Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 December 2017 to 09 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

In accordance with the OECD guideline for testing of chemicals No. 471 “Bacterial Reverse Mutation
Test”, adopted on 21st July 1997.

Deviations:

no

Principles of method if other than guideline:

- Principle of test:
- Short description of test conditions:
- Parameters analysed / observed:

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

EC Number:

947-922-8

Molecular formula:

n.a. (UVCB)

IUPAC Name:

Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

Test material form:

liquid

Details on test material:

clear yellowish liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:Clariant Produkte (Deutschland) GmbH,
- Expiration date of the batch: 2019-02-22
- Purity : 98.1%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility a test substance in the vehicle: Dimethly sulphoxide


OTHER SPECIFICS:

Method

Target gene:

Histidine Locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

1 mL of S9 homogenate was mixed with 9 mL co factor

Test concentrations with justification for top dose:

On the basis of test item solubility, precipitation and initial cytotoxicity test 2 µL/plate was considered as the highest test concentration for mutation assay and other concentrations tested are 0.02, 0.06, 0.20, 0.63 µL/plate

Vehicle / solvent:

The test item was miscible in dimethyl sulphoxide at 50 µL/mL- Vehicle(s)/solvent(s) used:Dimethyl sulphoxide
- Justification for choice of solvent/vehicle:The test item was miscible in dimethyl sulphoxide at 50 µL/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in

DURATION
- Preincubation period: 30 Minutes

NUMBER OF REPLICATIONS: Triplicates

CYTOTOXICITY :cytotoxicity by lawn evaluation & mutation assay by counting revertant colonies.

Rationale for test conditions:

Not applicable

Evaluation criteria:

The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.

Statistics:

Not applicable

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water
- Precipitation: The test item resulted in mild precipitation at 5 and 4 µL/plate, minimal precipitation at 3 µL/plate and no precipitation up to 2 µL/plate


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Plate incorporation method (WITH S9)
Tester Strain TA98 TA100 TA1535 TA 537 TA102
Mean 412. 0 385.5 143.2 118.2 606.2
±SD 21.7 20.9 9.6 8.7 27.6
Min 280 306 117 100 510
Max 440 419 292 149 670

Plate incorporation method (WITHOUT S9)
Tester Strain TA98 TA100 TA1535 TA 537 TA102
Mean 399.7 378.3 145.0 120. 2 613.4
±SD 36.2 23.9 12.6 8.5 22.6
Min 310 311 112 103 589
Max 428 446 190 158 694

Pre incubation method (WITH S9)
Tester Strain TA98 TA100 TA1535 TA1537 TA102
Mean 410.1 386.0 144.2 117.1 606.5
±SD 18.2 22.0 11.3 6.5 26.7
Min 290 298 103 102 508
Max 428 425 187 128 670

Pre incubation method (WITHout S9)
Tester Strain TA98 TA100 TA1535 TA1537 TA102
Mean 404.0 379.1 146.5 118.7 616.0
±SD 29. 0 23.7 14.5 5.5 25.9
Min 314 317 110 105 590
Max 428 450 200 134 699

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from the study, it is concluded that the test item, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration 2 µL/plate under the test conditions

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test as per the OECD guidelinefor testing of chemicalsNo. 471, “Bacterial Reverse Mutation Test”, adopted on21^stJuly 1997.

On the basis of test item solubility and precipitation tests, the initial cytotoxicity test was performed at 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5µL/plate. Initial cytotoxicity test was performed withSalmonella typhimuriumTA100 both in the presence and absence of metabolic activation system.

The tester strain,Salmonella typhimuriumTA100 treated with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. Those concentrations are graded as 1+(extremely reduced lawn) for 4 and5µL/plate,2+(moderately reduced lawn) for3µL/plate and 3 + (Slightly reduced lawn)for 2 µL/platewhen compared to vehicle control.

On the basis of cytotoxicity results 2 µL/plate was considered as the highest test concentration for mutation assay.

The test concentrations tested in the mutation assay were selected based on the results of solubility, precipitation and initial cytotoxicity test. The two independent trials (trial 1 and 2) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. In mutation assay the test item was tested at the concentrations of 0.02, 0.06, 0.20, 0.63 and 2µL/plate. Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

The tester strains used in the mutation assay wereSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537.

Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.

The number of revertant colonies in the positive controls resulted in 2.2 to 14.7 fold increase under identical conditions.