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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November 2017 - 10 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Product name: Sodium cocopropylenediamine propionate
CAS no (old) : 97659-50-2
CAS no (new): 2136366-30-6
Batch no.: 48724
Date of Production: 18.05.2017
Best before Date: 17.05.2020
Purity (certified): 29.7% w/w (UVCB) - Total solids (activity) % w/w 29.7 Lower limit: 29.0, Upper limit: 30.0

Main active ingredients
Dodecylpropylenediamine tripropionate: 64% w/w
Tetradecylpropylenediamine tripropionate: 16% w/w
(considering the composition of the other constituents it is considered justified that the two main constituents represent the whole test item)

Water solubility: soluble
Appearance: yellow, clear
State:liquid
Stability under test conditions: not specified

Viscosity at 20°C Cps 34, Upper limit:150
Color (20% aq solution) Hu 100, Upper limit: 250
pH (20% aq solution) pH 6.3, Lower limit: 6.0, Upper limit: 7.0
Recommended storage
Store container tightly closed in a dry, well-ventilated place

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without S9; tester strains TA1535, TA1537 and TA98): 17, 52, 164, 512, 1600 and 2500 μg/plate
First experiment (with S9; tester strains TA1535, TA1537 and TA98): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (without S9, tester strains TA1537, TA98 and TA100): 17, 52, 164, 512, 1600 and 2500 μg/plate
Second experiment (without S9, tester strains TA1535 and WP2uvrA): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (with S9, all tester strains): 17, 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Milli-Q water
- Justification for choice of vehicle: a previously performed solubility test showed that the test item was dissolved in Milli-Q water.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Remarks:
For details on positive control substances, see table 1
Details on test system and experimental conditions:
Two individual experiments were performed. The dose range-finding study with tester strains TA100 and TA1537 was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water or control solution and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. In the pre-incubation assay (second experiment) this procedure was preceeded by pre-incubation of the solutions.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded

COLONY COUNTING:
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times 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 strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times 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.

In addition to the criteria stated above, 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.

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 Charles River Den Bosch.
b) The selected dose-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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 512 (without S9) and at and above 1600 µg/plate (with S9)
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above1600 µg/plate (without S9) and at 5000 µg/plate (with S9)
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
Remarks:
at and above 1600 µg/plate (without S9) and at 5000 µg/plate (with S9)
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
Remarks:
at and above 1600 µg/plate (without and with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- In both experiments, in all tester strains in the absence and in the presence of S9-mix, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
- In both experiments, precipitation of the test item on the plates was not observed at the start end the end of the incubation period in any tester strain.

Any other information on results incl. tables

Table 2 Historical Control Data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 - 27

3 – 20

3 – 23

8 - 41

8 - 55

63 – 176

54 - 160

10 – 59

9 - 69

Mean

10

11

6

7

16

23

108

107

25

32

SD

3

4

2

3

5

7

19

20

7

8

n

2356

2336

2264

2235

2319

2360

2341

2336

2075

2078

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Table 3 Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

125 – 1248

73 – 1206

55 – 1353

54 – 1051

365 – 1995

250 – 1977

Mean

846

219

787

353

1406

887

SD

146

119

345

162

258

349

n

2348

2229

2003

2234

2200

2276

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1848

408 - 2651

93 – 1951

111 - 1359

Mean

901

1232

1094

437

SD

168

343

477

149

n

2335

2327

2021

2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD guideline 471 and GLP principles, Sodium cocopropylenediamine propionate was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with or without metabolic activation.