Reaction mass of disodium 2-[[2-hydroxy-3-[[methyl-(2-oxido-2-oxoethyl)amino]methyl]-5-nonylphenyl]methyl-methylamino and sodium 2-[(2-hydroxy-5-nonylphenyl)methyl-methylamino]acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential for the substance to cause genetic toxicity has been investigated in vitro is studies conducted using bacterial and mammalian cell systems. Negative results were obtained a reverse mutation assay (Ames test) using strains of Salmonella typhimurium and Escherichia coli conducted according to OECD TG 471; in a chromosome aberration test using human lymphocytes conducted according to OECD TG 473 and in a gene mutation using l5178Y TK+/- Mouse lymphoma cells conducted according to OECD TG 490, demonstrating that the substance lacks the potential for mutagenicity or clastogenicity in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start: 30 May 2018 Experimental Completion: 08 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Data Requirement

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Based upon a preliminary study (Experiment 1), the dose range used for the Main Study (Experiment 2) was 1.23, 4.1, 12.3, 41, 123, 410, 1230 and 4100 μg/plate.

Vehicle / solvent:

Tetrahydrofuran

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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:

Without Metabolic Activation:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the appropriate concentration of test item formulation or 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 plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation:
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation & Scoring:
All of the plates were incubated at 37±3°C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Without Metabolic Activation

S9 Mix	Dose (ug/plate)	No. of Revertant Colonies Per Plate (Mean ± SD)
		Base Pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent Control (THF)	130 ± 4.0	15 ± 2.1	21 ± 3.5	29 ± 10.3	9 ± 6.0
	1.23	121 ± 6.6	12 ± 3.5	21 ± 2.9	29 ± 0.0	13 ± 1.2
	4.1	127 ± 13.6	9 ± 4.6	21 ± 6.0	29 ± 3.6	12 ± 7.5
	12.3	113 ± 5.1	15 ± 6.1	28 ± 2.6^*	20 ± 6.0	14 ± 1.2
	41	117 ± 5.0	12 ± 3.2	24 ± 2.5	18 ± 3.6	12 ± 9.9
	123	83 ± 29.1	12 ± 7.2	25 ± 3.1	0 ± 0.0^V	5 ± 3.2
	410	0 ± 0.0^T	13 ± 3.6	27 ± 4.0	0 ± 0.0^T	0 ± 0.0^V
	1230	0 ± 0.0^T	0 ± 0.0^T	26 ± 2.0^S	0 ± 0.0^T	0 ± 0.0^T
	4100	0 ± 0.0^T	0 ± 0.0^T	0 ± 0.0^V	0 ± 0.0^T	0 ± 0.0^T
Positive Control	Compound	ENNG	ENNG	ENNG	4NQO	9AA
	ug/Plate	3	5	2	0.2	80
	Revertants/Plate	1120 ± 253.6	2601 ± 277.8	950 ± 33.1	403 ± 29.9	506 ± 54.0

^*p =≤0.05
^SSparse bacterial background lawn
^VVery weak bacterial background lawn
^TToxic – no bacterial background lawn

 

 

With Metabolic Activation

S9 Mix	Dose (ug/plate)	No. of Revertant Colonies Per Plate (Mean ± SD)
		Base Pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
+	Solvent Control (THF)	129 ± 8.5	13 ± 2.0	40 ± 8.5	31 ± 7	18 ± 3.8
	1.23	126 ± 28	15 ± 4.2	38 ± 1.7	28 ± 6.4	16 ± 3.2
	4.1	139 ± 9.5	12 ± 4.7	40 ± 5.5	26 ± 4.4	18 ± 1.2
	12.3	139 ± 3.0	17 ± 4.6	35 ± 3.5	30 ± 9.5	17 ± 4.2
	41	126 ± 8.6	13 ± 0.6	33 ± 7.2	29 ± 1.7	19 ± 2.5
	123	110 ± 14.7	14 ± 4.0	34 ± 6.9	26 ± 2.1	8 ± 1.5
	410	85 ± 11.4^S	11 ± 3.5	36 ± 3.5	29 ± 7.5	8 ± 2.1
	1230	0 ± 0.0^T	5 ± 1.5^S	28 ± 5.2	19 ± 3.5^S	0 ± 0.0^V
	4100	0 ± 0.0^T	0 ± 0.0^T	24 ± 8.0^S	15 ± 8.7^S	0 ± 0.0^T
Positive Control	Compound	2AA	2AA	2AA	BP	2AA
	ug/Plate	1	2	10	5	2
	Revertants/Plate	1959 ± 61.3	349 ± 13.1	282 ± 32.1	126 ± 7.4	346 ± 33

^SSparse bacterial background lawn
^VVery weak bacterial background lawn
^TToxic – no bacterial background lawn

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-mutagenic.

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, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods

 

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.23 to 4100 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (1.23 to 4100 μg/plate). Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

 

Results

 

The vehicle (tetrahydrofuran) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was 4100 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains (except TA98 dosed in the presence of metabolic activation) initially from 1230 μg/plate in both the absence and presence of metabolic activation.

 

Based on the results of Experiment 1, the same maximum dose level (4100 μg/plate) was employed in the second mutation test (pre-incubation method). In Experiment 2 (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 123 μg/plate (TA100 and TA98), 410 μg/plate (TA1535 and TA1537) and 1230 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 410 μg/plate (TA100), 1230 μg/plate (TA1535, TA98 and TA1537) and at 4100 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

 

A test item precipitate (greasy in appearance) was noted at and above 1230 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) only. 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 item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

 

Similarly, no biologically relevant 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 (S9-mix) in Experiment 2 (pre-incubation method). A minor statistical value was noted (WP2uvrAat 12.3 μg/plate in the absence of S9-mix (= p ≤0.05)), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

 

Conclusion

 

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start: 25 May 2018 Experimental Completion: 18 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Requirement

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test) (migrated information)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Cytokinesis block (if used):

Colcemid (0.1 ug/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The following main test concentrations were based upon the results of a preliminary toxicity test:

4-Hour (-S9): 0, 16.45, 32.9, 65.8, 98.6, 131.5, 164.4 & 263.0 ug/mL
4-Hour (+S9): 0, 40.0, 80.0, 160.0, 200.0, 240.0, 280.0 * 320.0ug/mL
24-Hour (-S9): 0, 2.47, 4.94, 8.20, 9.88, 16.45, 24.7 & 32.9 ug/mL

Vehicle / solvent:

MEM (Eagle's minimal essential medium)

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

The following were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed


Criteria for determining the Conclusion

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the Preliminary Study and therefore test in the Main Study at appropriate dose levels not causing cytotoxicity or precipitation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result is negative.

4 Hour Exposure Without Metabolic Activation (-S9)

^Treatment Group

^Replicate

^Mitotic Index (%)

^No. of Cells Scored

^Number of Aberrations

^Total No. of Aberrations

^Frequency of Aberrant Cells (%)

^Gaps

^Chromatid

^Chromosome

^Others

^Breaks

^Exchanges

^Breaks

^Exchanges

^X

^+ Gaps

^- Gaps

^+ Gaps

^- Gaps

^Vehicle Control (MEM)

^A

^3.35

^150

^0

^1

^0

^0

^0

^0

^1

^1

^1

^1

^B

^2.80

^150

^1

^0

^0

^0

^0

^0

^1

^0

^1

^0

^Total

^(100)

^300

^1

^1

^0

^0

^0

^0

^2

^1

^2 (0.7)

^1 (0.3)

^98.6ug/mL

^A

^1.75

^150

^1

^0

^0

^0

^0

^0

^1

^0

^1

^2

^B

^3.15

^150

^0

^2

^0

^2

^0

^0

^4

^4

^2

^2

^Total

^(80)

^300

^1

^2

^0

^2

^0

^0

^5

^4

^3 (1.0)

^2 (0.7)

^131.5ug/mL

^A

^2.40

^150

^1

^0

^0

^0

^0

^0

^1

^0

^1

^0

^B

^1.70

^150

^1

^1

^0

^0

^0

^0

^2

^1

^2

^1

^Total

^(67)

^300

^2

^1

^0

^0

^0

^0

^3

^1

^3 (1.0)

^1 (0.3)

^164.4ug/mL

^A

^1.25

^150

^0

^3

^0

^0

^0

^0

^3

^3

^3

^3

^B

^1.10

^150

^1

^2

^0

^0

^0

^0

^3

^2

^3

^2

^Total

^(38)

^300

^1

^5

^0

^0

^0

^0

^6

^5

^6 (2.0)

^5 (1.7)

^Positive Control (MMC)

^A

^1.25

^150

^3

^9

^1

^0

^0

^0

^14

^11

^12

^10

^B

^0.95

^150

^2

^4

^3

^0

^0

^0

^13

^11

^11

^9

^Total

^(36)

^300

^5

^13

^4

^0

^0

^0

^27

^22

^23 (7.7)

^19 (6.3)***

*** p = < 0.001
MMC Mitomycin C
MEM Eagle’s minimal essential medium

4 Hour Exposure With Metabolic Activation (+S9)

^Treatment Group

^Replicate

^Mitotic Index (%)

^No. of Cells Scored

^Number of Aberrations

^Total No. of Aberrations

^Frequency of Aberrant Cells (%)

^Gaps

^Chromatid

^Chromosome

^Others

^Breaks

^Exchanges

^Breaks

^Exchanges

^X

^+ Gaps

^- Gaps

^+ Gaps

^- Gaps

^Vehicle Control (MEM)

^A

^7.80

^150

^5

^1

^0

^0

^0

^0

^6

^1

^5

^1

^B

^4.90

^150

^2

^0

^0

^0

^0

^0

^2

^0

^2

^0

^Total

^(100)

^300

^7

^1

^0

^0

^0

^0

^8

^1

^7 (2.3)

^1 (0.3)

^80 ug/mL

^A

^7.05

^150

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^B

^6.65

^150

^0

^1

^0

^0

^0

^0

^1

^1

^1

^1

^Total

^(1080)

^300

^0

^1

^0

^0

^0

^0

^1

^1

^1 (0.3)

^1 (0.3)

^120 ug/mL

^A

^5.30

^150

^5

^2

^0

^2

^0

^0

^9

^4

^8

^4

^B

^4.40

^150

^0

^4

^1

^0

^0

^0

^5

^5

^4

^4

^Total

^(76)

^300

^5

^6

^1

^2

^0

^0

^14

^9

^12 (4.0)

^8 (2.7)*

^160 ug/mL

^A

^2.40

^150

^6

^1

^0

^2

^0

^0

^9

^3

^8

^3

^B

^2.70

^150

^3

^5

^0

^2

^0

^0

^10

^7

^10

^7

^Total

^(40)

^300

^9

^6

^0

^4

^0

^0

^19

^10

^18 (6.0)

^10 (3.3)*

^Positive Control (CP)

^A

^3.90

^98^

^10

^16

^3

^0

^0

^0

^29

^19

^22

^16

^B

^4.65

^72^

^3

^11

^5

^5

^0

^0

^24

^21

^18

^15

^Total

^(67)

^170

^13

^27

^8

^5

^0

^0

^53

^40

^40 (23.5)

^31 (18.2)***

* p = < 0.05
*** p = < 0.001
^ Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
CP Cyclophosphamide
MEM Eagle’s minimal essential medium

4 Hour Exposure With Metabolic Activation (+S9) – Confirmatory Experiment

^Treatment Group

^Replicate

^Mitotic Index (%)

^No. of Cells Scored

^Number of Aberrations

^Total No. of Aberrations

^Frequency of Aberrant Cells (%)

^Gaps

^Chromatid

^Chromosome

^Others

^Breaks

^Exchanges

^Breaks

^Exchanges

^X

^+ Gaps

^- Gaps

^+ Gaps

^- Gaps

^Vehicle Control (MEM)

^A1

^6.70

^150

^0

^1

^0

^1

^0

^0

^2

^2

^2

^2

^A2

^7.60

^150

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^B1

^7.20

^150

^1

^0

^0

^0

^0

^0

^1

^0

^1

^0

^B2

^7.80

^115

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^Total

^(100)

^600

^1

^1

^0

^1

^0

^0

^3

^2

^3 (0.5)

^2 (0.3)

^80 ug/mL

^A

^6.00

^150

^6

^1

^0

^0

^0

^0

^7

^1

^6

^1

^B

^6.20

^150

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^Total

^(83)

^300

^6

^1

^0

^0

^0

^0

^7

^1

^6 (2.0)

^1 (0.3)

^120 ug/mL

^A

^3.70

^150

^2

^2

^0

^1

^0

^0

^5

^3

^5

^3

^B

^3.80

^150

^0

^0

^2

^0

^0

^0

^2

^2

^2

^2

^Total

^(51)

^300

^2

^2

^2

^1

^0

^0

^7

^5

^7 (2.3)

^5 (1.7)

^160 ug/mL

^A

^1.60

^150

^2

^0

^0

^0

^0

^0

^4

^2

^4

^2

^B

^0.80

^150

^2

^0

^0

^3

^0

^0

^7

^5

^5

^4

^Total

^(16)

^300

^4

^0

^0

^3

^0

^0

^11

^7

^9 (3.0)

^6 (2.0)

^Positive Control (CP)

^A

^2.90

^87^

^6

^1

^1

^5

^0

^0

^25

^19

^20

^16

^B

^1.00

^52^

^3

^1

^1

^4

^0

^1

^21

^18

^16

^15

^Total

^(27)

^139

^9

^2

^2

^9

^0

^1

^46

^37

^36 (25.9)

^31 (22.3)***

*** p = < 0.001
^ Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
CP Cyclophosphamide
MEM Eagle’s minimal essential medium

24 Hour Exposure Without Metabolic Activation (-S9)

^Treatment Group

^Replicate

^Mitotic Index (%)

^No. of Cells Scored

^Number of Aberrations

^Total No. of Aberrations

^Frequency of Aberrant Cells (%)

^Gaps

^Chromatid

^Chromosome

^Others

^Breaks

^Exchanges

^Breaks

^Exchanges

^X

^+ Gaps

^- Gaps

^+ Gaps

^- Gaps

^Vehicle Control (MEM)

^A

^3.60

^150

^2

^0

^0

^0

^0

^0

^2

^0

^2

^0

^B

^5.25

^150

^4

^1

^0

^1

^0

^0

^6

^2

^6

^2

^Total

^(100)

^300

^6

^1

^0

^1

^0

^0

^8

^2

^8 (2.7)

^2 (0.7)

^8.2 ug/mL

^A

^3.40

^150

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^B

^3.80

^150

^0

^1

^0

^0

^0

^0

^1

^1

^1

^1

^Total

^(81)

^300

^0

^1

^0

^0

^0

^0

^1

^1

^1 (0.3)

^1 (0.3)

^9.88ug/mL

^A

^2.60

^150

^4

^2

^0

^0

^0

^0

^6

^2

^6

^2

^B

^3.20

^150

^1

^0

^0

^0

^0

^0

^1

^0

^1

^0

^Total

^(66)

^300

^5

^2

^0

^0

^0

^0

^7

^2

^7 (2.3)

^2 (0.7)

^16.45ug/mL

^A

^2.00

^150

^0

^0

^0

^0

^0

^0

^0

^0

^0

^0

^B

^1.30

^150

^0

^2

^0

^0

^0

^0

^2

^2

^2

^2

^Total

^(37)

^300

^0

^2

^0

^0

^0

^0

^2

^2

^2 (0.7)

^2 (0.7)

^Positive Control (MMC)

^A

^1.70

^98^

^1

^9

^8

^0

^0

^0

^18

^17

^16

^15

^B

^2.15

^107

^2

^9

^6

^6

^0

^0

^23

^21

^16

^15

^Total

^(44

^2025

^3

^18

^14

^6

^0

^0

^41

^38

^32 (15.6)

^30 (14.6)***

*** p = < 0.001
^ Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed
MMC Mitomycin C
MEM Eagle’s minimal essential medium

Conclusions:

The test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

 

This report describes the results of anin vitrostudy 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., 1991).

 

Methods

 

Replicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Experiment were as follows:

 

Exposure	Final Concentration of Test Item (ug/mL)
4 Hour (-S9)	0, 16.45, 32.9, 65.8, 98.6, 131.5, 164.4, 263
4 Hour (+S9)	0, 40, 80, 160, 200, 280, 320
24 Hour (-S9)	0, 2.47, 4.94, 8.2, 9.88, 16.45, 24.7, 32.9

 

Due to the results of the Main Experiment, a Confirmatory Experiment was conducted using 4 hours exposure in the presence of S9, at a 2% final concentration with cell harvest after a 20-hour expression period. The dose levels used in the Confirmatory Experiment were selected using data from the Main Experiment where, again, the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Confirmatory Experiment were as follows:

 

Exposure	Final Concentration of Test Item (ug/mL)
4 Hour (+S9)	0, 80, 120, 160, 180, 200, 240, 280

 

 

Results

 

Main Experiment

 

All vehicle (Eagle's minimal essential medium (MEM)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

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

In the Main Experiment, the test item was toxic to human lymphocytes andinduced modest but statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition in the presence of S9‑mix only. However, in the absence of metabolic activation the test itemdid not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that marginally exceeded 55±5% mitotic inhibition in both exposure groups.

 

Confirmatory Experiment

 

All vehicle (Eagle's minimal essential medium (MEM)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

The positive control item 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 was toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level thatexceeded 55±5% mitotic inhibitioninduced.

 

Conclusion

 

The test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start: 29 May 2018 Experimental Completion: 25 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Annex VIII Data Requirement.

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:

TK +/- Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Based upon a preliminary study, the main test was performed at the following concentrations:

4 Hours (-S9): 12.3, 24.6, 49.2, 98.4, 106.6, 114.8, 123, 131.2 ug/mL
4 Hours (+S9): 8.2, 16.4, 32.8, 49.2, 65.6, 98.4 ug/mL
24 Hours (-S9): 1.03, 2.05, 4.1, 8.2, 16.4, 24.6 ug/mL

Vehicle / solvent:

R0 Media

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at up to ten dose levels of the test item 3.08 to 131.2* μg/mL 4-hours –S9, 8.2 to 147.6* μg/mL 4-hours +S9, and 1.03 to 49.2* μg/mL 24-hours – S9, vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).

The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those that cover approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

None

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

Positive controls validity:

valid

4 Hour (-S9)

Treatment	% RSG	RTG	MF
0	100	1.00	164.87
3.08^	91	-	-
6.15^	88	-	-
12.3	94	0.87	128.81
24.6	90	0.88	128.14
49.2	54	0.49	233.84
98.4	47	0.45	209.52
106.6	53	0.49	200.43
114.8	53	0.49	209.37
123.0	34	0.31	199.81
131.2	18	0.12	198.42
MF Threshold for Positive Response: 290.87
EMS (400)	74	0.52	1586.99

% RSG = % relative suspension growth
RTG = relative total growth
MF = 5-TFT resistant mutants / 10⁶viable cells 2 days after exposure
^ = Not plated as surplus to requirements – excessive toxicity
EMS = Ethylmethanesulphonate

 

4 Hour (+S9)

Treatment	% RSG	RTG	MF
0	100	1.00	139.79
8.2	81	0.85	121.26
16.4	80	0.87	128.05
32.8	57	0.62	160.13
49.2	32	0.35	221.58
65.6	20	0.13	239.55
98.4	21	0.18	178.66
131.2^	8	-	-
147.6^	1	-	-
MF Threshold for Positive Response: 265.79
CP (1.5)	70	0.76	848.84

% RSG = % relative suspension growth
RTG = relative total growth
MF = 5-TFT resistant mutants / 10⁶viable cells 2 days after exposure
^ = Not plated as surplus to requirements – excessive toxicity
CP = Cyclophosphamide

 

24 Hour (-S9)

Treatment	% RSG	RTG	MF
0	100	1.00	138.00
1.03	89	1.04	106.47
2.05	77	0.99	106.19
4.1	88	1.11	105.85
8.2	57	0.84	115.32
16.4	25	0.27	231.53
24.6	12	0.21	191.78
32.8^	4	-	-
49.2^	0	-	-
MF Threshold for Positive Response: 264.0
EMS (150)	28	0.22	2032.08

% RSG = % relative suspension growth
RTG = relative total growth
MF = 5-TFT resistant mutants / 10⁶viable cells 2 days after exposure
^ = Not plated as surplus to requirements – excessive toxicity
EMS = Ethylmethanesulphonate

Conclusions:

The test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it was considered to be non-mutagenic in this assay.

Executive summary:

Introduction

 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods

 

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to 10 dose levels in duplicate, together with vehicle (R0 media), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test at a concentration range of 8.01 to 2050 μg/mL. The maximum dose level used in the Mutagenicity Test was limited by test item induced toxicity. The dose levels plated for viability and expression of mutant colonies were as follows:

 

Group	Concentration of test substance plated for viability and mutant frequency (μg/mL)
4 Hour (-S9)	12.3, 24.6, 49.2, 98.4, 106.6, 114.8, 123, 131.2
4 Hour (+S9)	8.2, 16.4, 32.8, 49.2, 65.6, 98.4
24 Hour (-S9)	1.03, 2.05, 4.1, 8.2, 16.4, 24.6

 

 

Results

 

The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency within the historical control data range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant increases in either of the three exposure groups. Optimum levels of toxicity were achieved in all three exposure groups. The GEF value was not exceeded at any test item concentration.

 

Conclusion

 

The test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it was considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based upon the in vitro data available, there is no requirement for an in vivo study to be conducted.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay (Ames test)

The potential for the substance to induce gene mutations in bacterial cells in vitro was investigated in a reverse mutation assay (Ames test) using Salmonella typhimurium and Escherichia coli conducted according to OECD TG 471. In the study, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the substance using both the Ames plate incorporation and pre-incubation methods at eight dose levels at concentrations up to 4100 µg per plate, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The substance did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix).  Under the conditions of the study, the substance was considered to be non-mutagenic to bacterial cells in vitro.

Clastogenicity assay – chromosome aberration test

The potential for the substance to induce structural chromosome aberrations in cultured mammalian cells in vitro was investigated in a chromosome aberration test using human lymphocytes conducted according to OECD TG 473. In the study, replicate cultures of human lymphocytes, treated with the substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

In the main experiment of the study, treated systems with a 4-hour exposure period (20-hour expression period) without S9 mix were dosed at 0, 16, 45, 32.9, 65.8, 98.6, 131.5, 164.4 and 263 µg/ml; treated systems with a 4-hour exposure period (20-hour expression period) with S9 mix (2%) were dosed at 0, 40, 80, 160, 200, 240, 280 and 320 µg/ml and treated systems with a 24-hour exposure period without S9 mix were dosed at 0, 2, 47, 4.94, 8.2, 9.88, 16.45, 24.7 and 32.9  µg/ml. Due to the results of the main experiment, a further confirmatory experiment was required in which systems treated with a 4-hourr exposure (20-hour expression period) were dosed at 0, 80, 120, 160, 180, 200, 240 and 280 µg/ml.

In the Main Experiment, the test item was toxic to human lymphocytes and induced modest but statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition in the presence of S9 mix only.  However, in the absence of metabolic activation the test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that marginally exceeded 55±5% mitotic inhibition in both exposure groups. In a further confirmatory test, the substance was found to be toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that exceeded 55±5% mitotic inhibition. Based on the findings of the study, the substance was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation (mouse lymphoma assay)

The potential for the substance to induce gene mutations in mammalian cells in vitro was investigated in l5178Y TK+/- Mouse lymphoma cells in a gene mutation assay conducted according to OECD TG 490. In the study, dose level for the main mutagenicity test were determined in a preliminary toxicity test using the substance at concentrations ranging from 8.01 to 2050 µg/plate. One main mutagenicity test was subsequently performed in which treated systems with a 4 hour exposure period, were dosed at 12.3, 24.6, 49.2, 98.4, 106.6, 114.8, 123 and 131.2 µg/ml in the absence of metabolic activation; test systems with a 4 hour exposure were dosed at 8.2, 16.4, 32.8, 49.2, 65.6 and 98.4 µg/ml in the presence of metabolic activation (S9) and treated systems with a 24 hr exposure were treated at 1.03, 2.05, 4.1, 8.2 ,16.4, 24.6 µg/ml in the absence of metabolic activation. The substance did not induce any toxicologically significant increases in either of the three exposure groups.  Optimum levels of toxicity were achieved in all three exposure groups.  The substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6 (i.e. the predefined mutant frequency threshold indicative of a positive response), in either the presence or the absence of metabolic activation, and was consequently considered to be non-mutagenic based on these findings.

Justification for classification or non-classification

Negative results were obtained a bacterial reverse mutation assay (Ames test) using strains of Salmonella typhimurium and Escherichia coli conducted according to OECD TG 471; in a chromosome aberration test using human lymphocytes conducted according to OECD TG 473 and in a gene mutation using l5178Y TK+/- Mouse lymphoma cells conducted according to OECD TG 490, demonstrating that the substance lacks the potential for mutagenicity or clastogenicity in vitro. The findings in studies in vitro do not trigger further investigations of genotoxicity in vivo. On this basis, the classification of the substance for genotoxicity according to Regulation (EC) 1272/2008 is not required.