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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From Sep. 4, 1985 to Sep. 14, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to a method comparable to OECD guideline 471 and conducted according to GLP. Deviations - strains E.coli WP2 or S. typhimurium TA102 were not included as recommended in the OECD Guideline. It is a read across study, hence maximum reliability rating of 2 assigned according to ECHA guidance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strains E.coli WP2 or S. typhimurium TA102 were not included as recommended in the guideline.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
A mixture of: hexyldioctylphosphineoxide; dihexyloctylphosphineoxide; trioctylphosphineoxide
IUPAC Name:
A mixture of: hexyldioctylphosphineoxide; dihexyloctylphosphineoxide; trioctylphosphineoxide
Constituent 2
Reference substance name:
A mixture of: hexyldioctylphosphineoxide; dihexyloctylphosphineoxide; trioctylphosphineoxide
EC Number:
403-470-9
EC Name:
A mixture of: hexyldioctylphosphineoxide; dihexyloctylphosphineoxide; trioctylphosphineoxide
IUPAC Name:
403-470-9
Details on test material:
-Name of test substance (as cited in study report): CYANEX® 923 Extractant
-Lot number: 860-12,13
-Physical state of the test substance: colorless mobile liquid
-Storage conditions of the test substance: room temperature
- Date of receipt: August 28, 1985

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats.
Test concentrations with justification for top dose:
30; 100; 300; 1,000 and 3,000 µg/plate
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: the test substance was found to be soluble in DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Anthramine
Details on test system and experimental conditions:
Test organism Storage and Maintenance: fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 mL of OXoid Nutrient Broth #2 and grown for approximately 10 h at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. After the 10 h incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer.

Negative and Positive Controls: tester strains were plated in triplicate with the appropriate solvent, both with and without metabolic activation, to obtain background lawn and revertant colony formation to serve as negative solvent controls. In order to validate the integrity of the test system, all tester strains were also run, in triplicate, with known positive response chemicals.

Preliminary Toxicity Screen: the preliminary toxicity screen for the Ames Assay performed without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, TA1538 and TAl00. The test compound was prepared to a concentration of 100 mg/mL and five different levels tested for toxicity.

Plate Incorporation Assay: top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 Ml/mL of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher IsotempR Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 mL aliquots of top agar solution, 0.1 mL of tester strain, and 0.1 mL of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.
Evaluation criteria:
A positive result was defined as a reproducible, dose-related increase in the number of histidine-independent colonies with at least one dose point inducing a mutant frequency value that is two-fold the solvent control value. Significance at the 95% confidence limit was determined by the program developed by Moore and Felton (1983). This program applies a linear regression analysis to the data points and any P value greater than 0.05 is considered significant. In addition, the greater the P value, the higher the probability that the data points fit a linear response. If the solvent control was within the 95% confidence limits of the historical mean for control values and the test chemical produced the highest increase equal to or greater than three times the solvent control value, the test chemical was considered positive. A negative result was defined as the absence of a reproducible increase in the number of histidine independent colonies.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Dose levels in a Preliminary Toxicity Screen were 100; 333; 1,000; 3,333 and 10,000 µg/plate. Strain TA1538 of Salmonella tyhimurium exhibited no inhibition of bacterial lawn growth at the levels tested. A reduced number of revertant colonies were observed in strain TA100 of Salmonella typhimurium at the 3,333 and 10,000 µg/plate. Based upon these findings, the top dose selected for the Plate Incorporation Mutation Assay was 3,000 µg/plate.

Any other information on results incl. tables

Table: Mean revertants per plate

 

a. Controls

 

 

Mean Spontaneous Revertants/Plate

Solvent control

Metabolic

activation

TA1535

TA1537

TA1538

TA98

TA100

DMSO

-

20 (SD.)7

16 (SD.)2

14 (SD.)6

33 (SD.)5

212 (SD.)8

DMSO

+

15 (SD.)3

15 (SD.)6

20 (SD.)2

54 (SD.)8

208 (SD.)18

Positive controls

 

 

 

 

 

 

Sodium Azide

-

1050 (SD.)84*

0 (SD.)0

0 (SD.)0

0 (SD.)0

910 (SD.)51*

9-Aminoacridine

-

0 (SD.)0

1364 (SD.)292*

0 (SD.)0

0 (SD.)0

0 (SD.)0

2-Nitrofluorene

-

0 (SD.)70

0 (SD.)0

177 (SD.)35*

452 (SD.)45*

0 (SD.)0

2-Anthramine

+

171 (SD.)4*

158 (SD.)21*

586 (SD.)76*

1296 (SD.)194*

1470 (SD.)12*

* Positive response

 

b. Test substance

 

 

Mean Total Revertant Colonies/Plate

Dose level (Test substance) µg/plate

Metabolic

activation

TA1535

TA1537

TA1538

TA98

TA100

30

-

22 (SD.) 8

15 (SD.) 3

16 (SD.)9

49 (SD.)7

208 (SD.)40

100

-

19 (SD.) 1

12 (SD.) 6

19 (SD.)5

38 (SD.)9

199 (SD.)11

300

-

15 (SD.) 6

9 (SD.) 4

14(SD.)6

37 (SD.)6

191 (SD.)12

1000

-

21(SD.) 10

6 (SD.) 2

15 (SD.)6

35 (SD.)5

166 (SD.)18

3000

-

22 (SD.) 2

8 (SD.) 3

12 (SD.)5

35 (SD.)5

147 (SD.)38

30

+

12 (SD.)3

15 (SD.)2

27(SD.)11

52 (SD.)6

149 (SD.)18

100

+

11 (SD.)6

16 (SD.)9

30 (SD.)9

57 (SD.)7

141 (SD.)18

300

+

12 (SD.)4

16 (SD.)2

26 (SD.)6

56 (SD.)6

137 (SD.)8

1000

+

13 (SD.)1

9 (SD.)2

26 (SD.)2

46 (SD.)7

129 (SD.)3

3000

+

11 (SD.)8

9 (SD.)3

23 (SD.)6

45 (SD.)7

129 (SD.)26

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the study conditions, the read across substance was considered non-mutagenic in this Salmonella typhimurium reverse mutation assay (Barfknecht TR, 1985).
Executive summary:

A study was conducted to evaluate the mutagenicity of the read across substance ‘A mixture of: hexyldioctylphosphineoxide; dihexyloctylphosphineoxide; trioctylphosphineoxide’ using a method similar to OECD Guideline 471, in compliance with GLP. The test substance was subjected to a preliminary toxicity screen at dose levels of 100, 333, 1,000, 3,333 and 10,000 µg/plate. Based upon the findings, the top dose selected for the plate incorporation mutation assay was 3,000 µg/plate. Test substance was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation at doses of 30, 100, 300, 1,000 and 3,000 µg/plate. The results were negative in all groups. All positive and solvent controls used in the evaluation of the test substance induced mean mutant frequency values which were within the acceptable range of mean historical data. Under the study conditions, the read across substance was considered non-mutagenic in this Salmonella typhimurium reverse mutation assay (Barfknecht TR, 1985).