 to 35 g.
- Assigned to test groups randomly: Yes using a random number table
- Housing: 5 per cage according to sex and dose group in stainless steel wire mesh cages
- Water: Tap water ad libitum
- Acclimation period: 33 days

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Lot No: 55F-0601 (Sigma Chemical Company)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
-The test material was weighed and diluted in corn oil. A good solution was obtained and maintained at all dose levels. Dosing solutions were prepared fresh and dosed within two hours of preparation. There was no apparent change in the physical state of the test material during the assay.
- Dose volume: 10 mL/kg

Duration of treatment / exposure:

The dosing solutions were administered in a single intraperitoneal dose.

Frequency of treatment:

Once

Post exposure period:

Groups were sacrificed 30, 48 and 72 hours after exposure.

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex per group

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: triethylenemelamine (TEM)
- Preparation: Dissolved in 0.9 % saline
- Route of administration: Intraperitoneal
- Doses / concentrations: 0.5 mg/kg/bw (dose volume 10 mL/kg)

Tissues and cell types examined:

Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose selection was determined as the result of the range finding test conducted at doses of 500, 1500, 3000, 4000 and 5000 mg/kg of body weight.

TREATMENT AND SAMPLING TIMES: A single dose was administered with sacrifice after 30, 48 or 72 hours.

DETAILS OF SLIDE PREPARATION: The femora were opened carefully at the proximal end with scissors until a small opening to the marrow canal became visible. A 1 mL tuberculin syringe filled with approximately 0.2 mL foetal bovine serum was inserted into the bone and the bone marrow was gently flushed (to assure maximum dispersion) into 1.0 mL of foetal bovine serum in a 3 mL conical centrifuge tube. The femora were flushed with foetal bovine serum until all the marrow was out and the bone appeared almost transparent. If necessary the distal ends were opened and flushed. The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of foetal bovine serum with the remaining cell button. The button was mixed with a Pasteur pipette to assure a homogenous mixture. A small drop of the mixture was immediately placed near the frosted end of a glass microscope slide previously cleaned in absolute ethanol and pulled behind a clean slide at a 45 ° angle. The slides were quick dried on a slide warmer at approximately 56 °C. Following preparation of the smears, they were dipped in absolute methanol and allowed to air dry.

STAINING: The slides were stained according to the following procedure: Fix in absolute methanol for 5 minutes and air dry. Remove metallic film from surface of Giemsa working solution (5 % Giemsa in pH 6.8 phosphate buffer solution) using a paper towel. Stain for 20 minutes in Giemsa working solution. Rinse twice. The first rinse is in deionised water adjusted to pH 4.0 to 4.5 and the second rinse in deionised water adjusted to pH 7.0. Clean back of slide with absolute methanol. Dry on 56 °C slide warmer. Clear in xylene. Mount in Permount with cover glass.

METHOD OF ANALYSIS: The slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained. One thousand (1000) PCE per animal were counted for the presence of micronuclei. The data were expressed as the number of micronucleated PCE versus total normal PCE in 1000 total PCE per animal. A total of 1000 polychromatic and normochromatic erythrocytes (NCE) was also counted per animal. These data were expressed as the ratio of polychromatic erythrocytes to normochromatic erythrocytes.

Evaluation criteria:

Micronuclei are uniform, darkly stained, typically round bodies in the cytoplasm of PCE. Occasionally, micronuclei appear almond, or tear drop shaped. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plane of the cell are judged to be artefacts and are not scored as micronuclei. Cells containing more than one micronucleus are only scored as micronucleated PCE.
Assessment of a test material as positive is based upon its ability to produce a statistically significant increase in the number of micronucleated polychromatic erythrocytes (PCE) as compared to the vehicle control.

CRITERIA FOR A VALID TEST
If the spontaneous rate of micronuclei in the polychromatic erythrocytes is less than 0.5 % and the positive control is statistically greater (p=0.05) than the spontaneous and at least seven animals per group survived the treatment, the results are deemed acceptable.

Statistics:

A one-tailed t-test was used to make pairwise comparisons between each treatment group with the vehicle control for statistically significant increases in the number of micronucleated PCE. The PCE/NCE ratio was also calculated based on 1000 erythrocytes for each animal. The proportion of PCE per 1000 erythrocytes per animal was evaluated by pairwise t-tests after an arcsin transformation was performed. Statistical significance was judged at the P=0.05 and P=0.01 levels.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Some animals exhibited decreased body tone and/or abdominal gait and/or body drop

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1500, 3000, 4000 and 5000 mg/kg/bw
- Clinical signs of toxicity in test animals: No signs were observed at any dose level immediately after administration. At 500 mg/kg/bw no signs were observed at 4 hours post-dose and only one male had a decrease in body tone at 24, 48 and 72 hours. Signs were observed at all other dose levels. One or more animals had a decrease in body tone at 4, 24, 48 and 72 hours in all test groups. Additional signs included abnormal gait at 72 hours in one female in each of the 1500, 4000 and 5000 mg/kg group. Body drop was observed at 72 hours in one male in the 3000 mg/kg group, in one male at 4000 mg/kg and in two males in the 5000 mg/kg group. No other signs were observed and no mortality occurred in the dose range study.

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.
Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.
The study fulfils the criteria of a valid test.

PHARMACOTOXIC EFFECTS OF TREATMENT IN DEFINITIVE STUDY
Immediately after dose administration, one male dosed with the test material exhibited decreased body tone. At 4, 24, 48 and 72 hours several animals exhibited decreased body tone and/or abdominal gait. Body drop was observed in a few animals at 48 hours and in two animals at 72 hours. No mortality occurred in this study.
No signs were observed in the animals administered the positive or negative controls.

Table 1: Micronucleated PCE per 1000 Polychromatic erythrocytes per animal

Animal number	Sex	Controls		Test material
		Corn oil (48 hours)	TEM (30 hours)	5000 mg/kg
		10 mL/kg	0.5 mg/kg	30 hours	48 hours	72 hours
1	M	0	48	0	0	0
2	M	0	46	0	0	0
3	M	0	97	1	1	0
4	M	1	52	0	0	2
5	M	0	42	0	0	0
6	F	0	47	0	0	0
7	F	0	65	1	0	0
8	F	0	40	0	0	1
9	F	0	74	0	1	0
10	F	0	45	2	0	0
Mean ± S.D.		0.10 ± 0.32	55.60 ± 18.01	0.40 ± 0.70	0.20 ± 0.42	0.30 ± 0.67
t value		-	9.744**	1.236	0.600	0.848

**Denotes statistical significance at P=0.01

Conclusions:

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

Executive summary:

An assay was conducted to evaluate the potential of the test material to induce micronuclei in mice following intraperitoneal administration using methodology equivalent to the standardised guideline OECD 474 under GLP conditions.

In a preliminary dose range finding test the test material was administered intraperitoneally at dose levels of 500, 1500, 3000, 4000 and 5000 mg/kg of bodyweight. Although some clinical signs were observed (decreased body tone, abnormal gait and body drop) the 5000 mg/kg of bodyweight dose was selected for the definitive study.

In the micronucleus test three groups of ten animals (5 males and 5 females per group) were given single doses of the test material at 5000 mg/kg in corn oil by intraperitoneal injection and sacrificed at 30, 48 or 72 hours. Similar groups (5 males and 5 females) were dosed with positive (triethylenemelamine) or vehicle (corn oil) controls and sacrificed at 30 and 48 hours respectively. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.

Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio.The study fulfils the criteria of a valid test.

The test material was negative in the micronucleus test at 5000 mg/kg of bodyweight. These findings are based upon the inability of the test material to produce a significant increase in the incidence of micronuclei per 1000 polychromatic erythrocytes per animal in the treated groups versus the vehicle control group. 

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test with 5 strains (Wisher, 2017)_Key study

A bacterial reverse mutation assay was carried out in accordance with the standardised guidelines OECD 471, EU Method B13/14, US EPA OCSPP 870.5100 and the testing published by the Japanese METI, MHLW and JMAFF under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in DMSO using both the Ames plate incorporation and pre-incubation methods at up to eight 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 Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Bacteria were also exposed to vehicle controls and appropriate positive controls.

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

In Experiment 1 there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test material precipitate (cream-coloured and particulate in appearance) was noted at and above 1500 µg/plate; this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain.

Under the conditions of this study the test material was considered to be non-mutagenic.

 

In vitro chromosomal aberration study on mammalian cells (Murli 1991)_Key study

A chromosomal aberration assay using human lymphocytes was carried out using methodology equivalent to the standardised guideline OECD 473 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Whole blood lymphocytes from a single human donor were used for the study. The test material was dissolved in ethanol which was then diluted in the medium for exposure to the cells.

A range finding test was performed to determine the concentrations to be used for testing, both with and without metabolic activation. Without metabolic activation, mitotic index was evaluated in cultures dosed with 167, 501, 1670, and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the non-activation aberrations assay. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500 and 5000 µg/mL with 24 and 48 hour harvests.

With metabolic activation, mitotic index was evaluated in cultures dosed with 50.1, 167, 501, 1670 and 5010 µg/mL. No reduction in mitotic index was evident; therefore a 24 hour harvest was selected for testing concentrations of 315, 630, 1260, 2510, and 5010 µg/mL in the aberrations assay with metabolic activation. The results from this assay were confirmed in independently conducted assays, testing concentrations of 313, 625, 1250, 2500, and 5000 µg/mL with 24 and 48 hour harvests.

Each concentration was tested in duplicate both with and without metabolic activation, in the form of S-9 mix. Negative (medium), solvent (ethanol) and positive controls (mitomycin C or cyclophosphamide) were also tested concurrently.

Following exposure the cells were harvested, fixed and stained with 5 % Giemsa solution. 100 cells, if possible, from each replicate culture at four test material dose levels and controls were analysed for chromosome aberrations. 25 cells were evaluated for the positive control.

No significant increase in the number of cells with chromosomal aberrations was observed at the concentrations analysed either with or without metabolic activation. Positive and negative controls were deemed to be valid.

Under the conditions of this study, the test material is considered to be negative for inducing chromosomal aberrations in cultured human whole blood lymphocyte cells with and without metabolic activation.

 

In vitro gene mutation test in mammalian cells (2021)_Key study

SpeedCure EHA was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β-Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.

A 3 hour treatment incubation period was used for each experiment.
In the cytotoxicity Range-Finder Experiment, nine concentrations were tested in the absence and presence of S-9 ranging from 0.7813 to 200 μg/mL (limited by expected solubility in the culture medium). The highest concentrations to provide >10% relative suspension growth (RSG) were 50 μg/mL in the absence of S-9 and
100 μg/mL in the presence of S-9, which gave 14% and 32% RSG, respectively.
In the Mutation Experiment, ten concentrations were tested, ranging from 10 to
100 μg/mL, in the absence of S-9 and ranging from 20 to 200 μg/mL in the presence of S-9. Two days after treatment, the highest concentrations selected to determine viability and TFT resistance were 40 μg/mL in the absence of S-9 and 120 μg/mL in the presence of S-9, which gave 16% and 6% RTG, respectively. Although 120 μg/mL in the presence of S-9 gave less than 10% RTG, the second highest concentration (100 μg/mL) gave 24% RTG and therefore both concentrations were analysed.
Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S-9). Therefore, the study was accepted as valid.

No increases in mutant frequency (MF), which exceeded the Global Evaluation Factor (GEF) of 126 mutants per 10^6 viable cells (compared to concurrent controls), were observed in any treated cultures and no significant linear trends were observed in either the absence or presence of S-9. The maximum concentration analysed in the presence of S-9 gave <10% RTG, however, no increases in MF were observed at this concentration and therefore it was considered to be a suitable maximum concentration.

It is concluded that SpeedCure EHA did not induce mutation at the tk locus of mouse lymphoma L5178Y cells when tested in the absence and presence of a rat liver metabolic activation system (S-9). The highest concentration analysed was limited by toxicity in both the absence and presence of S-9.

 

Ames test with E.coli (Dean, 1993)_Supporting study

The photo- mutagenic potential of the test material was investigated using the Escherichia coli strain WP2 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was dissolved in DMSO and the bacteria exposed in both the presence and absence of UV light using the plate incorporation method. Following a range-finding experiment, the dose levels selected for the assays were 8.0, 40.0, 200.0, 1000.0 and 5000.0 µg/plate for Experiment 1 and 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/plate for Experiment 2.

Negative controls comprised treatments with sterile DMSO, either un-irradiated or exposed to the chosen doses of unfiltered or glass-filtered UV light. Further negative controls comprised the photoactivating positive control, 8-methoxypsoralen (8-MOP), but in the absence of light treatment. The positive control chemicals were 8-MOP in the presence of light and 4-nitroquinoline1-oxide (NQO) in the absence of light.

In Experiment 1, precipitation of the test material was seen at 5000 and 1000 µg/plate. No significant increase in revertant colonies was seen at any dose when treated in the absence or presence of UV light.

For the second experiment, in which a top, precipitating dose of 1000 µg/plate and reduced dose intervals were used, no significant increase in the number of mutants was seen for any light treatment.

Negative control (solvent with or without light treatment, 8-MOP without light, test material without light) plates all gave low mean numbers of revertants. The mean number of revertants on the positive control (NQO, 8-MOP with light) plates were significantly elevated.

Under the conditions of the study the test material caused no reproducible increase in the reversion of E.coli WP2 to tryptophan independence when exposed to 2 doses of unfiltered and glass filtered UV light. It is concluded that the test material is not photomutagenic.

 

Dean (1993, CHO UV light)

The test material was investigated for its potential to cause gene mutations in cultured CHO cells with and without ultraviolet light under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested for toxicity in CHO cells in a range-finder experiment using final concentrations of 7.813, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL. The top three doses showed persistent precipitate. Mitotic inhibition was seen at concentrations of 62.5 µg/mL and above and 200 µg/mL was selected as the top dose concentration for the main study.

Part of Main study 1 was repeated in Main study 2 in order to achieve levels of mitotic inhibition consistent with demonstrating toxicity at the highest doses scored. For main study 1 final concentrations ranged from 3.125 to 200 µg/mL and for main study 2 from 10 to 100 µg/mL of test material in DMSO. Negative controls were treated with the solvent and 8-methoxypsoralen (8-MOP) but without UV irradiation. The positive controls were 8-MOP with irradiation and 4-nitroquinoline 1-oxide (NQO).

Concentrations of 50 µg/mL test material and above showed precipitation of the test material in the culture medium. Treatment of cultures with concentrations up to 50 µg/mL of test material in the presence of 200 mJ/cm2 UVA, 32 mJ/cm2 UVB (unfiltered) or 60 µg/mL in the presence of 700 mJ/cm2 UVA (glass filtered) did not show significant increases in the incidence of chromosome aberrations in CHO cells, compared with concurrent controls.

In the main study experiments the un-irradiated solvent control cultures showed frequencies of chromosome aberrations within the historical control ranges, as did the un-irradiated 8-MOP control and the un-irradiated test material cultures. The positive control treatments (NQO and 8-MOP plus light) induced large increases in the incidence of chromosome aberrations indicating that the test system was functioning.

Under the conditions of this study, when treated up to a precipitating concentration which also significantly reduced the mitotic index, the test material did not induce structural aberrations at a frequency significantly greater than the concurrent control values following exposure to unfiltered or glass filtered UV-light. It is concluded that the test material is not photoactivated to a clastogenic form.

 

 

In vivo Micronucleus test (San Sebastian, 1987)

An assay was conducted to evaluate the potential of the test material to induce micronuclei in mice following intraperitoneal administration using methodology equivalent to the standardised guideline OECD 474 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

In a preliminary dose range finding test the test material was administered intraperitoneally at dose levels of 500, 1500, 3000, 4000 and 5000 mg/kg of bodyweight. Although some clinical signs were observed (decreased body tone, abnormal gait and body drop) the 5000 mg/kg of bodyweight dose was selected for the definitive study.

In the micronucleus test three groups of ten animals (5 males and 5 females per group) were given single doses of the test material at 5000 mg/kg in corn oil by intraperitoneal injection and sacrificed at 30, 48 or 72 hours. Similar groups (5 males and 5 females) were dosed with positive (triethylenemelamine) or vehicle (corn oil) controls and sacrificed at 30 and 48 hours respectively. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal.

No statistically significant increases in the incidence of micronucleated PCE were detected in animals administered the test material, at any of the sacrifice times evaluated. No statistically significant differences were observed in the PCE/NCE ratio in any group of animals administered the test material.

Animals treated with the positive control gave a statistically significant increase in the incidence of micronucleated PCE and a statistically significant depression of the PCE/NCE ratio. The study fulfils the criteria of a valid test.

The test material was negative in the micronucleus test at 5000 mg/kg of bodyweight. These findings are based upon the inability of the test material to produce a significant increase in the incidence of micronuclei per 1000 polychromatic erythrocytes per animal in the treated groups versus the vehicle control group. 

Under the conditions of the study, the test material is not considered to be a clastogenic agent.

 

Justification for classification or non-classification

Based on the available data, no classification for genetic toxicity is required for 2-ethylhexyl 4-(dimethylamino)benzoate according to the Regulation (EC) No 1272/2008.