Nitrogen trifluoride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 1995 to 25 October 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and test guideline compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus 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

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Administration / exposure

Route of administration:

inhalation: gas

Duration of treatment / exposure:

4h /nose-only exposure

Frequency of treatment:

single administration

Post exposure period:

up to 72h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm

Doses / concentrations 2

Dose / conc.:

842 ppm

Doses / concentrations 3

Dose / conc.:

2 474 ppm

No. of animals per sex per dose:

5/sex/gp

Control animals:

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

Bone marrow PCE for MN

Details of tissue and slide preparation:

Range finder experiment:
Male and female rats received a single 4 h nose-only inhalation exposure to NF3 at 0, 219, 398, 854 and 1855 ppm.
Tissues and cells examined:Approximately 72 h post dosing peripheral blood and bone marrow was sampled, smears produced to determined PCE:RBC ratio. At least 1000 PCE and 200 erythrocytes/animal were examined in peripheral blood and bone marrow.

Main experiment:
Treatment:single 4 h nose-only inhalation exposure.
Sampling times: Mice were sacrifice at 24, 48 and 72 h post the single administration.
Number of animals/group:5 animals/sex/group/time point
Tissues and cells examined:Bone marrow; 1000 PCEs examined/animal for the presence of micronuclei. For toxicity assessment, the proportion of PCE to total erythrocytes was recorded (number of PCE in 200 erythrocytes scored).
Dose groups:single 4 h nose-only inhalation exposure at 0, 840, 1274 and 2469 ppm. The positive control was urethane (300 mg/kg) dosedviaIP to males.

Details of slide preparation:
Bone marrow cells were collected from the femurs and cell smears were prepared and stained according to conventional cytological procedures.
Coded slides were scored for the presence of micronuclei in 1000 PCE/ animal. The ratio of PCEs to total erythrocytes was also recorded for each animal, as an indication of cytotoxicity to the target tissue.

Evaluation criteria:

The test article was considered positive if the incidence of MN PCE was significantly higher than that in the air control group (p<0.05).

Statistics:

Each individual test animal was the unit used for analysis of MN PCE frequency. Chi square was used to assess each test material dose group separately against the vehicle control. No linear trend analysis was conducted.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TEST:
Three animals/sex/gp were exposed to NF3 as a single 4 h treatmentat 0, 219, 398, 854 and 1855 ppm. No deaths occurred. Clinical signs of toxicity included discoloured feet in the 3rdhour of exposure in all mice at 854 ppm. Hypoactivity at 1855 ppm in all mice was observed after completion of exposure. 
Peripheral blood analysis 72 h post dosing confirmed no evidence of bone marrow toxicity, but rather an increase in the PCE:NCE ratio, suggestive of increased RBC turnover, erythropoiesis.
Based on these data the maximum dose selected for the micronucleus assay was 2500 ppm, with the intention of obtaining evidence of bone marrow toxicity (i.e. suppression in PCE, rather than an increase). Other dose levels were 625 and 1250 ppm
 
MICRONUCLEUS ASSAY
Clinical observations:
No mortalities were observed in the main experiment. Clinical signs reported were similar to those already noted in the RF at the high dose group only during exposure. By 24 h all animals appeared normal. No effect on body weight was observed.
 
PCE ratio:
Evidence of increased erythropoiesis was observed in females at 48 h post dosing and males and females 72 h post dosing. The latter time point is now no longer considered compliant with current guidance, therefore the data will not be discussed.
 
Micronucleated polychromatic erythrocytes (MN PCE):
Analysis of the mean MN PCE group data individually and combined indicated that there was no statistically significant increases in MN PCE frequency compared to concurrent control values for either males at the 24 or 48 h time points. For females a dose dependent increase in MN PCE frequency was observed at the 48 h time point, with % mean MN PCE of 0.06, 0.12, 0.24 and 0.46% in the control, 300, 842 and 2464 ppm groups respectively. In hindsight the number of PCE scored for MN were insufficient (1000 /animal). As a result the OECD guidance for this assay stipulates that 2000 PCE are scored for MN. A further 1000 PCE were scored and the data combined. As a result the dose related increase in MN PCE disappeared, with % mean MN PCE of 0.06, 0.12, .24 and 0.46% in the control, 300, 842 and 2464 ppm groups respectively.
 
PCE/NCE ratio confirmed increase an in the PCE population in females, which is suggestive of erythropoiesis (i.e. increased PCE production). See table 7.6.2.1 in any other information.
Due to the dose related increase in MN PCE frequency in females at the 48 h time point the sample size was increased to 2000 PCE/animal (as per current recommendations). See table 7.6.2.2 in any other information.

Any other information on results incl. tables

Table 7.6.2 -1: Summary of micronucleus results in male and female mice (24 and 48hr sample)

Dose (ppm)	Males				Females
	PCE / NCE (%)	No. of PCE	Mean %MN PCE / 1000 PCE	PCE / NCE (%)		No. of PCE	Mean %MN PCE / 1000 PCE
24 h sample
Vehicle	56.66	5004	0.20 ± 0.07	56.45		5007	0.22 ± 0.07
NF3 (300)	63.62	5006	0.12 ± 0.05	60.00		5007	0.22 ± 0.06
NF3 (842)	56.43	5006	0.12 ± 0.04	50.86		5007	0.08 ± 0.02
NF3 (2474)	50.53	5007	0.22 ± 0.10	59.31		5008	0.26 ± 0.07
Urethane (300 mg/kg)	59.51	5005	1.42 ± 0.11*	-		-	-
48 h sample
Vehicle	62.31	5006	0.14 ± 0.05	61.29		5005	0.06 ± 0.02
NF3 (300)	59.67	5005	0.16 ± 0.04	61.05		5006	0.12 ± 0.04
NF3 (842)	54.04	5006	0.28 ± 0.12	64.26		5007	0.24 ± 0.09*
NF3 (2474)	52.15	5005	0.18 ± 0.05	73.74		5008	0.46 ± 0.19*
Urethane (300 mg/kg)	44.74	5007	0.40 ± 0.10*	-		-	-

* p<0.01

   

Table 7.6.2 -2: Summary of 48 h micronucleus results in female mice following increased sample size

Dose (ppm)	PCE / NCE (%)	No. of PCE	Mean %MN PCE / 1000 PCE
Vehicle	58.75	10006	0.16 ± 0.03
NF3 (300)	62.63	10004	0.17 ± 0.06
NF3 (842)	60.69	10006	0.14 ± 0.03
NF3 (2474)	62.94	10009	0.21 ± 0.04

Applicant's summary and conclusion

Conclusions:

Interpretation of results negative
Based on the results from this studyNF3did not exhibit an increase in the frequency of micronucleated polychromatic erythrocytes in male or female mouse bone marrow cells when tested up to a dose of 2474 ppm (considered to be a MTD, due to evidence of bone marrow effects [erythropoiesis]) when sampled at 24 and 48 h post a single 4 h nose-only inhalation exposure.

Executive summary:

In a bone marrow micronucleus assay using Swiss Webster mice, a single, 4 h nose-only exposure to the gas,NF3at concentrations of 0, 300, 842 and 2474 ppm was undertaken. Animals were sampled for bone marrow at 0, 24, 48 and 72 h post the single administration. Each group contained 5 animals/sex/gp. Doses were selected from a pilot toxicity study where no effect on bone marrow toxicity / proliferation was observed up to a dose of 1855 ppm. For the micronucleus study the intention was to increase the dose in order to have an effect on bone marrow proliferation / toxicity. For the purpose of this study discussion results from the 72 h time point have been omitted as they not consistent with the current guidance. AsNF3did not prolong the cell cycle the requirement for a latter time point is unnecessary.

 

Mice were treatedNF3and sampled at as previously stated. A negative control group was treated with vehicle only (air). To examine the sensitivity of the analyst to detect MN PCE a positive control group was included. Male mice only were dosed with urethane,ipat a dose of 300 mg/kg. Slides of bone marrow cells were prepared from five animals/sex/time point for each group and scored for the occurrence of micronucleated polychromatic erythrocytes (MN PCE) and PCE/total erythrocyte ratios.

 

Whilst no mortality was observed, clinical signs of toxicity reported in the micronucleus test were similar to those observed in the dose range finding experiment (hypoactivity and discoloured feet. The latter is a potential sign of methaemoglobinaemia).

 

Analysis of the mean MN PCE group data individually and combined indicated that there was no statistically significant increases in MN PCE frequency compared to concurrent control values for either males at the 24 or 48 h time points. For females a dose dependent increase in MN PCE frequency was observed at the 48 h time point, with % mean MN PCE of 0.06, 0.12, 0.24 and 0.46% in the control, 300, 842 and 2464 ppm groups respectively. In hindsight the number of PCE scored for MN were insufficient (1000 /animal). As a result the OECD guidance for this assay stipulates that 2000 PCE are scored for MN. A further 1000 PCE were scored and the data combined. As a result the dose related increase in MN PCE disappeared, with % mean MN PCE of 0.06, 0.12, .24 and 0.46% in the control, 300, 842 and 2464 ppm groups respectively.

 

PCE/NCE ratio confirmed increase an in the PCE population in females, which is suggestive of erythropoiesis (i.e. increased PCE production).

 

Based on the results from this study NF3 did not exhibit an increase in the frequency of micronucleated polychromatic erythrocytes in male or female mouse bone marrow cells when tested up to a dose of 2474 ppm (considered to be a MTD, due to evidence of bone marrow effects [erythropoiesis]) when sampled at 24 and 48 h post a single 4 h nose-only inhalation exposure.