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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay, test article did not meet the criteria for a potential mutagen.

 

In an in vitro TK mutation bioassy, the test article did not induce a biologically relevant increase in the mutation frequency in the, either in the presence or absence of a metabolic activation system (S9-mix).

 

In an in vitro chromosome aberration assay, the test article was not considered to be genotoxic when exposed to Chinese Hamster Ovary (CHO) cells either in the presence and absence of S9 metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March to 04 May, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes
Remarks:
US FDA (21 CFR Parts 58, 210, 211, and 820) Regulations
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test Article: Agent 447C; 1-octanol, reaction products with phosphorus oxide (P2Os), potassium salts (CAS 111062-42-1 ; EC 807-789-8)
Species / strain / cell type:
S. typhimurium, other: TA97A, TA98, TA100, TA1535
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S-9, rat liver
Test concentrations with justification for top dose:
5 μL, 1.6 μL, 0.5 μL, 0.16 μL, 0.05 μL/plate

The concentrations tested were based on the OECD 471 highest recommended concentration for non-cytotoxic compounds.
Vehicle / solvent:
The test article was diluted in sterile water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-0-phenylenediamine (tested without S-9 metabolic activation only), 2-aminofluorene (tested with S-9 metabolic activation only), 2-aminoanthracene (tested with S-9 metabolic activation only)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: the titers of each strain culture had concentrations of approximately 10E8 CFU/mL or higher

DURATION
- Exposure duration: The plates were incubated for growth at 37 ± 2°C for 48-72 hours.

NUMBER OF REPLICATIONS: 3 replicates for each test article or control were prepared.
Evaluation criteria:
Criteria for a Mutagen:
1) A reversion rate greater than 200% of the solvent control in strains TA97a and TA 100. A reversion rate greater than 300% of the solvent control in strains TA98,TA1535, and WP2.
2) Demonstration of a clear dose related response when dilutions are tested.

Criteria for a Non-Mutagen:
1) A reversion rate less than or equal to 200% of the solvent control in strains TA97a and TA 100. A reversion rate less than or equal to 300% of the solvent control in strains TA98, TA 1535, and WP2.
2) No dose related response when dilutions are tested.
Statistics:
The results were calculated using a validated computer program. Manual calculations may have differed slightly due to rounding. All results greater than 300 colony forming units (CFU) were considered estimates.
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest test article concentration (5 µL/plate) without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

The criteria for acceptance of the test and criteria for determination of a mutagen were:

1) Tested strains for phenotype verification and achieved the appropriate responses.

2) All chemical controls included in the test gave the appropriate responses.

               a) for TA97a: % of control results >200 qualify as a positive

               b) for TA98: % of control results >300 qualify as a positive

               c) for TA100: % of control results >200 qualify as a positive

               d) for TA1535: % of control results >300 qualify as a positive

               e) for WP2: % of control results >300 qualify as a positive

3) The reversion rates for each tester strain were within the historical ranges (from 2016)

Conclusions:
The test article concentrations of 5 μL, 1.6 μL, 0.5 μL, 0.16 μL, 0.05 μL/plate did not produce a two-fold or three-fold increase in the number of revertants nor produce a clear dose related response in any of the 5 tester strains (S. typhimurium tester strains TA97a, TA98, TA100, and TA1535, and E. coli test strain WP2). The spot tests showed no zone of increased reversion or of toxicity. In summary, the test article concentrations tested against the five strains did not meet the criteria for a potential mutagen.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2018 to 14 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
OECD 473
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2016
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Test material identity: Agent 447C; 1-Octanol, reaction products with phosphorus oxide (P2O5), potassium salts (CAS 111062-42-1; EG 807-789-8)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- CHO cells were obtained from ATCC and were certified mycoplasma-free.
- cell line has an average generation time of approximately 12-13 hours.
- cells were passed a maximum of 14 times at Nelson Laboratories.
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate; obtained from Moltox; prepared from 8-10 week old Sprague Dawley male rats injected intraperitoneally with Aroclor 1254.
Test concentrations with justification for top dose:
0.05 μL/mL, 0.016 μL/mL, and 0.005 μL/mL. Dose determination studies were performed to find concentrations that were compatible with the test system.
Untreated negative controls:
yes
Remarks:
USP Sterile Water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- incubated with Kaighn's Modification of Ham's F-12 Medium + 10% Fetal Bovine Serum (F12K10) until 40-60% confluent.
- Cell density at seeding (if applicable): Approximately 9.5x10^5 - 2.0x10^6 cells were seeded into 75 cm2 cell culture flasks.

DURATION
- Exposure duration:
For the non-activated system: flasks incubated for 16-18 hours at 37 ± 1°C with 5 ± 1% CO2.
For the metabolic activation system: flasks exposed for 3-4 hours at 37 ± 1°C with 5 ± 1% CO2. Cells then incubated at 37 ± 1°C with 5 ± 1% CO2 for 15-21 hours.
- Expression time (cells in growth medium): At the end of the exposure and expression periods the cells were arrested in metaphase with colcemid for 2 hours ± 30 minutes.

STAIN (for cytogenetic assays):
- chromosomes were stained with Giemsa stain

NUMBER OF REPLICATIONS: The negative control, positive control, and test articles were tested in duplicate.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were removed from the flasks and treated with a 75 mM KCI hypotonic solution and fixative. The cells were dropped onto clean and cold microscope slides. The chromosomes were stained with Giemsa stain and coverslips were secured to the slides with mounting medium. The cells were then examined microscopically for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: degree of discernable morphological cytotoxicity on a relative scale of 0 to 4.
Evaluation criteria:
- Cells with 20 ± 2 chromosomes, with minimal overlapping, were scored for aberrations.
- Cells scored based on number of aberrant cells. Total cells scored = 300.
- Cytotoxicity assessed via types of aberrations assessed included: gaps, simple breaks, complex aberrations (e.g., dicentric, quadradial, ring, interstitial deletion, complex rearrangement), and other (pulverized, >10).
Statistics:
Results were evaluated with the chi-square calculation using a validated spreadsheet. The critical value for the chi-square test is 3.841 at a confidence level of 95%.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
test article is not considered to be genotoxic when exposed to CHO cells
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
cytotoxicity data showed no significant reactivity to the test article solution when placed on CHO cells
Untreated negative controls validity:
valid
Positive controls validity:
valid

Test Method Acceptance Criteria:

The following conditions were met, thus the data was considered acceptable:

1. Positive results compared to the negative control were higher than the critical chi-square value of 3.841 at a confidence interval of 95%.

2. The average percent aberrations for the controls were within the historical control limits set in the test procedure(s).

Conclusions:
1-Octanol, reaction products with phosphorus oxide (P2O5), potassium salts is not considered to be genotoxic at concentrations of 0.05 µL/mL, 0.016 µL/mL, and 0.005 µL/mL when exposed to Chinese Hamster Ovary (CHO) cells both in the presence and absence of S9 metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar 2019 to May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Purity/Composition: UVCB; solid matter 41.4%
Target gene:
forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: American Type Culture Collection
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes from Trinova Biochem GmbH, Giessen, Germany from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)
- method of preparation of S9 mix: S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP, 4 μmol HEPES. The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 4% (v/v)
Test concentrations with justification for top dose:
Without activation: 1, 5, 10, 30, 60, 90, 100, 110 µg/mL
With activation: 1, 5, 10, 30, 60, 100, 120, 140 µg/mL
corrected for the % solid matter (41.4%)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water

- Justification for choice of solvent/vehicle: marketed as an aqueous solution
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
In the first experiment, the test item was tested up to concentrations of 110 μg/mL and 140 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours.

In the second experiment, the test item was tested up to concentrations of 170 μg/mL in the absence of S9-mix.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The cloning efficiency (CE) was then calculated as follows: CE = -ln P(0)/number of cells plated per well
The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture: RTG = RSG x RCE/100
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CE-day2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10E-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10E-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10E-6).
Statistics:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The suspension growth over the two-day expression period for cultures treated with Milli-Q Water was between 12 and 13 (3 hour treatment) and 83 and 90 (24 hour treatment).
Conclusions:
The test item is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the mutagenic potential of 1-Octanol, reaction products with phosphorus oxide (P2O5), potassium salts by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

The study procedures were based on the most recent OECD guideline.

The test item was a colourless-light yellowish liquid. A correction factor of 2.42 was used to correct for the % solid matter (41.4%). The vehicle of the test item was Milli-Q water.

In the first experiment, the test item was tested up to concentrations of 110 μg/mL and 140 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 14% and 8% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 170 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8%.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency.

In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under these experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The in vitro testing available did not meet the criteria for a classification as a potential mutagen. Therefore, the substance does not meet the criteria for classification as a germ cell mutagen according to CLP (EC 1272/2008 as amended).