Soybean oil, reaction products with ethaneperoxoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1-23 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/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 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, reaction products with ethaneperoxoic acid

Cas Number:

693217-63-9

Molecular formula:

C59 H106 O11

IUPAC Name:

Soybean oil, reaction products with ethaneperoxoic acid

Test material form:

liquid: viscous

Details on test material:

The test article Agrol® Polyol, CAS No. 693217-63-9, was a hydroxylated soybean oil, manufactured by BioBased Technologies LLC. The Lot No. 09-3.6-ASO-010, assigned ISI Sample No. 101-149-1, was a clear, yellow, viscous liquid with minimum 99% purity and an expiry date of September 17, 2010. Agrol® Polyol had a specific gravity of 0.97 to 1.1 at 25°C. It was stored under nitrogen, in a dry place, in a tightly closed container at room temperature (15 to 30ºC) until dissolved in dimethyl sulfoxide (DMSO) for the experiments.

Method

Target gene:

S. typhimurium strains, TA98, TA100, TA1535, TA1537, are histidine auxotrophs (Maron, D.M. and Ames, B.N., 1983). The E. coli strain, WP2 uvrA, is tryptophan-deficient (Green, M.H.L. and Muriel, W.J., 1976).

References:
Green, M.H.L. and Muriel, W.J., Mutagen testing using TRP reversion in Escherichia coli. Mutat. Res. 38:3-32, 1976.

Maron, D.M. and Ames, B.N., Revised methods for Salmonella mutagenicity test. Mutat. Res. 113:173-215, 1983.

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 was purchased from Molecular Toxicology Inc., Boone, NC, U.S.A. and was stored at -80°C. It corresponds to the 9,000 x g fraction of liver homogenate from male Sprague-Dawley rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

For all strains, the concentrations tested in the plate incorporation test (with and without S9) and preincubation test (without S9) were 0, 0.062, 0.19, 0.56, 1.7, and 5.0 mg per plate.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Positive controls for experiments without S9 were sodium azide; 2-nitrofluorene, methyl methanesulfonate and 9-aminoacridine (9-AA). For experiments with S9, benzo[α]pyrene, 2-aminoanthracene and cyclophosphamide monohydrate were used.

Details on test system and experimental conditions:

S. typhimurium strains, TA98, TA100, TA1535, TA1537, are histidine auxotrophs (Maron, D.M. and Ames, B.N., 1983). The E. coli strain, WP2 uvrA, is tryptophan-deficient (Green, M.H.L. and Muriel, W.J., 1976). All strains were purchased as dried discs from Molecular Toxicology Inc., Boone, NC, U.S.A. and stored in a refrigerator between 2 to 8°C. Frozen permanent stocks were prepared from fresh cultures of the discs and stored at -80°C in the presence of 9 % DMSO.

Media
For daily growth of the tester strains, 2.5% Oxoid Nutrient Broth No. 2 was used (Oxoid Ltd., Basingstoke, Hampshire, England). Bottom agar plates for S. typhimurium were made of minimal glucose agar that was based on a standard formula: 2 % glucose, Vogel-Bonner medium E and 1.5% BactoTM agar (Becton Dickinson and Company, Sparke, MD, U.S.A.). Bottom agar plates for E. coli contained 1.44% agar, 0.38% glucose, 0.24% casamino acids, 0.23 µg per mL tryptophan and 23.9% v/v Davis Mingioli Salt Solution.

Top agar for the selection of S. typhimurium revertants was 0.6% BactoTM agar, containing 0.5% NaCl and supplemented with histidine and biotin to 50 µM each (Maron, D.M. and Ames, B.N. 1983). Top agar for the selection of E. coli revertants contained 0.7% agar only (Green, M.H.L. and Muriel, W.J., 1976).

Metabolic Activation

S9 was purchased from Molecular Toxicology Inc., Boone, NC, U.S.A. and was stored at -80°C. It corresponds to the 9,000 x g fraction of liver homogenate from male Sprague-Dawley rats treated with Aroclor 1254. Immediately prior to use, an S9 mix was constituted by the standard formula: 0.1 M NaH2PO4 / Na2HPO4, pH 7.4; 5 mM Glucose-6-phosphate; 4 mM NADP; 33 mM KCl; 8 mM MgCl2 and 10% rat liver S9 (Maron, D.M. and Ames, B.N., 1983).

Controls

The negative control was DMSO. Positive controls for experiments without S9 were aqueous solutions of sodium azide (NaAz) (CAS No.: 26628-22-8); and DMSO solutions of 2-nitrofluorene (2-NF) (CAS No.: 607-57-8), methyl methanesulfonate (MMS) (CAS No.: 66-27-3) and 9-aminoacridine (9-AA) (CAS No.: 52417-22-8). For experiments with S9, benzo[α]pyrene (B[α]P) (CAS No.: 50-32-8) and 2-aminoanthracene (2-AMA) (CAS No.: 613-13-8) were dissolved in DMSO; and cyclophosphamide monohydrate (CP) (CAS No.: 6055-19-2), in water. DMSO was purchase from Caledon Laboratories Ltd., Georgetown, ON, Canada and all other chemicals were purchased from Sigma-Aldrich Canada Ltd., Oakville, ON, Canada.

Preparation of Cultures

Master plates were prepared from frozen permanent stocks and were stored between 2 to 8°C for up to 2 months. The genotypes were confirmed (Appendix II). For S. typhimurium strains, the plates were made of minimal glucose agar plus 250 µM histidine, 3 µM biotin and, additionally for TA98 and TA100, 25 µg per mL ampicillin. For the E. coli strain, nutrient agar no. 2 plates were used. Overnight cultures were grown in Oxoid Nutrient Broth No. 2 at 37 ± 2°C with shaking at 210 rpm for 12 to 16 hours. For the E. coli strain WP2 uvrA, the overnight culture was diluted and incubated for approximately 2.5 hours to the desired density. The density of the cultures ranged from 0.1 to 2.0 x 109 colony-forming units per mL as measured by absorbance readings at 650 nm.

Preparation of Test Article
On each day, the test article was dissolved in DMSO at a concentration of 50 mg Agrol® Polyol per mL. Serial dilutions were performed in DMSO to 17, 5.6, 1.9, and 0.62 mg Agrol® Polyol per mL. These preparations were not corrected for purity (≥99% Agrol® Polyol).

The remainder of the test article preparations not used in treatment was stored at 15 to 30°C. Duplicate one mL aliquots of each preparation were sent to Investigative Science Incorporated for analysis by U.V. spectrophotometry using a validated method (2010-26 471 Method). The analytical results of the dosing formulations showed that the doses prepared were generally consistent with nominal concentrations for both June 11, 2010 and June 15, 2010 preparations except for marginally low recovery at the highest concentrations.

Treatment with Test Article
A plate incorporation test and a preincubation test were performed in the absence of S9 on June 11, 2010 and in the presence of S9 on June 15, 2010. In the plate incorporation test, the test components: 0.1 mL of a test article solution, 0.5 mL of 0.1 M phosphate buffer, pH 7.4, or 0.5 mL of S9 mix and 0.1 mL of an overnight bacterial culture and 2 mL of appropriate molten top agar were combined. The contents were mixed and layered onto the appropriate bottom agar. In the preincubation test, these test components were pre-incubated at 37°C for 20 minutes prior to adding the top agar and plating (Maron, D.M. and Ames, B.N., 1983). Negative and positive control tubes and plates were handled in the same manner. Triplicate plates were used for each dose level.

Incubation and Colony Counting

All plates were incubated at 37 ± 2°C for 48 to 72 hours. The numbers of revertant colonies were counted. The plates were also examined for the health status of the bacterial background lawn and possible precipitates of the test article. Results were recorded in tabular forms.

Evaluation criteria:

No additional information available.

Statistics:

The colony counts were transformed to normalize the data for statistical analysis. The one-sided Dunnett’s test was used to aid data analysis (Mahon, G.A.T., et al., 1989). All colony counts were compared to the historical data from this laboratory.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

For all strains with and without S9, the concentrations tested in the plate incorporation test were 0, 0.062, 0.19, 0.56, 1.7, and 5.0 mg per plate. All five concentrations used in the experiment were analyzable. Slight precipitate, which did not interfere with counting, was visible with the unaided eye only at the highest concentration of 5.0 mg per plate although slight precipitate was visible at 1.7 and 0.56 mg per plate under a microscope. No reduction in the background lawn was observed at any concentration nor was there any reduction in the number of resultant colony counts observed at the highest concentration.

The dose design of the plate incorporation test was repeated in the preincubation test. Again all five concentrations used in the experiment were analyzable. Precipitation in the preincubation test was similar to the precipitation observed in the plate incorporation test. Slight reduction of the background lawn was only observed at 5.0 mg per plate for all S. typhimurium strains in the absence of S9. All other concentrations for all the strains produced a normal background lawn both in the presence and absence of metabolic activation. There was no obvious reduction in the number of resultant colony counts observed at the highest concentration for all conditions.

The colony counts from the plate incorporation test are presented in Table 1. There was a clear overlap between the colony counts per plate of Agrol® Polyol (mean ± S.D.) and the upper limit for the concurrent negative control (mean + S.D.), for most tester strains with a few exceptions. Both TA100 with S9 at 0.56 mg per plate, and WP2 uvrA without S9 at 1.7 mg per plate exceeded this limit but there were no statistically significant increases in revertants (p>0.05). Although the number of TA100 revertant colonies at 0.56 mg per plate (162 ± 5) exceeded the range with 95% confidence of the historical data of 134 – 144, the historical data does show that occasionally the number of revertants exceeded the range.

E. coli WP2 uvrA at 1.7 mg/plate had a mean of 31 ± 2 revertant colonies per plate which exceeded the range of the historical data of 23 – 27. However, the historical data shows that occasionally the number of revertants exceeded this range.
.
The colony counts from the preincubation test confirmed the results of the plate incorporation test and are presented in Table 2. There was a clear overlap between the colony counts per plate of Agrol® Polyol (mean ± S.D.) and the upper limit for the concurrent negative control (mean + S.D.), for all tester strains.

The negative controls for each tester strain were similar to the historical negative control data. All concurrent positive controls induced at least 5 fold increase in colony counts per plate when compared to the corresponding negative controls (Tables 1 and 2) and were at levels similar to the historical positive control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Plate Incorporation Assay

Agrol Polyol mg per plate	Mean + S.D.
	TA98	TA100	TA1535	TA1537	E. coli WP2 uvrA
Without S9
0	22 + 5	114 + 9	14 + 2	10 + 4	26 + 2
0.062	21 + 4	105 + 5	16 + 1	11 + 1	25 + 1
0.19	21 + 6	108 + 2	13 + 6	9 + 3	31 + 4
0.56	19 + 4	107 + 6	11 + 2	7 + 3	22 + 1
1.7	19 + 6	103 + 9	14 + 2	10 + 4	31 + 2
5.0	23 + 6	115 + 5	12 + 5	9 + 2	25 + 7
2 -NF, 5 ug	928 + 178	N.A.	N.A.	N.A.	N.A.
NaAz, 5 ug	N.A.	1961 + 13	1681 + 92	N.A.	N.A.
9 -AA, 100 ug	N.A.	N.A.	N.A.	2377 + 48	N.A.
MMS, 1 ul	N.A.	N.A.	N.A.	N.A.	497 + 4
With S9
0	29 + 4	141 + 6	13 + 3	14 + 2	39 + 9
0.062	34 + 9	136 + 11	13 + 2	15 + 4	39 + 6
0.19	35 + 4	137 + 10	16 + 1	13 + 3	33 + 4
0.56	30 + 6	162 + 5	14 + 6	15 + 6	44 + 4
1.7	27 + 2	143 + 8	15 + 4	11 + 2	43 + 12
5.0	32 + 4	139 + 22	15 + 5	16 + 11	44 + 2
BP, 5 ug	612 + 11	1526 + 48	N.A.	252 + 36	N.A.
CP, 100 ug	N.A.	N.A.	100 + 18	N.A.	N.A.
2 -AMA, 100 ug	N.A.	N.A.	N.A.	N.A.	262 + 11

Note:9-AA,9-aminoacridine;2-AMA,2-aminoanthracene;2-NF,2-nitrofluorene;NaAz,sodiumazide; BP,benzo[α]pyrene;CP,cyclophosphamide;MMS,methylmethanesulfonate

N.A. Not Applicable

Table 2: Pre-Incubation Assay

Agrol Polyol mg per plate	Mean + S.D.
	TA98	TA100	TA1535	TA1537	E. coli WP2 uvrA
Without S9
0	20 +6	123 +11	17+2	10+3	26+4
0.062	21+4	96 +5	18+1	9 +3	23+4
0.19	23 +5	102 +19	15+3	10+3	26+5
0.56	22+2	100+2	18 +6	8+2	23+3
1.7	19+1	111+3	14+3	7 +1	30+1
5.0	18+3	93+8	14+4	7+5	27+8
2 -NF, 5 ug	1010+69	N.A.	N.A.	N.A.	N.A.
NaAz, 5 ug	N.A.	2019+57	1855+139	N.A.	N.A.
9 -AA, 100 ug	N.A.	N.A.	N.A.	1194+241	N.A.
MMS, 1 ul	N.A.	N.A.	N.A.	N.A.	851+58
With S9
0	33+7	151+15	16+3	11+5	43 +4
0.062	34+8	136+11	13+6	14+1	41+11
0.19	33 +5	141+4	12+3	15+2	36+6
0.56	27+5	143+11	16+6	16+3	47+5
1.7	28+6	148 +13	13+7	16+4	42+1
5.0	20+6	136+5	16+3	13+4	45+4
BP, 5 ug	683+11	1281+61	N.A.	232+8	N.A.
CP, 100 ug	N.A.	N.A.	335+20	N.A.	N.A.
2 -AMA, 100 ug					217+14

Note:as perTable1.

N.A. Not Applicable

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Agrol® Polyol was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain,WP2uvrA, under the test conditions.

Executive summary:

The test article, Agrol^® Polyol, was evaluated for its potential to induce point mutations in Salmonella typhimurium strains, TA98, TA100,TA1535, TA1537 and Escherichia coli strain WP2uvrA. The experimental design followed the “OECD Guideline for Testing of Chemicals – 471, Bacterial Reverse Mutation Test” (OECD, 1997).

 

Agrol^® Polyol, CAS No. 693217 -63 -9, was a hydroxylated soybean oil manufactured by BioBased Technologies LLC. The Lot No.09 -3.6 -ASO-010 was a clear, yellow, viscous liquid with minimum 99% purity and an expiry date of September 17, 2010. It was stored under nitrogen in a tightly closed container at 15 to 30ºC until dissolved in dimethylsulfoxide for the experiments.The concentrations of Agrol^® Polyol investigated were 0, 0.062, 0.19, 0.56, 1.7 and 5.0 mg per plate for all strains with and without the presence of S9. Triplicate plates were used for each dose level.

 

Both plate incorporation and preincubation experiments were performed in the presence and absence of S9. Throughout the study,slight precipitate was observed with the unaided eye at the highest concentration of 5.0 mg per plate. Slight reduction of the background lawn was only observed in the preincubation test at 5.0 mg per plate for all S.typhimurium strains in the absence of S9.

 

In the plate incorporation test both in the presence and absence of S9, the number of revertant colonies overlapped the concurrent negative control range with a few exceptions. Despite the exceptions, the test article did not produce any statistically significant increases in revertants (p>0.05), overlapped the historical negative control data for all strains and did not produce any dose-responses. The preincubation experiment confirmed the negative results of the plate incorporation experiment as the number of revertants were all within the ranges of the concurrent negative controls. All concurrent positive controls induced at least 5 fold increase in colony counts per plate when compared to the corresponding negative controls.

 

Thus, it was concluded that Agrol^® Polyol was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain,WP2uvrA, under the test conditions.