Lecithins, hydrogenated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lecithins, hydrogenated

EC Number:

295-786-7

EC Name:

Lecithins, hydrogenated

Cas Number:

92128-87-5

Molecular formula:

not applicable - UVCB substance

IUPAC Name:

Lecithins, hydrogenated

Specific details on test material used for the study:

Container : plastic bag
Quantity : 64.7091 g (content + container)
Category : cosmetic ingredient
Date of reception* : 30.04.2018
LEMI Code : LM-18/0143
Concentration :Not provided (UVCB substance)
Composition : Complex mixture of Phospholipids, free fatty acids and triglycerides (more details are given in SIP (see annexes))
Purity : Not provided (UVCB substance)

Method

Target gene:

TA98: his D
TA 1537: his C
TA 100 : his G
TA 1535 : his G
WP2 : tryp E

Species / strain

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 mix

Test concentrations with justification for top dose:

The test item was tested at these doses (5 000, 1 500, 500, 150 and 50 μg/plate).

Vehicle / solvent:

water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-anthramine for TA 98, TA 100, TA 1535 and TA 1537 with metabolic activation ; For E. coli WP2: without metabolic activation: Cis-platinum (II) Diammine Dichloride, with metabolic activation : dimethyl-benzanthracene.

Details on test system and experimental conditions:

-> Assay without metabolic activation
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x109 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) / 50 μL (in non-aqueous or non-oily vehicule as DMSO ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 109 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) / 50 μL (in non-aqueous or non-oily vehicule as DMSO ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.
Plates are incubated at 37° C over a 48-72 hour period. The number of revertant colonies per plate is counted.

Moreover the following controls are carried out:
- Negative controls :
-absolute negative control containing no test item corresponding to the spontaneous reversion rate,
-solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M
-Vehicle used to solubilize test item : water
-Positive control

-> Assay with metabolic activation
Two protocols can be used:
- either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ;
-or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 μL of S9-mix fraction are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate.
This method is known to increase the detection sensitivity of a number of promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471).
For the first assay direct incorporation method is used.


-> Assay repetition
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.

PRESENTATION OF DATA
After a 48-72 hour incubation period at 37° C, revertant colonies are counted in each plate.
Data are presented as the number of revertant colonies (mean  standard deviation) per plate.
The following ratio is calculated:
R= Number of revertant colonies in the presence of the test item/Number of revertant colonies in the absence of the test item

Evaluation criteria:

EVALUATION CRITERIA
Ensure that the criteria of validity of the study are well respected namely :
-the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
-the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
-the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
-the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
-Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 100, TA 98, E. Coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Executive summary:

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) performed from suspensions of the test substance do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.