reaction product of: saturated, monounsaturated and multiple unsaturated long-chained partly estrified alcohols of vegetable origin (Brassica napus L., Brassica rapa L., Helianthus annuus L., Glycine hispida, Gossypium hirsutum L., Cocos nucifera L., Elaeis guineensis) with O,O-diisobutyldithiophosphate and 2-ethylhexylamine and hydrogen peroxide

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:

July 20 to August 3, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline, but did not includ an E. coli strain.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-630-5

EC Name:

-

Molecular formula:

Not applicable

IUPAC Name:

reaction product of Z-9-octadecen-1-ol and O,O-diisobutyl hydrogen dithiophosphate

Details on test material:

- Name of test material (as cited in study report): BECROSAN 6920
- Physical state: BECROSAN 6920
- Lot/batch No.:4567
- Storage condition of test material:room temperature, protected from light
- Expiration date of the lot/batch: December 1993

Method

Target gene:

Not required

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Range-finding test
Concentrations ranging from 10 to 10 OOO pg per plate were employed in the preliminary toxicity test.

Experiment 2: Main test
The frrst main test (4% S9) employed the following concentrations: 8,40,200,1 OOO and 5 OOO pdplate.
The second main test was performed using the same concentrations as in the first main test. The S9 concentration was increased to 10%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The solvent was acetone.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 10 ug/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 50 ug/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

no

Positive controls:

yes

Remarks:

Concurrent

Positive control substance:

other: 4-Nitro-l,2-phenylene diamine, 10 ug/plate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (3 ug/plate),

Evaluation criteria:

The following criteria were used for the acceptance of an assay:
- The negative controls had to be within the expected range as defined by published data (Maron and Ames 1983).
- The positive controls had to show sufficient effects as defined by the laboratory's experience.
- The titer determination has revealed a sufficient bacterial density in the susperision.

Assessment of mutagenicity and bacteriotoxicity
A reproducible and dose-related increase of mutant counts for at least one strain is considered positive. For TA 98 and TA 1535 a twofold increase of revertants compared to the negative controls should be reached, whereas for TA 1537 a threefold increase should be attained. For TA 100 a 1.5-fold increase is regarded as an indication of potential mutagenicity. Otherwise the results are considered to be negative.
The criterion for a biologically significant bacteriotoxic effect is a reduction in the number of colonies/plate or revertants/plate or in background growth by
more than 50% relative to the respective negative control.

Statistics:

NDA

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary test

No mutagenic and bacteriotoxic effects were observed at any concentrations.

The total number of viable cells, the number of revertants and the background growth were in the range of biological variability.

The test article precipitated in the range from 1000to 10000 ug/plate. This effect was observed either after addition of the test article solution to the top agar or during the evaluation of the test plates.

 

Main test I (4% S9)

The results of the first main test in the concentration range of 8 to 5000 ug/plate using the four bacterial strainsTA1535,TA1537,TA 98andTA100, are as follows:

• With test organismTA98 an increased count of revertants as an indication for a possibly mutagenic effect was observed at 5000 ug/plateinthe absence of metabolic activation
• No evidence of a biologically significant mutagenic effect was found with the test organimsTA1535,TA1537 andTA100.
• No bacteriotoxic effects were caused at any concentration.
• The test article precipitated in the range from 1000to 5000 ug/plate.

 

Main test II(10%S9)

The same concentrations were used as in main test I. TheS9concentration was increased to 10%. The resultsareas follows:

• No significant mutagenic effect was observed. Neither with nor without S 9 mix was a biologically relevant or a dose-related increase of the reversion rates observed.when compared to the negative control (solvent). The result of the first main test withTA98 without metabolic activation was not reproducible.
• The absence of bacteriotoxic effects and the precipitation of the test article in the range of 1OOOto 5000 ug/plate was confirmed.

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.

Executive summary:

INTRODUCTION

The mutagenic potential of the test article was assessed in the Salmonella / microsome test as described by Ames et al. (1973a, 1975) and Maron and

(1983).

The Salmonella / microsome test is an in vitro screening method to detect point mutations caused by chemicals. As tester strains, histidine auxotrophic mutants of Salmonella typhimuriumareused. To demonstrate the induction of point mutations, the reversion ratetoprototrophy in negative controls and treated groups is determined. If this rate increases sufficiently in the treated groups, a mutagenic effect is assumed.

Possible effects of mammalian metabolismaresimulated in this test by the addition of the 9000 x g fraction of homogenized mammalian livers and supplementary cofactors (which constitute the "S9 mix"). It is possible to examine .chemicals for intrinsic mutagenic activity in this test. The Ames test can also detect potential carcinogens with a reasonable level of reliability (Ames 1979, Andxews et al. 1978, Clayson 1980, Glatt et al. 1979, McCann and Ames 1976,Rinkusand Legator 1979 and Zeiger 1987).

SUMMARY

The test articleBECROSAN 6920was investigated in the Salmonella/ microsome test for point mutations using four Salmonella typhimuriumLT2mutants. These tester strains were the histidine auxotrophic strainsTA 1535, TA 1537, TA 98andTA 100.

Concentrations ranging from10to 10,000ug per plate were employed in the

preliminary toxicity test. The solvent was acetone.

Nobacteriotoxic effects were observed. The total number of viable cells, the count of revertants and the background growth were within the range of biological variability.

On the basis of these results, the frrst main test(4% S9)employed the following concentrations:8, 40, 200, 1000and5000 ug/plate.

No evidence of biologically significant mutagenic activity ofBECROSAN 6920was found with three of the four test organisms. WithTA 98a slightly increased count of revertants, indicating a possible mutagenic effect, was observed in the absence of metabolic activation at the highest test concentration of5000 ug/plate (Q:2.0).

The second main test was performed using the same concentrations as in the first main test. TheS9concentration was increased to10%.No evidence of a biologically significant mutagenic effect was found. Neither with nor without metabolic activation(10% S9)was a biologically relevant or dose-related increase in reversion rates observed, in comparison with the negative control (solvent). The result with test organismTA 98regarding the increased count of revertants was not reproducible.

In order to clarify the result of first main test with test organismTA 98,a repetition test(4% S9)with smaller intervals between the test concentrations (1000,2000,3000,4000and5000ug/plate) was performed.Asin the main testE,the increased count of revertants with test organismTA 98in the absence of metabolic activation was not observed.

Thus for TA 98, the criterion for existence of mutagenic activity, dose-relation and reproducibility, were also not fulfilled and a mutagenic effect could be excluded.

Summarizing, bacteriotoxic effects were not observed at any concentration.

Test article precipitation was observed either after addition to the top agar or during the evaluation in the range from 1000to 10000ug/plate.

The sensitivity of the test system was demonstrated by the marked mutagenic effects exerted on each strain by 2 of the following positive controls:9-aminoacridine hydrochloride, sodium azide, 4-nitro-1 ,a-phenylene diamine and 2-aminoanthracene.

Insummary,it may be concluded that the test article BECROSAN 6920 caused no mutagenic effect at concentrations ranging from 8 to5000 ug/plate.