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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Ames test no increase in mutations were observed in Salmonella typhimurum strains TA98, TA100, TA1535, and TA1537 and E. coli WP2 uvrA with and without metabolic activation up to 5000 µg/plate.

In a key in vitro chromosome aberration test in human lymphocytes, the test item did not induce structural aberration in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 365 and 295 µg/mL, respectively. 

In a gene mutation test ( HPRT) in mouse lymphoma L5178Y cells, the test item did not induce mutations in the absence and presence of metabolic activation (read-across: 2,4,7,9-Tetramethyl-5-decyne-4,7-diol).

Additionally, no hints for gentoxic activity were derived from QSAR models.

Link to relevant study records

Reference

Reference 1

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

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

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.)

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.

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.

please refer to attachment

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: purchased from Molecular Toxicology, Inc. (Boone, NC)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix
The liver metabolic activation system is prepared and used as described (Ames
et al., 1975; Maron and Ames, 1983). The 9000 × g liver supernatant fraction (S9) from phenobarbital/5,6-benzoflavone-treated male rats is obtained from Molecular Toxicology, Inc. (Boone, NC).

Test concentrations with justification for top dose:

Initial assay (plate incorporation):
25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate in triplicate with and without S9.
Confirmatory assay (preincubation):
25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strains
TA98, TA100, TA1535, and TA1537 and 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate
for strain WP2 uvrA in triplicate with and without S9.
Repeated Confirmatory assay with strain TA 100 (without metabolic activation) due to insufficient number of non-cytotoxic concentrations:
2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: two (plate incorporation and preincubation method)
A repeat was conducted in strain TA100 without metabolic activation due to an insufficient
number of non-cytotoxic concentrations per OECD

METHOD OF TREATMENT/ EXPOSURE:
-Overnight cultures used
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min (preincubation method)
- Exposure duration/duration of treatment: 48 h


FOR GENE MUTATION:
After the 2 day incubation period, plates were evaluated macroscopically for precipitation of test article and microscopically for thinning of the background lawn. The number of revertant colonies was counted by hand or with an automatic colony counter and then recorded. Plates that have a cytotoxic reduction in background lawn growth (>50% reduction in the background lawn) were not counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Methods and concentrations are according current OECD method and based on results of the pretest.

Evaluation criteria:

Criteria for Positive Response
The test article is considered positive for mutagenicity if it induces an increase of mean revertants per concentration with increasing test article concentration. The increases should be at least 2 times the vehicle control background frequency for strains with high spontaneous levels (i.e., TA100) and 3 times for those with low spontaneous levels (TA98, TA1535, and TA1537
and WP2 uvrA). These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.


Criteria for Negative Response
The test article is considered negative for mutagenicity if it does not induce a response which fulfills the above criteria.


Criteria for Equivocal Response
Cases which do not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results.

Statistics:

Mean colony counts and standard deviation

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 1000 µg/plate (repeat confirmatory assay)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 1000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initial Assay
The concentrations tested in the initial assay were 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate. Precipitates were observed at ≥ 2500 μg/plate in WP2 uvrA without metabolic activation; at ≥ 2500 μg/plate in strains TA98 and TA100 with and without metabolic activation; and at 5000 μg/plate in strain WP2 uvrA with metabolic activation and in strains TA1535 and TA1537 with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 2500 μg/plate in strains TA98 and TA1535 without metabolic activation and strains TA100 and TA1537 with and without metabolic activation.

Confirmatory Assay
The concentrations tested in the confirmatory assay were the test substance was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strains TA98, TA100, TA1535, and TA1537 and 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strain WP2 uvrA using the plate incorporation method. Precipitates were observed at ≥ 2500 μg/plate in strain TA1535 without metabolic activation and strain WP2 uvrA with and without metabolic activation; and at 5000 μg/plate in strain TA1535 with metabolic activation and in strains TA98, TA100, and TA1537 with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/plate in strain TA1537 with metabolic activation; at ≥ 1000 μg/plate in strain TA1537 with metabolic activation and in strain TA100 with and without metabolic activation; at ≥ 2500 μg/plate in strains TA98 and TA1535 with metabolic activation; and at 5000 μg/plate in strain TA1535 without metabolic activation.
The plate incorporation method was used to dose the confirmatory assay instead of the preincubation method, therefore, the assay was repeated. In the repeat confirmatory assay, the test item was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strains TA98, TA100, TA1535, and TA1537 and 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strain WP2 uvrA using the preincubation method. Precipitates were observed at ≥ 2500 in all strains with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/mL in strain TA100 without metabolic activation; at ≥ 1000 μg/mL in strain TA98 and TA1535 without metabolic activation and in strain TA1537 with and without metabolic activation; and at ≥ 2500μg/mL in strains TA98, TA100, and TA1535 with metabolic activation.
A repeat was conducted in strain TA100 without metabolic activation due to an insufficient number of non-cytotoxic concentrations per OECD. In the second repeat confirmatory assay, the test item was tested at 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/plate using the preincubation method. Precipitates were not observed in strain TA100 without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/mL without metabolic activation.

please refer to attachment

Conclusions:

The data from the vehicle and positive controls demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation.
Mean increases in the number of revertant colonies indicative of a positive response were not observed with the test substance in the S. typhimurium strains TA98, TA100, TA1535, and TA1537 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay. Therefore, the test substance is considered to be negative for inducing mutagenicity in this assay.

Executive summary:

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated was evaluated for mutagenic activity in the in vitro bacterial reverse mutation assay. Four tester strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and 1 Escherichia coli strain (WP2 uvrA) were used for mutagenicity testing.The test substance was prepared as a stock formulation in dimethylsulfoxide (DMSO) at concentrations up to 50 mg/mL for the initial, confirmatory, and repeat confirmatory assays and up to 10 mg/mL for the second repeat confirmatory assay. Mutagenicity testing was performed in triplicate at each concentration with and without a phenobarbital/5,6-benzoflavone-induced rat liver S9 metabolic activation system.

In the initial assay, the test substance was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate using the plate incorporation method. Precipitates were observed at ≥ 2500 μg/plate in WP2 uvrA without metabolic activation; at ≥ 2500 μg/plate in strains TA98 and TA100 with and without metabolic activation; and at 5000 μg/plate in strain WP2 uvrA with metabolic activation and in strains TA1535 and TA1537 with and without metabolic activation.
Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 2500 μg/plate in strains TA98 and TA1535 without metabolic activation and strains TA100 and TA1537 with and without metabolic activation.

In the confirmatory assay, the test substance was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strains TA98, TA100, TA1535, and TA1537 and 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strain WP2 uvrA using the plate incorporation method. Precipitates were observed at ≥ 2500 μg/plate in strain TA1535 without metabolic activation and strain WP2 uvrA with and without metabolic activation; and at 5000 μg/plate in strain TA1535 with metabolic activation and in strains TA98, TA100, and TA1537 with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/plate in strain TA1537 with metabolic activation; at ≥ 1000 μg/plate in strain TA1537 with metabolic activation and in strain TA100 with and without metabolic activation; at ≥ 2500 μg/plate in strains TA98 and TA1535 with metabolic activation; and at 5000 μg/plate in strain TA1535 without metabolic activation.

The plate incorporation method was used to dose the confirmatory assay instead of the preincubation method, therefore, the assay was repeated. In the repeat confirmatory assay, the test substance was tested at 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strains TA98, TA100, TA1535, and TA1537 and 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate for strain WP2 uvrA using the preincubation method. Precipitates were observed at ≥ 2500 in all strains with and without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/mL in strain TA100 without metabolic activation; at ≥ 1000 μg/mL in strain TA98 and TA1535 without metabolic activation and in strain TA1537 with and without metabolic activation; and at ≥ 2500μg/mL in strains TA98, TA100, and TA1535 with metabolic activation.

A repeat was conducted in strain TA100 without metabolic activation due to an insufficient number of non-cytotoxic concentrations per OECD. In the second repeat confirmatory assay, the test substance was tested at 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/plate using the preincubation method. Precipitates were not observed in strain TA100 without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was observed at ≥ 500 μg/mL without metabolic activation.

In all assays, criteria for a negative response were met for all tester strains with and without metabolic activation. The data from the vehicle and positive controls demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation.

These data support the conclusion that the test substance is negative for mutagenic activity in the S. typhimurium strains TA98, TA100, TA1535, and TA1537 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar precursors under similar conditions
- they share structural similarities with common functional groups: the substances start with an acetylene group as core structure; geminal hydroxyl groups on the alpha carbon atoms; distal to the geminal hydroxyl groups is an isobutyl group (methyl isopropyl); the target substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (1.3) is further functionalised with ethylene oxide and has an ethoxylation degree of 1.3; the source substance 2,4,7,9-Tetramethyl-5-decyne-4,7-diol, ethoxylated (3.8) has an ethoxylation degree of 3.8
- they have similar physicochemical properties and thus, show a similar toxicokinetic behaviour
- they are expected to undergo similar metabolism: oxidation of the terminal methyl groups to result in alcohol, aldehyde and finally the corresponding acid

Therefore, read-across from the existing toxicity, ecotoxicity, environmental fate and physicochemical studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Type of assay:

mammalian cell gene mutation assay

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Two independent experiments wre performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozyous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the asence and presence of metabolic activation (2% S9). In experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 -hour exposure group in the absence of metabolic activation.
The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 280 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 210 µg/ml in the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative non-mutagenic

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Additional information

Bacterial reverse mutation test

2,4,7,9-Tetramethyldec-5-yne-4,7-diol, ethoxylated was evaluated for mutagenic activity in the in vitro bacterial reverse mutation assay. Four tester strains of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and 1 Escherichia coli strain (WP2 uvrA) were used for mutagenicity testing. The data from the vehicle and positive controls demonstrated the validity and sensitivity of this test system for detecting chemical mutagens with and without metabolic activation.
Mean increases in the number of revertant colonies indicative of a positive response were not observed with the test substance in the S. typhimurium strains TA98, TA100, TA1535, and TA1537 and in the E. coli strain WP2 uvrA, with and without metabolic activation, under the conditions of this assay. Therefore,the test substance is considered to be negative for inducing mutagenicity in this assay.

 

Chromosome aberration test

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 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.

 

Gene mutation study in mammalian cells (read across and QSAR modelling; for read across justification please refer to Chapter 13) 

Based on the comparable chemical structure, physico-chemical, ectoxicological and toxicological properties a read across to the results of an in vitro gene mutation study in mammalian cells performed with the source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol is performed. Since the results of the newly performed Ames test and the cytogenicity study in human lymphoblasts derived with 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol, ethoxylated provide the same negative results for gene mutations and cytogenicity it was decided to conduct an analogy approach to the results derived with the source substance, 2,4,7,9-Tetramethyl-5-dcyne-4,7-diol. This read across is supported by the results of the applied QSAR models (DEREK, SARAH).

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material 2,4,7,9-Tetramethyl-5-decyne-4,7-diol on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and the Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9). In experiment 2, the cells were treated with the test material at up to eight dose levels using a 4 -hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 280 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 4.38 to 140 µg/ml in the absence of metabolic activation, and 17.5 to 210 µg/ml in the presence of metabolic activation.

Results:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of the test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

Justification for classification or non-classification

According to the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Part 3 Chapter 3.5 this substance is not causing concern to be mutgenetic/genetic toxic.