Poly(oxy-1,2-ethanediyl), .alpha.-[3-[(carboxymethyl)methylamino]-2-hydroxypropyl]-.omega.-(4-nonylphenoxy)-, sodium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-13 till 2012-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

disodium 2-({2-hydroxy-3-[2-(4-nonylphenoxy)ethoxy]propyl}(methyl)amino)acetate 2-{[3-({1-chloro-3-[2-(4-nonylphenoxy)ethoxy]propan-2-yl}oxy)-2-hydroxypropyl](methyl)amino}acetate

EC Number:

616-248-3

Cas Number:

75627-31-5

Molecular formula:

UVCB not applicable

IUPAC Name:

disodium 2-({2-hydroxy-3-[2-(4-nonylphenoxy)ethoxy]propyl}(methyl)amino)acetate 2-{[3-({1-chloro-3-[2-(4-nonylphenoxy)ethoxy]propan-2-yl}oxy)-2-hydroxypropyl](methyl)amino}acetate

Details on test material:

Identity: Nonylphenol ethoxylate, sarcosine derivative
Batch No.: 120229AAH
Purity: 81.7 weight %, dose calculation adjusted to purity
Storage: At room temperature
Expiration Date: February 28, 2017
Stability in Solvent: Not indicated bythe sponsor

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in deionised water
- Justification for choice of solvent/vehicle: better than others

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: suspended in deionised water
- Precipitation:
The test item precipitated in the overlay agar in the test tubes 2500 and 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 2500 - 5000 5000 1000 - 5000 1000 - 5000
TA 1537 2500 - 5000 5000 1000 - 5000 1000 - 5000
TA 98 / / 2500 - 5000 5000
TA 100 5000 2500 - 5000 1000 - 5000 2500 - 5000
WP2 uvrA / / 5000 /
/ = no reduced background growth
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 1000 - 5000 2500 - 5000 2500 - 5000 1000 - 5000
TA 1537 1000 - 5000 2500 - 5000 1000 - 5000 1000 - 5000
TA 98 5000 5000 2500 - 5000 5000
TA 100 333 - 5000 2500 - 5000 333 - 5000 2500 - 5000
WP2 uvrA / / 5000 /
/ = no Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1.1     Summary of Results Pre-Experiment and Experiment I

 

Study Name: 1483004	Study Code: Harlan CCR 1483004
Experiment: 1483004 VV Plate	Date Plated: 13/08/2012
Assay Conditions:	Date Counted: 16/08/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			13 ± 5	10 ± 3	22 ± 2	217 ± 14	48 ± 2
	Untreated			10 ± 3	7 ± 1	25 ± 3	203 ± 9	58 ± 8
	Test Item	3 µg		14 ± 1	10 ± 1	21 ± 6	219 ± 17	47 ± 2
		10 µg		14 ± 4	10 ± 2	25 ± 2	225 ± 13	45 ± 2
		33 µg		11 ± 3	11 ± 3	23 ± 4	217 ± 14	51 ± 7
		100 µg		14 ± 2	10 ± 2	22 ± 0	127 ± 5	50 ± 6
		333 µg		8 ± 4	8 ± 1	25 ± 3	78 ± 4	49 ± 13
		1000 µg		3 ± 2	2 ± 1	21 ± 6	55 ± 6	39 ± 3
		2500 µg		3 ± 0^R	1 ± 1^R	12 ± 2	21 ± 7	39 ± 8
		5000 µg		2 ± 1^P R M	1 ± 1^P R	9 ± 2^P M	15 ± 0^P R	30 ± 6^P
	NaN3	10 µg		1728 ± 76			1600 ± 25
	4-NOPD	10 µg				298 ± 23
	4-NOPD	50 µg			77 ± 9
	MMS	3.0 µL						1128 ± 47
With Activation	Deionised water			16 ± 2	11 ± 2	36 ± 2	208 ± 10	59 ± 9
	Untreated			15 ± 5	18 ± 1	35 ± 4	215 ± 12	64 ± 4
	Test Item	3 µg		18 ± 1	12 ± 1	37 ± 6	208 ± 17	65 ± 11
		10 µg		15 ± 5	11 ± 4	34 ± 4	210 ± 19	69 ± 1
		33 µg		14 ± 5	11 ± 2	32 ± 4	210 ± 13	61 ± 11
		100 µg		13 ± 5	12 ± 2	33 ± 4	230 ± 8	57 ± 2
		333 µg		15 ± 7	8 ± 3	32 ± 3	200 ± 6	56 ± 13
		1000 µg		11 ± 4	6 ± 0	32 ± 4	90 ± 12	55 ± 7
		2500 µg		6 ± 1	2 ± 0	22 ± 2	33 ± 4^R	52 ± 6
		5000 µg		1 ± 1^P R	1 ± 1^P R M	15 ± 1^P	17 ± 2^P R	49 ± 5^P
	2-AA	2.5 µg		316 ± 5	212 ± 14	1200 ± 22	2187 ± 29
	2-AA	10.0 µg						235 ± 26

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P R M	Precipitate Reduced background growth Manual count

 

1.2     Summary of Results Experiment II

 

Study Name: 1483004	Study Code: Harlan CCR 1483004
Experiment: 1483004 HV2 Pre	Date Plated: 22/08/2012
Assay Conditions:	Date Counted: 29/08/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			13 ± 4	16 ± 7	23 ± 6	139 ± 11	44 ± 2
	Untreated			15 ± 2	19 ± 5	29 ± 6	116 ± 7	44 ± 3
	Test Item	3 µg		12 ± 2	15 ± 2	21 ± 6	155 ± 15	43 ± 6
		10 µg		15 ± 1	18 ± 5	28 ± 5	159 ± 8	36 ± 8
		33 µg		11 ± 3	15 ± 7	24 ± 5	104 ± 10	37 ± 7
		100 µg		11 ± 3	17 ± 4	25 ± 8	63 ± 12	40 ± 9
		333 µg		8 ± 2	11 ± 3	15 ± 5	59 ± 11	40 ± 5
		1000 µg		7 ± 2^R	4 ± 2^R	14 ± 3	34 ± 6^R	39 ± 4
		2500 µg		5 ± 2^M R	2 ± 1^R M	8 ± 2^M R	21 ± 2^R	33 ± 2
		5000 µg		3 ± 1^P M R	1 ± 1^P M R	7 ± 2^P M R	9 ± 3^P M R	18 ± 5^P M R
	NaN3	10 µg		1692 ± 26			1635 ± 67
	4-NOPD	10 µg				272 ± 18
	4-NOPD	50 µg			70 ± 12
	MMS	3.0 µL						445 ± 14
With Activation	Deionised water			26 ± 3	22 ± 4	30 ± 3	164 ± 10	56 ± 1
	Untreated			19 ± 3	19 ± 3	31 ± 6	174 ± 18	56 ± 13
	Test Item	3 µg		22 ± 2	26 ± 2	37 ± 7	163 ± 13	52 ± 14
		10 µg		15 ± 1	20 ± 4	35 ± 10	149 ± 3	59 ± 2
		33 µg		18 ± 7	26 ± 3	30 ± 3	159 ± 17	55 ± 9
		100 µg		22 ± 7	22 ± 8	30 ± 1	155 ± 12	50 ± 2
		333 µg		15 ± 1	17 ± 4	33 ± 4	145 ± 18	54 ± 4
		1000 µg		7 ± 1^R	8 ± 1^R	32 ± 9	78 ± 2	47 ± 11
		2500 µg		4 ± 1^M R	7 ± 1^R	22 ± 6	26 ± 6^R	39 ± 2
		5000 µg		1 ± 1^P M R	3 ± 1^P M R	6 ± 1^P M R	10 ± 2^P M R	43 ± 7^P
	2-AA	2.5 µg		226 ± 37	163 ± 6	1395 ± 224	1654 ± 174
	2-AA	10.0 µg						218 ± 38

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R P M	Reduced background growth Precipitate Manual count

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of Nonylphenol ethoxylate, sarcosine derivate to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth in all strains in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Nonylphenol ethoxylate, sarcosine derivative at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.