C8-18 (even numbered) and C18 (unsaturated) sarcosinates, 2-hydroxypropylammonium salt

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:

22 Feb 2016 to 11 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 21 Jul 1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Official Journal of the European Union No. L142, 31 May 2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(N-methyldodecanamido)acetic acid 2-hydroxypropan-1-aminium 2-(N-methyldodecanamido)acetate

EC Number:

943-406-1

Molecular formula:

Not applicable. This substance is UVCB.

IUPAC Name:

2-(N-methyldodecanamido)acetic acid 2-hydroxypropan-1-aminium 2-(N-methyldodecanamido)acetate

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): MIPA-Cocoyl-Sarkosinat
- Substance type: organic (UVCB)
- Physical state: clear yellow-brown viscous liquid
- Analytical purity: 100%, according to definition of UVCB
- Impurities (identity and concentrations): no impurities, UVCB
- Lot/batch No.: 070715
- Expiration date of the lot/batch: 31 Dec 2016
- Stability under test conditions: stable in vehicle water
- Storage condition of test material: at room temperature
- Other: pH 5.7 - 6.0 (1% in water)

Method

Target gene:

His-operon, Trp-operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa, gal, chl, bio, uvrB

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix (5 or 10%, depending on strain and experiment)

Test concentrations with justification for top dose:

Exp 1: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate ± S9 (TA100, WP2uvrA); 5.4, 17, 52, 164, 512, 1600 µg/plate - S9, 17, 52, 164, 512, 1600 µg/plate + S9 (TA1535, TA1537, TA98)
Exp 2: 154, 275, 492, 878, 1568, 2800 µg/plate ± S9 (TA1535, TA1537, TA98, TA100); 492, 878, 1568, 2800, 5000 µg/plate ± S9 (WP2uvrA)
Exp 3: 48, 87, 154, 275, 492 µg/plate - S9 (TA1535, TA1537, TA98, TA100); 48, 87, 154, 275, 492 µg/plate + S9 (TA1537)
Exp 4: 48, 87, 154, 275, 492, 878 µg/plate - S9 (TA98)
Exp 5: 154, 275, 492, 878, 1568, 2800, 5000 µg/plate - S9 (TA98)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: - S9: sodium azide (TA1535, 5 µg/plate); ICR-191 (TA1537, 2.5 µg/plate); 2-nitrofluorene (TA98, 10 µg/plate); methylmethanesulfonate (TA100, 650 µg/plate); 4-nitroquinoline N-oxide (WP2uvrA, 10 µg/plate)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: + S9: 2-aminoanthracene (TA1535, 2.5 µg/plate; TA1537 [5% S9], 2.5 µg/plate; TA1537 [10% S9], 5 µg/plate; TA98, 1 µg/plate; TA100 [5% S9], 1 µg/plate; TA 100 [10% S9], 2 µg/plate; WP2uvrA, 15 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h

SELECTION AGENT (mutation assays): culture plates lacking histidine (Salmonella) or tryptophan (E. coli)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction of revertant numbers, reduction of background lawn, size increase of microcolonies

OTHER:
In the combined range finder/experiment 1 a concentration of 5% S9-mix was used for metabolical activation. In the repeat experiments an S9-mix concentration of 10% was chosen, which is considered to be an appropriate variation of experimental parameters for repeat experiments, according to the experience of the testing laboratory.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected concentration range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is not greater than two (2) times the revertant number in the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the revertant number in the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is greater than two (2) times the revertant number in the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the revertant number in the concurrent vehicle control.
b) In case a follow-up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow-up experiment.

Statistics:

None

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Range finder was performed in TA100 and WP2uvrA, which was reported within the scope of Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed results were in accordance with the laboratory historical control data ranges, indicating that the test conditions were adequate and and that the metabolic activation system functioned properly. The only deviation from the historical control data ranges was observed for the response of WP2uvrA (+ S9) in the second experiment, where the observed numbers of revertants exceeded the historical control data ranges. The positive control serves as a reference for the test system where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values and even exceeded the historical control range, this deviation in the mean plate count of the positive control did not affect the integrity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Exp 1 (µg/plate): - S9: TA100 ≥512, WP2uvrA =5000, TA1535 ≥1600, TA1537 ≥512, TA98 ≥1600; + S9: TA100 ≥1600, TA1535 ≥512, TA1537 ≥1600, TA98 ≥ 1600
Exp 2: ± S9: No cytotoxicity
Exp 3 (µg/plate): - S9: TA1535≥492, TA1537≥492, TA100 ≥492; +S9: TA1537 ≥275
Exp 4 + 5 (µg/plate): - S9: TA98 ≥878

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 (incl. results of dose range finding test with TA100 and WP2uvrA)

Dose (µg/plate)	Mean number of revertants ± SD
	TA100	WP2uvrA	TA1535	TA1537	TA98
Without S9-mix
Pos. control	1094 ± 67	1614 ± 69	916 ± 16	678 ± 31	1621 ± 55
Solv. control	136 ± 6	16 ± 4	8 ± 6	5 ± 1	16 ± 2
1.7	134 ± 6	17 ± 2	---	---	---
5.4	120 ± 9	20 ± 7	9 ± 4	6 ± 5	13 ± 6
17	118 ± 8	16 ± 6	7 ± 4	4 ± 2	17 ± 5
52	103 ± 20	27 ± 6	4 ± 1	3 ± 1	17 ± 4
164	115 ± 9 n	20 ± 4	6 ± 3	4 ± 2 n	11 ± 2
512	39 ± 7 m	20 ± 2	2 ± 1	1 ± 1 s	11 ± 1
1600	^e MC	15 ± 3	^NP e MC	^NP e MC	6 ± 2^NP n
5000	0 ± 0^a NP	10 ± 2^n NP	---	---	---
With S9-mix (5%)
Pos. control	1404 ± 107	578 ± 49	325 ± 34	451 ± 85	1320 ± 28
Solv. control	124 ± 6	21 ± 9	6 ± 2	5 ± 2	23 ± 2
1.7	139 ± 2	29 ± 7	---	---	---
5.4	130 ± 19	34 ± 7	---	---	---
17	142 ± 12	19 ± 3	7 ± 3	3 ± 2	19 ± 1
52	128 ± 12	27 ± 7	9 ± 2	3 ± 3	23 ± 4
164	138 ± 14	34 ± 11	2 ± 1^n	2 ± 2	14 ± 3
512	104 ± 14^n	35 ± 11	1 ± 1^s	1 ± 1^n	14 ± 3
1600	9 ± 4^m	21 ± 3	3 ± 2^NP m	^NP e MC	8 ± 2^NP n
5000	0 ± 0^a NP	14 ± 5^n NP	---	---	---

MC: Microcolonies
NP: No precipitate
a: Bacterial background lawn absent
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

Table 2: Experiment 2

Dose (µg/plate)	Mean number of revertants ± SD
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Pos. control	814 ± 31	1005 ± 133	1862 ± 314	746 ± 44	538 ± 10
Solv. control	9 ± 3	4 ± 2	14 ± 4	94 ± 13	30 ± 4
154	4 ± 3	2 ± 1	10 ± 5	105 ± 6	---
275	3 ± 2^n	1 ± 1^n	9 ± 2^n	56 ± 5^n	---
492	0 ± 1^m	^e MC	5 ± 1^m	^e MC	24 ± 1
878	^e NP MC	^e MC	^e MC	^e MC	19 ± 3
1568	^e NP MC	^e MC	^e MC	^e MC	28 ± 6
2800	^e NP MC	^e NP MC	^e NP MC	^e NP MC	34 ± 11
5000	---	---	---	---	32 ± 4^n NP
With S9-mix (10%)
Pos. control	173±16	411±52	657±75	1287±119	1500±104
Solv. control	5±2	4±1	13±1	88±19	15±5
154	4±1	4±3	20±3	94±1	---
275	3±1	1±1^n	13±3	99±7^n	---
492	2±1^n	1±0^s	15±3	86±22^s	16±1
878	1±1^s	^e MC	11±2	^e MC	18±2
1568	0±1^m	^e MC	8±2^n	^e MC	12±5
2800	^e NP MC	^e NP MC	1± 2^s NP	^e NP MC	13±8
5000	---	---	---	---	12±8^n NP

MC: Microcolonies
NP: No precipitate
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

Table 3: Experiment 3

Dose (µg/plate)	Mean number of revertants ± SD
	TA1535	TA1537	TA98	TA100
Without S9-mix
Pos. control	1060 ± 79	880 ± 265	1827 ± 118	1005 ± 33
Solv. control	6 ± 3	6 ± 2	18 ± 4	96 ± 16
48	11 ± 2	8 ± 4	20 ± 13	111 ± 9
87	6 ± 3	6 ± 2	21 ± 10	89 ± 5
154	5 ± 2	8 ± 3	19 ± 3	83 ± 22
275	6 ± 4^n	5 ± 2^n	59 ± 9	68 ± 15
492	1 ± 2^s NP	2 ± 1^s NP	42 ± 23^n NP	11 ± 8^n NP
With S9-mix (10%)
Pos. control	---	136 ± 12	---	---
Solv. control	---	12 ± 3	---	---
48	---	11 ± 6	---	---
87	---	9 ± 2	---	---
154	---	10 ± 3	---	---
275	---	5 ± 4	---	---
492	---	3 ± 2^n NP	---	---

NP: No precipitate
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

Table 4: Experiment 4 and 5

Dose (µg/plate)	Mean number of revertants ± SD
	TA98 Exp. 4	TA98 Exp. 5
Without S9-mix
Pos. control	1830 ± 182	1684 ± 46
Solv. control	13 ± 4	16 ± 6
48	11 ± 3	---
87	13 ± 3	---
154	10 ± 5	12 ± 1
275	9 ± 3	14 ± 1
492	9 ± 3	13 ± 2
878	10 ± 4	8 ± 4^n
1568	---	6 ± 1^s
2800	---	4 ± 3^m
5000	---	^e NP MC

MC: Microcolonies
NP: No precipitate
e: Bacterial background lawn extremely reduced
m: Bacterial background lawn moderately reduced
n: Normal bacterial background lawn
s: Bacterial background lawn slightly reduced
---: not tested

 

CONCLUSION:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative