1-Octadecanamine, reaction products with 1,1'-methylenebis[4-isocyanatobenzene] and 1-octanamine

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2017 - 28 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: white powder
- Test item storage: at room temperature

Method

Target gene:

Thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) 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).

Test concentrations with justification for top dose:

The test item precipitated in the exposure medium at concentrations of 12.5 µg/ml and above. The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 50 µg/mL.
- Dose-range finding test (with and without S9; cytotoxicity test): 3.13, 6.3, 12.5, 25 and 50 µg/mL
- Experiment 1 (with and without S9) and experiment 2 (without S9): 0.1, 0.2, 0.4, 0.8, 1.6, 3.1, 6.3 and 12.5 µg/mL.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: as recommended in OECD test guideline 490

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL

DURATION
- Exposure duration:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT: 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- test concentrations: 1 per test concentration
- positive control: 1
- solvent control: 2

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

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 mutation frequency of more than the mutation frequency in the 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 the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls 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 10^6 survivors and = 170 per 10^6 survivors.
c) The suspension growth 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 10^-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 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING STUDY
- The test item precipitated in the exposure medium at the test item concentration of 12.5 µg/mL and above.
- Both in the absence and presence of S9-mix, in the 3-hour exposure period, no toxicity in the relative suspension growth was observed up to a test item concentration of 50 µg/mL compared to the solvent control.
- In the 24-hour exposure period (without S9-mix), no toxicity in the relative suspension growth was observed up to a test item concentration of 50 µg/mL compared to the solvent control.

FIRST AND SECOND EXPERIMENT (Mutation experiments)
- The test item precipitated in the exposure medium at the test item concentration of 12.5 µg/mL.
- No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL DATA: see table 1 and table 2

ACCEPTABILITY CRITERIA
- The positive controls both produced significant increased in the mutation frequency, which was within the 95% control limits of the historical data range.
- The absolute cloning efficiency of the solvent controls (CEday2) was 108 and 115 (without S9) and 70 and 93 (with S9) in experiment 1 and 76 and 93 in experiment 2.
- The spontaneous mutation frequency in the solvent control was 99 and 87 (without S9) and 97 and 64 (with S9) per 10^6 survivors in experiment 1 and 76 and 85 per 10^6 survivors in experiment 2. These frequencies were within the 95% control limits of the historical data range.
- The suspension growth over the two-day expression period for cultures treated with ethanol was between 17 and 23 (3 hour treatment) and 57 and 70 (24 hour treatment).

Any other information on results incl. tables

Table 1Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	86	81	87
SD	23	26	28
n	220	202	273
Upper control limit (95% control limits)	135	135	145
Lower control limit (95% control limits)	37	28	28

SD = Standard deviation; n = Number of observations

Table 2Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	857	688	1710
SD	246	187	815
n	110	102	139
Upper control limit (95% control limits)	1425	1124	4214
Lower control limit (95% control limits)	289	253	-793

SD = Standard deviation; n = Number of observations

Distribution historical control data from experiments performed between January 2013 and November 2016. 

Applicant's summary and conclusion

Conclusions:

KY-UN is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of KY-UN to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. The test item was tested up to and including a dose of 12.5 µg/mL in the absence of S9 in a 3 and 24 hour treatment period and in the presence of S9 in a 3 hour treatment period.

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 test item precipitated at the highest dose level. No toxicity was observed up to and including the highest dose level, in the absence and in the presence of S9. The test item did not induce an increase in the mutation frequency. Therefore, KY-UN is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.