2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2021 - March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

EC Number:

500-022-5

EC Name:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated

Cas Number:

9014-85-1

Molecular formula:

variable

IUPAC Name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated

Constituent 2

Reference substance name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol , ethoxylated

IUPAC Name:

2,4,7,9-Tetramethyl-5-decyne-4,7-diol , ethoxylated

Test material form:

liquid: viscous

Method

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

Human venous blood will be drawn into sterile, heparinized vacutainers from a healthy donor,
non-smoker, between the ages of 21 - 35, no history of radiation therapy, chemotherapy or illegal
drug use. Donor must be free of any known viral infections.

Cytokinesis block (if used):

Colcemid® (0.1 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic Activation System
The 9000 × g liver supernatant fraction (S9) from phenobarbital/5,6-benzoflavone-treated male rats, glucose-6-phosphate and MgCl2-KCl in 0.1M phosphate buffer (Regensys “A”), and lyophilized nicotinamide adenine dinucleotide phosphate (NADP; Regensys “B”) were obtained from Molecular Toxicology, Inc. (Boone, NC). These reagents were combined to create the metabolic activation mixture (S9), maintained at 2°C to 8°C or on ice prior to use, on the day of testing.

Test concentrations with justification for top dose:

Range-Finding Cytotoxicity Assay
1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL

Chromosome Aberration Assay
- 3h Treatment without metabolic activation (with percent cytotoxicity): 194 (23%), 239 (25%) and 365 (55%) µg/mL
- 22h Treatment without metabolic activation (with percent cytotoxicity): 67.6 (1%), 103 (34%) and 215 (66%) µg/mL
- 3h treatment with metabolic activation (with percent cytotoxiciy): 239 (9%), 266 (33%) and 295 (48%) µg/mL

Vehicle / solvent:

DMSO (1% max.)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (max. 1%)

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate). duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
Cultures were prepared by placing 0.65 mL whole blood and 0.2 mL phytohemagglutinin per culture in RPMI-1640 complete media (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2mM L-glutamine or Glutamax) in 25 cm2 flasks with a final volume of 10 mL. These cultures were incubated at 36°C to 38°C in a humidified atmosphere with 4% to 6% CO2 for approximately 2 days before exposure to the test or control article.
- Test substance added in medium/1% DMSO

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
with metabolic actiovation: 3h
without metabolic activation: 3 and 22h
- Harvest time after the end of treatment (sampling/recovery times): 22h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays):Colcemid® (0.1 μg/mL), 1.5 to 2.5 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
At 22 hours, cells will be collected by centrifugation, resuspended in hypotonic solution and fixed (approximately 3:1 methanol:glacial acetic acid mix) before being stored in the refrigerator. Fixed cells will be washed twice more with fresh fixative prior to slide preparation. The final concentrated cell suspensions will be dropped onto clean, cold wet glass slides, dried prior to staining with DiffQuik solution at room temperature, and coverslipped. At least 2 slides will be prepared for each culture.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
Cytotoxcity: least 1000 cells from each culture
Chromosome Aberration: least 300 metaphase cells (150 from each culture) or ≥ 50 aberrant cells (≥ 25 from each culture) from each test article concentration or control; Numerical aberrations (endoreduplication and polyploidy) were evaluated from 400 cells from each concentration
(200 from each culture).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
A standard form was used to record chromatid breaks, deletions, exchanges, rings, dicentrics, and other changes in chromosomal morphology. In addition, chromatid or chromosome gaps and uncoiled chromosomes were recorded but not included in the aberrations frequency because they are not considered to represent true breaks.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Rationale for test conditions:

according to current OECD guideline and based on results of pretest

Evaluation criteria:

Assay Acceptance Criteria
Each treatment condition (e.g., with or without metabolic activation, short or long incubations) was considered independent with regard to assay acceptance and was repeated independently as necessary to satisfy the acceptance criteria.

Acceptable Controls
The positive control results for each treatment condition had to be statistically significant (p ≤ 0.05) when compared with the relevant vehicle controls. In addition, the vehicle and positive control results should be comparable to relevant historical control data.

Criteria for Positive Response
As a general guideline, a test article will be considered positive for inducing chromosomalaberrations if a significant increase (p ≤ 0.05) in the mean percentage of cells with chromosomal aberrations is observed at 1 or more dose levels. If a significant increase is seen at 1 or more dose levels, a dose-response should be observed. A response will be considered statistically significant for dose-response trend in the Cochran-Armitage test if p ≤ 0.05. At least 1 concentration should be associated with an increase that is outside the historical control range of the vehicle control.

Criteria for Negative Response
The test article will be considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤ 0.05) is observed in the mean percentage of cells with chromosomal aberrations at any of the test concentrations and there is no concentration-related increase when evaluated in the Cochran-Armitage test. All results should be comparable to the historical control range of the vehicle control.

Criteria for Equivocal Response
Cases which do not clearly fit into the positive or negative criteria may be judged equivocal (e.g., a response is statistically significant but not dose dependent). In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results.

Statistics:

The test article was considered negative for inducing chromosomal aberrations if no statistically significant increase (p ≤ 0.05) is observed in the mean percentage of cells with chromosomal aberrations at any of the test concentrations and there is no concentration-related increase when evaluated in the Cochran-Armitage test. All results should be comparable to the historical control range of the vehicle control.

Results and discussion

Test results

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity and Test System Evaluations
The test item concentrations tested in the range-finding assay were 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL, up to the highest concentration recommended by OECD Guideline No. 473. Precipitates were observed at 500 µg/mL in in the 22-hour treatment without metabolic activation the end of the test article treatment. Changes in the pH (as indicated by color change of the media) were not observed in any treatment at the end of test article treatment.
Based on results of the range-finding assay, the concentrations tested in the aberration assay were at 194, 215, 239, 266, 295, 328, 365, 405, 450, 500 μg/mL for the 3 hour treatment without metabolic activation; at 60.8, 67.6, 75.1, 83.4, 92.7, 103, 114, 127, 141, 174, 215, and 266 μg/mL for the 22 hour treatment without; and at 60.5, 75.1, 103, 114, 127, 141, 157, 174, 194, 215, 239, 266, 295, 328 μg/mL for the 3 hour treatment with metabolic activation. Precipitates were not observed at the end of test article treatment in any treatment condition. Changes in the pH (as indicated by color change of the media) were not observed in any treatment at the end of test article treatment.

Chromosome Aberration Evaluation
The highest concentration chosen for evaluation of chromosome aberrations were from cultures with cytotoxicity at or near the target range of 50% to 60% or the lowest concentration tested at which precipitates were observed. The low and middle concentrations evaluated for chromosome aberrations displayed minimal or intermediate cytotoxicity relative to the respective high concentration.
Data from the vehicle and positive controls were comparable to expected historical control ranges and yielded expected results. These results demonstrated the validity and sensitivity of the test system for detecting chemical clastogens in the presence and absence of metabolic activation.

3-Hour Treatment without Metabolic Activation
In the 3-hour treatment without metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity): 194 µg/mL (23%), 239 µg/mL (25%), and 365 µg/mL (55%). No statistically significant test article-related increase in structural chromosome aberrations (the percent of cells with aberrations or the percent of cells with > 1 aberration) was noted. In addition, no increase in numerical aberrations (percent of cells with polyploidy and/or endoreduplication) was observed compared to vehicle control samples.

22-Hour Treatment without Metabolic Activation
In the 22-hour treatment without metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity): 67.6 µg/mL (1%), 103 µg/mL (34%), and 215 µg/mL (66%). No statistically significant test article-related increase in structural chromosome aberrations (the percent of cells with aberrations or the percent of cells with > 1 aberration) was noted. In addition, no increase in numerical aberrations (percent of cells with polyploidy and/or endoreduplication) was observed compared to vehicle control samples.

3-Hour Treatment with Metabolic Activation
In the 3-hour treatment with metabolic activation, the concentrations selected for chromosome aberration analysis were as follows (with percent cytotoxicity): 239 µg/mL (9%), 266 µg/mL (33%), and 295 µg/mL (48%). No statistically significant test article-related increase in structural chromosome aberrations (the percent of cells with aberrations or the percent of cells with > 1 aberration) was noted. In addition, no increase in numerical aberrations (percent of cells with polyploidy and/or endoreduplication) was observed compared to vehicle control samples.

Any other information on results incl. tables

please refer to attachment

Applicant's summary and conclusion

Conclusions:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated was considered negative for inducing structural aberrations in HPBL with or without metabolic activation under the conditions of this test system. In addition, no increases in numerical aberrations (polyploidy or endoreduplication) were observed in treated cultures

Executive summary:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated was evaluated for the potential to induce chromosome aberrations in human peripheral blood lymphocytes during short (3-hour) and long (22-hour) incubations with or without an exogenous metabolic activation system.

The test item was prepared as a stock formulation in DMSO at target concentrations up to 200 mg/mL in the range-finding assay, up to 50.0 mg/ml in definitive Trial 1, and up to 32.8 mg/mL in definitive Trial 2. Human peripheral blood lymphocytes cultures were treated with the test article, positive control, or vehicle control in the presence and absence of phenobarbital/5,6‑benzoflavone-induced rat liver S9 microsomal fraction. The DMSO concentration in the culture medium was 1% v/v. Concentrations tested in the range-finding assay ranged from 1.95 to 2000 µg/mL, up to the highest concentration recommended by current OECD Guideline No. 473. Precipitates were observed at 500 µg/mL in in the 22-hour treatment without metabolic activation the end of the test article treatment. Based on the results of the range-finding assay, concentrations used during the chromosome aberration assay ranged from 194 to 500 µg/mL for the 3-hour treatment without metabolic activation, 60.8 to 266 µg/mL for the 22-hour treatment without metabolic activation, and 60.8 to 328 µg/mL for the 3-hour treatment with metabolic activation.

The concentrations selected for evaluation of chromosome aberrations in the aberration assay were based on cytotoxicity and are as follows (with percent cytotoxicity): a) 3‑hour treatment without metabolic activation, 194 µg/mL (23%), 239 µg/mL (25%), and 365 µg/mL (55%); b) 22‑hour treatment, 67.6 µg/mL (1%), 103 µg/mL (34%), and 215 µg/mL (66%); and c) 3-hour treatment with activation, 239 µg/mL (9%), 266 µg/mL (33%), and 295 µg/mL (48%). These cultures, along with the vehicle and one concentration of positive control for each treatment condition, were analyzed for aberrations. Structural chromosome aberrations were scored for each concentration from a total of 300 metaphase cells or ≥50 aberrant cells. Numerical aberrations were evaluated in 400 metaphase cells per concentration.

No statistically significant differences in the percent of cells with structural chromosome aberrations or the percent of cells with greater than one aberration were noted under any assay condition. In addition, there was no statistically significant test article-related increase in numerical aberrations (polyploidy or endoreduplication) in any treatment compared to the vehicle controls. The data from the vehicle and positive control substances demonstrated the validity and sensitivity of this test system.

The test substance was considered negative for inducing structural aberrations in human peripheral blood lymphocytes with or without metabolic activation under the conditions of this test system. In addition, no statistically significant increases in numerical aberrations (polyploidy or endoreduplication) were observed in treated cultures.