Nitrogen trifluoride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2012 to 27 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and TG compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrogen trifluoride

EC Number:

232-007-1

EC Name:

Nitrogen trifluoride

Cas Number:

7783-54-2

Molecular formula:

F3N

IUPAC Name:

trifluoroamine

Test material form:

gas

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / 5,6-benzoflavone induced rat liver

Test concentrations with justification for top dose:

First test: 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100% (v/v)
Second test: 0, 6.25, 12.5, 25, 50 and 100% (v/v)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: dichloromethane (-S9); vinyl chloride monomer (+S9)

Details on test system and experimental conditions:

Initial toxicity-mutation assay:
The five tester strains were exposed toNF3 in stainless steel vessels at 7 concentrations (see above).

Aliquots (0.1 mL) of a 10 hour bacterial culture and 10% S9 mix (0.5 mL) or 0.1M pH 7.4 phosphate buffer (0.5 mL) were placed in glass vessels and 2 mL of agar containing histidine (0.5 mM), biotin (0.5 mM) and tryptophan (0.5 mM) were added. The mixtures were thoroughly shaken and overlaid onto previously prepared Petri dishes containing approximately 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were prepared for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and phosphate buffer. The seeded plates were placed in stainless steel vessels. The plates were supported, inverted with lids removed, on stainless steel racks inside the vessels, so as to permit circulation of the test substance. Plates containing S9 mix and buffer were placed in separate vessels. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance or vapour-phase positive controls were injected via a needle valve or septum. The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air. The plates were incubated for ca 48 hours in the vessels at 37°C and then removed from the vessels under air extraction. The plates were incubated for a further period ofca. 24 hours at 37°C to permit the growth of revertant colonies. Further sets of plates were prepared for the liquid positive control compounds. Aliquots of 0.1 mL of the positivecontrol solutions were added to the plates together with the bacteria, buffer or S9 mix andagar overlay. These plates were incubated at 37°C for 48-72 hours (not in stainless steel vessels). After this period the appearance of the background bacterial lawn was examined and revertant colonies were counted using an automated colony counter (Perceptive Instruments Sorcerer).

Any toxic effects of the test substance would be detected by a substantial (ca. 50%) reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the maximum concentration used in the second test was the same as that used in the first. If toxic effects had been observed at more than one concentration, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally a minimum of four non-toxic concentrations should beobtained.

Confirmatory mutagenicity assay:
The second test was conducted using exactly the same procedures as the first test. The maximum test substance concentration chosen was again 100% v/v (nominal), but only five concentrations were used.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

Toxicity was assessed by reduction in the background lawn; dose-dependent reduction in the mutant count/plate (concurrent vehicle control).

Statistics:

No statistical analysis was performed on the data.

Results and discussion

Test results

Test results 1

Species / strain:

other: TA100, TA1535, WP2uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

other: TA98 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Six doses of the test material ranging from 16 to 5000 ug/plate±S9 were evaluated in the plate incorporation test. No precipitation or toxicity was observed up to the limit dose, 5000 ug/plate.
No evidence of toxicity was observed in either the presence or absence of S9 in either test.

Any other information on results incl. tables

INITIAL TOXICITY-MUTATION ASSAY:

 

Table 7.6.1 -1: Bacterial mutation assay, summary of results– first test

Dose (%)	0	1.56	3.13	6.25	12.5	25	50	100	+ve control^a
REVERTANTS/PLATE –S9
TA98	27.7 ± 4.2	26.0 ± 2.6	27.3 ± 0.6	23.3 ± 6.0	28.7 ± 2.1	29.0 ± 3.5	27.7 ± 1.5	25.7 ± 2.5	374.0 ± 47.9
TA100	121.7 ± 12.3	104.0 ± 2.6	127.3 ± 11.9	115.3 ± 19.3	110.0 ± 3.5	138.3 ± 10.5	169.7 ± 11.9	385.7 ± 11.1	1004.7 ± 19.1
TA1535	23.3 ± 0.6	24.3 ± 2.5	25.7 ± 4.7	30.3 ± 3.1	37.0 ± 2.6	67.7 ± 5.9	124.7 ± 10.6	247.7 ± 15.6	909.3 ± 175.2
TA1537	10.3 ± 1.5	12.7 ± 2.1	13.0 ± 1.7	11.0 ± 4.6	10.0 ± 1.0	9.7 ± 0.6	9.3 ± 3.1	15.3 ± 3.8	571.3 ± 114.6
WP2uvrA	134.7 ± 5.8	137.3 ± 1.5	148.3 ± 11.0	162.7 ±5.9	167.7 ± 8.5	212.3 ± 9.0	357.7 ± 29.3	708.3 ± 32.1	2047.0 ± 210.5
REVERTANTS/PLATE +S9
TA98	36.7 ± 1.5	39.7 ± 3.1	40.0 ± 5.2	37.7 ± 1.2	36.0 ± 1.7	39.7 ± 2.1	37.3 ± 2.1	34.3 ± 7.4	338.7 ± 8.6
TA100	125.3 ± 9.3	135.0 ± 1.7	163.3 ± 11.0	213.3 ± 20.3	307.3 ± 5.7	520.7 ± 4.9	599.0 ± 21.6	595.3 ± 39.2	1254.7 ± 118.3
TA1535	21.0 ± 0.0	24.7 ± 1.2	62.3 ± 8.7	169.7 ± 11.1	211.3 ± 24.3	239.0 ± 7.0	303.0 ± 5.3	313.0 ± 6.9	245.7 ± 43.9
TA1537	27.0 ± 0.0	22.0 ± 4.6	25.3 ± 2.3	26.7 ± 2.5	25.7 ± 1.5	23.3 ± 2.3	25.3 ± 3.2	23.0 ± 1.0	183.0 ± 7.9
WP2uvrA	151.3 ± 9.1	150.7 ± 6.7	184.0 ± 18.0	214.7 ± 16.3	339.3 ± 13.6	406.0 ± 12.1	484.7 ± 14.6	402.0 ± 60.6	2258.7 ± 113.9

a – standard liquid positive controls were used; the dose for these therefore was ug/plate

 

Table 7.6.1 -2: Bacterial mutation assay, summary of results– second test

Dose (%)	0	6.25	12.5	25	50	100	+ve control^a
REVERTANTS/PLATE –S9
TA98	34.7 ± 0.6	27.3 ± 7.1	26.3 ± 2.1	24.7 ± 2.9	31.7 ± 9.3	26.0 ± 1.7	383.7 ± 11.6
TA100	129.3 ± 2.3	135.7 ± 4.9	148.7 ± 10.1	186.3 ± 6.7	374.3 ± 27.0	489.0 ± 76.2	1052.3 ± 40.5
TA1535	20.7 ± 2.5	26.0 ± 5.0	32.3 ± 4.5	60.7 ± 6.5	146.0 ± 42.3	237.0 ± 24.6	849.3 ± 200.7
TA1537	13.0 ± 0.0	10.7 ± 1.5	12.7 ± 3.1	10.0 ± 1.0	11.3 ± 0.6	12.3 ± 3.2	702.0 ± 103.1
WP2uvrA	134.7 ± 13.6	171.0 ± 20.7	193.0 ± 7.0	254.0 ± 11.8	404.0 ± 20.1	878.7 ± 66.1	2510.3 ± 355.3
REVERTANTS/PLATE +S9
TA98	40.0 ± 4.4	41.0 ± 6.6	37.0 ± 3.0	35.7 ± 2.1	39.3 ± 3.2	30.7 ± 1.5	361.7 ± 29.2
TA100	122.0 ± 8.5	248.7 ± 16.6	352.3 ± 34.1	434.3 ± 13.8	583.0 ± 22.5	624.7 ± 19.6	889.0 ± 33.4
TA1535	22.3 ± 1.2	161.3 ± 17.1	252.7 ± 10.1	303.0 ± 21.0	348.3 ± 20.6	396.3 ± 9.1	224.0 ± 19.0
TA1537	20.7 ± 1.2	27.0 ± 1.0	28.7 ± 4.6	31.0 ± 4.6	31.7 ± 8.7	24.0 ± 3.6	170.3 ± 7.1
WP2uvrA	147.0 ± 7.9	245.0 ± 2.6	392.7 ± 3.8	441.7 ± 4.9	479.0 ± 8.7	430.3 ± 17.6	2140.7 ± 109.5

 

Table 7.6.1 -3: Bacterial mutation assay, summary of gaseous positive controls – first and second test

Dose (%)	Positive control	Revertants / plate
		1st test	2nd test
TA98 – S9	DCM	120.0 ± 13.1	366.7 ± 44.5
TA100 – S9	DCM	597.7 ± 66.2	794.7 ± 42.0
TA100	VCM	393.7 ± 35.2	417.0 ± 41.3
TA1535	VCM	136.3 ± 5.5	161.0 ± 4.4
WP2uvrA	VCM	808.0 ± 22.9	855.0 ± 70.8

DCM - dichloromethane

VCM – vinyl chloride monomer

 

Applicant's summary and conclusion

Conclusions:

It was concluded that NF3 showed evidence of mutagenic activity in this bacterial system with increases in revertants observed in twoSalmonella typhimuriumstrains (TA100 and TA1535) and oneEscherichia colistrain (WP2uvrA(pKM101)) in the absence and presence of metabolic activation. In the remaining two Salmonella typhimurium strains (TA98 and TA1537) in the absence and presence of metabolic activation no evidence of mutagenic activity was observed when tested up to the maximum practicable concentration (100% v/v), under the test conditions employed.

Executive summary:

In a reverse gene mutation assay in bacteria, fiveSalmonella typhimuriumstrains (TA98, TA100, TA1535, and TA1537) and oneE.coli(WP2uvrA(pKM101) were exposed to NF3 gas diluted in atmospheric air. Atmospheric air was also used as a vehicle control. To confirm sensitivity of the test system standard liquid positive controls were along with gaseous positive controls (dichloromethane in the absence of S9 and vinyl chloride monomer).

 

Concentrations of NF3 up to 100% (v/v) were tested (the maximum practicable concentration).

 

No signs of toxicity were observed towards the tester strains in either mutation test following exposure toNF3.

 

In both mutation tests in the absence of S9 mix, substantial, concentration-related increases in revertant colony counts were obtained in strains TA1535 and WP2uvrA (pKM101). Smaller increases were obtained in strain TA100.

 

In both mutation tests in the presence of S9 mix, substantial, concentration-related increases in revertant colony counts were obtained in strains TA1535 and TA100. Smaller increaseswere obtained in strain WP2uvrA (pKM101).

 

These data suggest that NF3 is mutagenic in bacteria, inducing base changes. The effect of metabolic activation did not alter the mutagenic potential in these strains.

 

It was concluded that NF3 showed evidence of mutagenic activity in this bacterial system with increases in revertants observed in twoSalmonella typhimuriumstrains (TA100 and TA1535) and oneEscherichia colistrain (WP2uvrA(pKM101)) in the absence and presence of metabolic activation. In the remaining two Salmonella typhimurium strains (TA98 and TA1537) in the absence and presence of metabolic activation no evidence of mutagenic activity was observed when tested up to the maximum practicable concentration (100% v/v), under the test conditions employed.