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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471:

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.

OECD 490:

In the framework of OECD 490 under the described experimental conditions, solutions obtained from the test item do not induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence or in the presence of metabolic activation (2.5% S9-mix) at these doses.

OECD 473:

According to the criteria of conclusion of the study protocol and OCDE 473, solutions obtained from the test item are not considered clastogenic in the test system used (CHO) in the conditions of the assay.

Link to relevant study records

Referenceopen allclose all

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
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
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)
Target gene:
TA98: his D
TA 1537: his C
TA 100 : his G
TA 1535 : his G
WP2 : tryp E
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
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).
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
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.

Endpoint:
in vitro gene mutation study in mammalian cells
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Date of reception: 30.04.2018
Stability: stable under normal storage conditions and handling
Batch production date: 28.07.2017
Expiry date: 19.03.2019
Aspect: light yellow to pale yellow powder
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse lymphoma L5178Y TK+/-cells (ATCC-CRL-9518) purchased from ATCC (American Type Culture Collection-Rockeville, MD 20852 – USA) have been used successfully in “in vitro” experiments for many years. These cells are characterized by their high proliferation rate (10 h – 12 h doubling time of the stock cultures) and their cloning efficiency, usually more than 50 %. They possess a nearly diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the thymidine kinase (TK) locus which allows to detect mutation events at the TK-locus.
Cells from the cell bank stored at- 80°C are systematically checked to be free from mycoplasma contamination
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 – USA (S9 Moltox-11101-5-3919 validated on 06.07.2018 – expiry date: 07.02.2020).

S9-mix is prepared at 4 °C on the day of use, as presented below. The final concentration of cofactors and salts is as follow:
S9 fraction: 2.5 % (v/v)
MgCL2-6H2O: 8 mM
KCl: 33 mM
Glucose-6-Phosphate Na2: 5 mM
NADP Na2: 4 mM
Phosphate buffer pH 7.4: 0.1 M
Test concentrations with justification for top dose:
Test item is poorly soluble in water: 5 mg/L (provided by the sponsor). A non-GLP test conducted by LEMI pre-determined value of 10 μg/mL in culture medium (no precipitate visible by eye).
Test item is not soluble in solvent (DMSO or Ethanol) at concentration that would allow to obtain a final concentration in contact with the cells higher than 10 μg/mL.
For the assay, to obtained a final concentration at 25 μg/mL (slightly higher than the maximum soluble concentration visible to the naked eye, in accordance with OECD requests) we used a homogenous suspension of the test item, at 100 μg/mL, in sterile conditions. The highest concentration chosen is slightly higher than the maximum soluble concentration visible to the naked eye, in accordance with OECD 490 requests (§29 “For poorly soluble test chemicals that are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analyzed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test chemical.”).
Vehicle / solvent:
Complete culture medium (CCM): Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX – I supplemented with 10 % (v/v) (GIBCO – ref. 31966021 - batch 2007769) inactivated horse serum (10 %), (GIBCO – ref. 16050-130 - batch 1861235) and 1 % (v/v) antibiotics (GIBCO – ref. 15240-096 - batch 1981203/1953089).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMEM glutamax (Dulbecco's Modified Eagle’s Medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: assays are run independently using duplicate cultures.

METHOD OF TREATMENT/ EXPOSURE:
The cell density is determined every day, and adjusted to 2 x 105 cells/mL, if necessary.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
Evaluation criteria:
-Acceptability criteria for the assay
A gene mutation assay is considered acceptable if it meets the following criteria:
- the test must be conducted under two experimental conditions (short treatment without and with metabolic activation) unless one resulted in positive results.
- adequate number of cells (a minimum of 6 x106 cells) and concentrations should be analysable

-Acceptability criteria for negative and positive controls
Every experiment should be evaluated as to whether the untreated control meets the IWGT MLA Workgroup acceptance criteria, below:
• Mutant Frequency 50 – 170 x 10-6
• Cloning Efficiency 65 – 120%
• Suspension Growth:
− 8 – 32 fold (3-4-hour treatment)
− 32 – 180 fold (24-hour treatment, if conducted)
Every experiment should also be evaluated as to whether the positive controls meets at least one of the following two acceptance criteria:
- The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40 % of the IMF should be reflected in the small colony MF.
- The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated control (a small colony IMF of 150 x 10-6).
The upper limit of cytotoxicity observed in the positive control culture should be the same as of the experimental cultures. In other words, the RTG/RS should not be less than 10 %.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Executive summary:

In the framework of OECD 490 under the described experimental conditions, solutions obtained from the test item do not induce a mutagenic effect in L5178Y TK+/-Mouse lymphoma cells in the absence or in the presence of metabolic activation (2.5% S9-mix) at these doses.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Organoleptic specifications:
. Aspect:
Light yellow to pale yellow powder
. pH:
Not provided
Other physical properties:
. sterility:
No sterile
. density:
NA
. CAS N°:
92128-87-5
. EC N° / EINECS:
295-786-7
Storage conditions:
room temperature** - keep away from light - hygroscopic
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
-Source of cells: ATCC CCL 61, ECACC 85051005

-Suitability of cells: Chinese Hamster Ovary (CHO) cultures are recommended by the OECD guideline n° 473. Moreover, CHO are currently used in standard protocols for in vitro cytogenetic tests. CHO are tested for absence of mycoplasma and population doubling.

-Cell cycle length: 17.4h

-Methods for maintenance in cell culture: Mc Coy’s + 10 % FCS

-Modal number of chromosomes: 20

-Passage number: 21 (for assay 1) - 27 (for assay 2)

-Caryotype: stable


MEDIA USED
- Type and identity of media:
Cells are seeded in a 25 cm² culture flask at the starting density of 10.103 cells/cm² into 5 ml of complete culture medium* (Mc Coy’s supplemented with 10 % (v/v) Fetal Calf Serum (FCS)).
Cell cultures are incubated at 37°C in a humid atmosphere containing 5% (v/v) CO2, for 40 hours.

- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix

Preparation of S9 fraction
S9 (microsome fraction from the liver of Sprague Dawley rats treated with Aroclor 1254 (500 mg/kg over a 5 day period)) is prepared in compliance with Ames and al4, and provided by MOLTOXTM (POB Box 1189 – 157 Industrial Park Dr – Boone, NC 28607 – USA). The S9 fraction (Ref: 11-101.5 – Batch: 3919 – Expiry date: 07.02.2020) was previously validated on 06.07.2018 in the laboratory according to the LEMI SOP n° MB06/09.
Test concentrations with justification for top dose:
25 μg/mL - 10 μg/mL - 4 μg/mL - 1.6 μg/mL
The highest concentration chosen is slightly higher than the maximum soluble concentration visible to the naked eye, in accordance with OECD 473 requests
Vehicle / solvent:
Mc Coy’s supplemented with 10 % (v/v) Fetal Calf Serum (FCS), 1 % (v/v) antibiotics (penicillin 10 000 U/mL, streptomycin 10 000 μg/mL, amphotericin B 25 μg/mL).
True negative controls:
yes
Remarks:
Mc Coy's
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Selected concentrations of test item solutions are placed in contact with the test system. Two independent tests are carried out.
- Assay 1: short-term treatment
- Without metabolic activation: 4 hours exposure followed by 18 hours of expression.
-With metabolic activation (S9-mix 10 % (v/v)): 3 hours exposure followed by 18 hours of expression.
- Assay 2: long-term treatment
- Without metabolic activation: 20 hours of exposition.

1. assay without metabolic activation
40 hours after the seeding, the complete cell culture is removed and replaced by:
Complete culture medium + vehicle (mc coy's) + test item (25 μg/mL - 10 μg/mL - 4 μg/mL - 1.6 μg/mL)

-Short-term treatment (Assay 1): 4 hours later at 37° C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is discarded and the cells washed twice with culture medium. 5 mL of fresh complete culture medium are added and the cells incubated, 18 hours at 37° C in a humidified atmosphere containing 5 % (v/v) CO2.
-Long-term treatment (Assay 2): the cells are incubated, 20 hours at 37° C in a humidified atmosphere containing 5 % (v/v) CO2.

2. assay with metabolic activation (Assay 1)
40 hours after the seeding, the complete cell culture is discarded, cells layer washed with culture medium and incubated with reaction mixture composed by culture medium supplemented with 10 % (v/v) S9-mix:S9 medium + vehicle (Mc Coy's) + test item (25 μg/mL - 10 μg/mL - 4 μg/mL - 1.6 μg/mL)
3 hours later at 37° C in a humidified atmosphere containing 5 % (v/v) CO2, the culture medium is discarded and the cells layer washed twice with culture medium. 5 mL of fresh complete culture medium are added and the cells layer incubated, 18 hours at 37° C in a humidified atmosphere containing 5 % (v/v) CO2.


Harvest and microscope slides preparation
At the end of the incubation period (18h and 20h), Colcemid® (Demecolcine SIGMA-D1925-RNBG7053) (0.15 μg/mL) is added in each flask. Cells are incubated at 37° C for 2.5 hours in a humidified atmosphere containing 5 % (v/v) CO2 in order to block the cell division in the metaphase stage, then collected:
– culture medium is removed
– cells layer washed once with PBS
– cells are detached (about 2 minutes at 37°C) using 0.5 mL trypsin (0.05 % (w/v) in Hank's balanced solution Ca2+ and Mg2+ free supplemented with 1 mM EDTA)
– then 4.5 mL of Mc Coy’s supplemented with 5 % (v/v) Fetal Calf Serum (FCS) are added
– 100 μL of cell suspension and 100μL of trypan blue solution at 0.2 % (w/v) in 0.15 M NaCl are added (incubation for 2 minutes).
– thereafter living cells (uncolored) are counted using an haemocytometer (Malassez cell) (LEMI SOP n° MB08/23).
– the remaining cell suspension is centrifuged (200 g, 6 min)
– hypotonic shock (KCl 0.075 M) at 37° C for 10 minutes
– fixation (2 to 3 x 5 min) using the Carnoy mixture (methanol: acetic acid, 3:1)
– spread on coded microscope slides
– stained using Giemsa stain at 0.4 % (w/v) in phosphate buffer (0.01 M, pH 6.8)
Metaphases are analyzed under a microscope (Zeiss), magnification x1000 for the detection of chromosomal aberrations, polyploidy and endoreduplications.

Evaluation criteria:
see below in the section 'any other information on materials and methods incl. tables'
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Executive summary:

According to the criteria of conclusion of the study protocol and OCDE 473, solutions obtained from the test item are not considered clastogenic in the test system used (CHO) in the conditions of the assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification