Dehydrated sorbitol, C18 (unsaturated) fatty acid esters, ethoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Sorbitan monolaurate, ethoxylated, < 2.5 EO (CAS 9005-64-5). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan monolaurate, ethoxylated

EC Number:

500-018-3

EC Name:

Sorbitan monolaurate, ethoxylated

Cas Number:

9005-64-5

Molecular formula:

Molecular formula cannot be given as substance is a mixture.

IUPAC Name:

Sorbitan monolaurate, ethoxylated (1-6.5 moles ethoxylated)

Test material form:

liquid: viscous

Details on test material:

- Name of test material: PC-2012-412
- Molecular formula: UVCB
- Physical state: yellow viscous liquid
- Analytical purity: 100%
- Batch No.: ES61C86614
- Expiration date of the batch: 05 January 2014
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Basic medium
RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium
For 3 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum.
For 24 hour exposure:
Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

First experiment:
Without S9-mix: 0.3, 1, 3, 10, 33, 100, 125, 150, 175, 200, 225, 250 and 275 μg/mL (analysed concentrations: 0.3, 1, 3, 10, 33, 100 and 125 μg/mL)
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 33, 100, 200 and 300 μg/mL

Second experiment:
Without S9-mix: 0.3, 1, 3, 10, 33, 66, 100, 110, 120, 130, 140 and 150 µg/mL (analysed concentrations: 0.3, 3, 10, 33, 100, 150 and 190 μg/mL)
With 12% (v/v) S9-mix: 0.3, 1, 3, 10, 33, 100, 200, 300 and 350 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (test substance), Hanks’ balanced salt solution (positive controls)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

-S9: methylmethansulfonate (15 and 5 μg/mL for the 3 and 24 hours treatment period, respectively); +S9: cyclophosphamide (10 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h (first experiment), 3 and 24h (second experiment)
- Selection time (if incubation with a selection agent): 48h
- Fixation time (start of exposure up to fixation or harvest of cells): after 3 to 4 days, depending on exposure

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two independent experiments

NUMBER OF CELLS EVALUATED: 10E+006 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; relative survival

Evaluation criteria:

Acceptability of the assay
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls was between 65 and 120%. An acceptable number of surviving cells could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E+006 survivors and ≤ 170 per 10E+006 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of methylmethanesulfonate should not be below 500 per 10E+006 survivors, and for cyclophosphamide not below 700 per 106 survivors.

Data evaluation and statistical procedures
In addition to the statistical criteria, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency (MF) of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reached a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the exposure medium at concentrations of 100 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10E+006 cells (10E+006 cells/mL for 3 hours treatment) or 5 x 10E+006 cells (1.25 x 10E+005 cells/mL for 24 hours treatment) with different test substance concentrations increasing with approximately half log steps. Since the test substance was poorly soluble in exposure medium, the highest tested concentration was 2500 μg/mL exposure medium. Cell cultures were exposed to the test substance in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted and subcultured again for another 24 hours before the cells were counted. The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10E+005 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

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 I - 3 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (Ethanol)	101	100	56	1
0.3	79	83	67	1.20
1	98	108	50	0.89
3	93	112	54	0.96
10	97	110	49	0.88
33	84	83	64	1.14
100*	104	95	47	0.84
125*	93	53	46	0.82
MMS, 15	77	62	811	14.48

MMS: methylmethanesulfonate

*: precipitation

Table 2: Experiment I - 3 h exposure - With Metabolic Activation (8% S9)

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	100	100	59	1
0.3	111	93	80	1.36
1	88	67	79	1.34
3	98	82	93	1.58
10	80	79	88	1.49
33	95	71	84	1.42
100*	95	71	100	1.69
300*	139	52	127	2.15
CP, 10	50	26	1165	19.75

CP: cyclophosphamide

*: precipitation

Table 3: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (Ethanol)	100	100	53	1
0.3	101	114	93	1.75
3	101	102	81	1.53
10	81	80	100	1.89
33	118	90	66	1.25
100*	118	67	71	1.34
150*	95	53	85	1.60
190*	121	40	88	1.66
MMS, 5	86	65	840	15.85

MMS: methylmethanesulfonate

*: precipitation

Table 4: Experiment II - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	100	100	85	1
0.3	99	122	80	0.94
3	89	99	50	0.59
10	93	101	81	0.95
33	90	101	100	1.18
100*	70	66	102	1.2
200*	83	82	91	1.07
300*	102	94	77	0.91
350*	75	66	114	1.34
CP, 10	48	31	1093	12.86

CP: cyclophosphamide

*: precipitation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative