Castor oil, sulfated, ammonium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

From 10 August to 04 October 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oils, fish, oxidized, bisulfited, sodium salts

EC Number:

307-037-4

EC Name:

Oils, fish, oxidized, bisulfited, sodium salts

Cas Number:

97488-98-7

IUPAC Name:

Oils, fish, oxidized, bisulfited, sodium salts

Details on test material:

- Name of test material (as cited in study report): FLL sample 3
- Physical state: viscous liquid
- Lot/batch No.: LS06921
- Expiration date of the lot/batch: 13 October 2010
- Storage condition of test material: room temperature in the dark
- Other: amber color
- All other template details: Not reported

Method

Target gene:

thymidine kinase, TK +/-, locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media: RPMI 1640 culture medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37 degrees C with 5% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/BNF S9 prepared from livers of male Sprague-Dawley rats weighing approximately 250g

Test concentrations with justification for top dose:

preliminary test (µg/ml): 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 3750
main test (µg/ml): 0, 75, 150, 300, 600, 700, 800, 900, 1000, 1100, 1200

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

ethylmethanesulphonate : Sigma batch 0001423147 for positive control in absence of metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Cyclophosphamide (CP) Acros batch A0277203 was used as the positive control in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with metabolic activation, 4 and 24 hours without metabolic activation
- Expression time (cells in growth medium): two days
- Fixation time (start of exposure up to fixation or harvest of cells): 52-72 hours

SELECTION AGENT (mutation assays): 4 µg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1 x 10^6 cells/ml

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, viability

OTHER EXAMINATIONS:
- Other: determination of mutation frequency

OTHER: not applicable

Evaluation criteria:

Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive.

Statistics:

The UKEMS statistical package was used to determine significant increases in mutant colonies.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity observed at highest tested concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the first study, a precipitate of test material was observed at and above 150 μg/ml in the absence of metabolic activation, and at and above 300 μg/ml in the presence of metabolic activation. In the second study, a precipitate of test material was observed at and above 150 µg/ml in both exposure groups.
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: the preliminary study showed a marked reduction in relative suspension growth % between 625 and 1250 µg/ml. In the subsequent mutagenicity test, the maximum dose level was limited by test material-induced toxicity. A precipitate was observed at and above 156.25 µg/ml in the 4-hour exposure groups, and at and above 78.13 µg/ml in the 24-hour exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: the positive and vehicle control responses were within historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Summary of results are shown in Tables 1 and 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of Results: Experiment 1

Treatment (µg/ml)	4-Hours without S-9			Treatment (µg/ml)	4-Hours with S-9
	% RSG	RTG	MF		% RSG	RTG	MF
0	100	1.00	86.67	0	100	1.00	109.06
75	114			75	97
150	111			150	105
300	113			300	102
600	99	1.00	132.66	600	94	0.94	140.40
700	95	1.11	84.16	700	74	0.79	133.39
800	91	1.03	103.56	800	78	0.87	117.68
900	89	0.87	109.87	900	74	0.87	95.67
1000	69	0.69	119.94	1000	46	0.41	113.54
1100	9	0.13	108.70	1100	4	0.04	179.53
1200	1			1200	2
Linear Trend = Not Significant				Linear Trend = Not Significant
EMS				CP
400	76	0.4	859.95	2	63	0.37	1185.45

 

Table 2. Summary of Results: Experiment 2

Treatment (µg/ml)	4-Hours without S-9			Treatment (µg/ml)	4-Hours with S-9
	% RSG	RTG	MF		% RSG	RTG	MF
0	100	1.00	114.51	0	100	1.00	161.92
75	95			75	112
150	89			150	112
300	87	0.96	100.01	300	108	1.14	117.09
600	56	0.59	131.16	600	96	1.10	101.92
700	40	0.49	147.69	700	92	1.16	87.96
800	36	0.40	160.16	800	78	0.86	107.66
900	28	0.30	177.82	900	52	0.57	105.15
1000	18	0.18	175.16	1000	10	0.08	116.36
1100	14	0.12	140.86	1100	0
1200	7			1200	0
Linear Trend = Not Significant				Linear Trend = Not Significant
EMS				CP
150	55	0.47	1668.52	2	94	0.74	669.89

 

** RSG = Relative Suspension Growth Rate

** RTG = Relative Total Growth

** MF = Mutation Frequency

Applicant's summary and conclusion

Conclusions:

The substance was tested for OECD 476. Under the experimental conditions the test item was considered to be non-mutagenic to L5178Y cells.

Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

 

Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at ten dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test material was selected following the results of a preliminary toxicity test and was 75 to 1200 μg/ml, in both the absence and presence of metabolic activation, for both the first and second experiments.

 

Results. The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. A precipitate of test material was observed at and above 150 μg/ml in the absence of metabolic activation, and at and above 300 μg/ml in the presence of metabolic activation in the first experiment. A precipitate of test material was observed at and above 150 μg/ml in both the absence and presence of metabolic activation in the second experiment. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

 

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.