Alcohols, C6-8, ethoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2021 To 25 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 476

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

To evaluate the mutagenic potential of the test item quantitation of forward mutations at the Hprt locus of CHO AA8 cells was performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C6-C8-(even numbered, linear)-ethoxylated (<2,5 EO)

EC Number:

800-182-9

Cas Number:

1426148-68-6

Molecular formula:

R-O-R' , whereas R=C6-8-(even numbered, linear, saturated)-alkyl and R'=H or xEO wheras x=1-12, mean=2,3

IUPAC Name:

Alcohols, C6-C8-(even numbered, linear)-ethoxylated (<2,5 EO)

Test material form:

liquid

Details on test material:

Chemical Name (IUPAC): Alkyl polyglycol ether; Alcohols, C6-8-alkyl-(even numbered), ethoxylated (˂2.5EO)
CAS No.: 1426148-68-6
Physical appearance (with color): Clear liquid
Based on the qualitative and quantitative information on the composition, the sample used is representative of the boundary composition.

Method

Target gene:

Hprt locus of CHO AA8 cells.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

AA8 Celles

Details on mammalian cell type (if applicable):

CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. Batch of S9 homogenate was assessed for sterility, protein content (Modified Lowry assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.
One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.37 and 7.34.
The details of S9 homogenate and activation mixture used in the study are given below:
Details of S9 homogenate:
Date of preparation:
09/12/2021
Batch No.:
S9-XI/21
Date of characterization
Sterility check:
09/12/2021
Activity check:
10/12/2021
Protein estimation:
13/12/2021
Expiry date:
08/12/2023

Test concentrations with justification for top dose:

Based on precipitation results, 0.125, 0.25, 0.5, 1, and 2 µL/mL were used for initial cytotoxicity testing.
Based in cytotoxicity results, 0.125, 025, 0.5, and 1 µL/mL were used for gene mutation testing with the survival rate being > 10 % at 1 µL/mL.

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of vehicle: Based in solubility testing

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: tetra plate cultures, each tetra plate treatments were pooled into pre-labeled tube and centrifuged at 800 rpm for 10 mintes. Supernatant was discarded, and cell pellet was retained and resuspended in culture media. Each treatment replicate was plated in a triplicate with cell concentration of 200 cells/5 mL media in 25 cm² flasks and incubated at 37 +/- 1 °C with 5 +/- 1 % CO2 for 9 days.
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
Approximately 2×106 (Initial cytotoxicity test and Gene mutation test) cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% Penicillin and Streptomycin). Four additional flasks were seeded and kept for incubation along with flasks for treatment to determine cell count at the beginning of the treatment to determine the Adjusted Cloning Efficiency. The flasks were incubated at 37±1oC with 5±1% CO2 for 23 hours (Initial cytotoxicity test) and 23 hours and 15 minutes (Gene mutation test). Cells free of mycoplasma were used for the experiment.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2.
- Harvest time after the end of treatment (sampling/recovery times): 8 days

FOR GENE MUTATION:
Post expression period of 8 days of mutant phenotype, each replicate treatment cultures were pooled and sub cultured in quintuplicates at a density of 4×105 cells per 25 cm2 flask with culture media containing 10 μM of 6-Thioguanine and 200 cells /25 cm2 flask in triplicates without 6-Thioguanine for determination of cloning efficiency. Flasks were incubated at 37±1°C with 5±1% CO2 for 9 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 μL of vehicle/different concentrations of test item was added to tetra plate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2. For tests without exogenous metabolic activation, a volume of 100 μL of vehicle/different concentrations of test item was added to tetra plate culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 18 minutes at 37±1oC with 5±1% CO2. Post incubation period (Set 1 and 2), medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinized by adding trypsin-EDTA. Trypsinization was stopped by adding culture media followed by collecting the media with cells. Tetra plate treatments were pooled and collected in prelabelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded, and cell pellet was retained and resuspended in culture media.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells / 5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 9 days. Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually. Relative Survival Rate was used to determine cytotoxicity.

Evaluation criteria:

Acceptability Criteria
Acceptance of a test is based on the following criteria:
1 The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
2 Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
3 Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
4 Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
5 The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Interpretation of Results
A test chemical is clearly positive if, in any of the experimental conditions examined:
1 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2 The increase is concentration-related when evaluated with an appropriate trend test.
3 Any of the results are outside the distribution of the historical negative/vehicle control data.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
4 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
5 There is no concentration-related increase when evaluated with an appropriate trend test.
6 All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=(X+A)B
Where,
BIO-GNT 1805 Study Report Page 18 of 35
Y = transformed mutant frequency, X = observed mutant frequency
[Where X=No. of mutant colonies per replicateACE value×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance is significant at p < 0.05, Dunnett’s test will be conducted, comparing each treatment group and the positive control to the vehicle control.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

AA8

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 μL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 1 μL/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item was found miscible in DMSO at 200 μL/mL. Precipitation test was conducted at 0.125, 0.25, 0.50, 1 and 2 μL/mL concentrations. Post 3 hours and 45 minutes of incubation, no precipitation observed at any of the test concentrations. No change in pH was observed at the tested concentrations up to 2 μL/mL.
Based on precipitation results, 2 μL/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.125, 0.25, 0.5, 1 and 2 μL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 18 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10 % at 1 μL/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 1 μL/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 0.125, 0.25, 0.5 and 1 μL/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (4 hours and 15 minutes).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test.
Cytotoxicity as Relative Survival was 17.05 to 18.39% in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 1 μL/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.