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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,5-Naphthylene diisocyanate
- Physical state: solid
- Purity: 99.7 %
- Lot/batch No.: B3YE591000
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl: NMRI BR
- Source: Charles River (Sulzfeld, Germany)
- Age at study initiation: approx. 8-12 weeks
- Weight at study initiation: 32-45 g
- Assigned to test groups randomly: yes
- Housing: individual in type II cages (positive control)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose-only

EXPOSURE CONDITIONS:
Animals were exposed to the aerosolized test substance in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animal's size. These restrainers were designed so that the animal's tail remained outside the restrainer, thus restrained-induced hyperthermia can be avoided. This type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, 1994) and is preferable to whole-body exposure on scientific (Pauluhn, 1984; 1988) and technical reasons (rapid attainment of steady-state concentrations, no technical problems with regard to test atmosphere inhomogeneities, better capabilities to control all inhalation chamber parameters, easier cleaning of exhaust air, and lower consumption of test substance). The micronized test substance was aerosolized as dry powder.

AEROSOL GENERATION AND EXPOSURE TECHNIQUE:
- Aerosol generation:
For atmosphere generation the Wright dust feeder was utilized. This dust generator was delivered from BGI Inc. Waltham MA, (USA) and is used for dry powder dispersion with conditioned compressed air (28 I/min). The micronized test substance is first filled into a reservoir of the device and is then compressed to a pellet using approximately 0.5 metric ton by a carver laboratory press (F.S. Carver INC. Wabash, IN 46992, USA). From this pellet defined amounts of the test substance were scraped off with 0.1 to 0.42 revolution/min and were then effectively dispersed using pressurized air (approx. 140 kPa). As this generator uses dry air, the humidity in the inhalation chamber is influenced. The scrape off velocity is the main parameter to
adjust the atmosphere concentration. Under dynamic conditions the target dust concentrations of the test substance were generated entraining the dust into a cyclone for optimization of particles distribution and optionally dilution with dry air by the dilution device. The dust is then entrained into the intake of the cylindrical inhalation chamber.
- Optimization of respirability:
In order to increase the efficiency of the generation of respirable particles and to prevent larger particles from entering the chamber a cyclone was used. Additionally the test substance was micronized.
- Inhalation chamber:
The aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 I).
- Inhalation chamber steadv-state concentration:
The test atmosphere generation conditions provide an adequate number of air exchanges per hour (> 200 x, continuous generation of test atmosphere). Under such test conditions steady state is attained within approximately one minute of exposure (t99 % = 4.6 x chamber volume/flow rate; McFarland, 1976). The ratio between the air supplied and exhausted was chosen so that approximately 90 % of the supplied air is removed from the chamber as exhaust. The remainder provides adequate deadspace ventilation for the exposure tubes. At each exposure port a minimal air flow rate of 0.75 I/min was provided. The test atmosphere can by no means be diluted by bias-air-flows. The inhalation chamber was operated in a weil venlilaled chemical fume hood.
- Conditioning the compressed air:
Compressed air was supplied by Boge compressors and was conditioned (Le. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Air flows:
During the exposure period air flows were monitored continuously and, if necessary, readjusted to the conditions required. Air flows were measured with calibrated flowmeters. The proper performance of mass flow controller used was validated utilizing a digital precision flow-meter calibratlon device (Bios DryCal Defender 510).
- Treatment of exhaust air:
The exhaust air was purified via cotton-wool/HEPA filters. These filters were disposed of by Bayer Schering Pharma AG.
- Inhalation chamber temperature and humidity:
Temperature and humidity measurements were made using a computerized system (Hydra, Fluke-Philips). The values were recorded at intervals of 5 min (eomputerized recording). The test atmosphere temperature and humidity were measured at the exposure location. Humidity and temperature were measured using a FTF-sensor (Elka-Elektronik, Lüdenscheid). The sensor was calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33 % (MgCI2 ) and at 75 % (NaCI) relative humidity. The calibration of the temperature sensor is also checked at two temperatures using reference thermometer. The measured values were evaluated using spreadsheet software.

ANALYSIS OF THE TEST ATMOSPHERE:
- Nominal concentration:
A nominal concentration was not calculated since the construction and the heavy weight of the dust generator and its reservoir, respectively, were not suitable for a precise measurement of the sum of weight loss.
- Gravimetric evaluation:
The test-substance concentration was determined by gravimetric analysis (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
Chamber samples were taken in the vicinity of the breathing zone. The number of samples taken was sufficient to characterize the test atmosphere and was adjusted so as to accommodate the sampling duration and/or the need to confirm specific concentration values. Optimally, samples were collected after the equilibrium concentration had been attained in hourly intervals. All gravimetrical concentrations reported refer to mg of test substance/m3 air.
- Characterization of aerodynamic particle-size distribution:
The samples for the analysis of the particle-size distribution were also taken in the vicinity of the breathing zone. During the exposure two samples were taken, which was the maximum feasible due to the gravimetrical samples taken. The particle-size distribution was analyzed using a Berner type impactor. The individual impactor stages (aluminium foil with silicone layer) were subjected to gravimetric analysis. The parameters characterizing the particle-size distribution were calculated according to the following procedure:
Mass Median Aerodynamic Diameter (MMAD): Construct a 'Cumulative Percent Found - Less Than Stated Partiele Size' table; calculate the total mass of test substance collected in the cascade impactor. Start with the test substance collected on the stage that captures the smallest particle-size fraction, and divide this mass of the test substance by the total mass found above. Multiply this quotient by 100 to convert to percent. Enter this percent opposite the effective cut-off diameter of the stage above it in the impactor stack. Repeat this step for each of the remaining stages in ascending order. For each stage, add the percentage of mass found to the percentage of mass of the stages below it. Plot the percentage of mass less than the stated size versus particle size in a probability scale against a log particle-size scale, and draw a straight line best fitting the plotted points. A weighted least square regression analysis may be used to achieve the best fit. Note the particle size at which the line crosses the 50 % mark. This is the estimated Mass Median Aerodynamic Diameter (MMAD).
Calculation of Geometric Standard Deviation (GSD): Refer to the log probability graph used to calculate the Mass Median Aerodynamic Diameter. Provided that the line is a good fit to the data, the size distribution is log normal, and the calculation of the Geometrie Standard Deviation is appropriate.
- Stability of the test atmosphere:
The integrity and stability of the aerosol generation and exposure system was measured by using a Microdust pro (Casella) real-time aerosol photometer. Samples were taken continuously from the vicinity of the breathing zone. This chamber monitoring allows for an overall survey of toxicologically relevant technical parameters (inlet and exhaust flows as well as atmosphere homogeneity, temporal stability, and generation performance). Interruptions in exposure (e.g. resulting from obstruction of nozzles or other technical mishaps) are recorded and, if applicable, a commensurate interval is added to the exposure duration for compensation.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
single exposure
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 5, 25, 50, 70 mg/m3
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 5.5, 26.8, 51.9, 71.7 mg/m3
Basis:
other: gravimetric concentration
No. of animals per sex per dose:
5 males
Control animals:
yes, sham-exposed
Positive control(s):
Cyclophosphamide: dissolved in physiological saline, single i.p. injection with 10 ml/kg bw

Examinations

Tissues and cell types examined:
The bone marrow was prepared by centrifugation. The numbers of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) with and without micronuclei (MN) were counted.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In an acute study with rats 6 hours inhalation of 56 mg/m³ induced no lethality. At 140 mg/m³ two of 18 rats succumbed. Hence 93 mg/m³ are in the range of the lethal threshold concentration. Therefore, based on lethality, 70 mg/m³ are considered to be the MTD for the present study with mice.

DETAILS OF SLIDE PREPARATION:
Schmid's method was used to produce the smears (Mutation Res. 31, 9-15, 1975).

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. In addition, the number of normochromatic erythrocytes per 2000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 6000 normochromatic erythrocytes per 2000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omit-ted from the evaluation. A relevant, treatment-related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.


Evaluation criteria:
A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible.
Statistics:
The 1,5-Naphthylene diisocyanate group(s) with the highest mean per time point (provided this exceeded the respective negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5 % and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5 % and the treatment group figure was higher than that of the negative control.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see "Additional information on results"
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENERAL TOXICITY:
All mice survived the exposure period. Comparison between the exposure groups revealed a concentration-dependent decrease in body weights. Thebody weight loss on post exposure day in the exposure groups was significant compared to controls. The mice showed the following clinical signs: bradypnea, labored breathing patterns, breathing sounds, reduced motility, tremor, gait high-Iegged, gait staggering, piloerection, stridor and hypothermia. Most signs were transient within the short observation period. At 5 mg/m3 mice were without findings, whilst at 25, 50 and 70 mq/m3 concentration-dependent hypothermie effects (intensity and/or duration) occurred. Necropsy revealed at 70 mg/m3 a whitish pasty content in the nose as distinguishable finding.

Any other information on results incl. tables

Table 1: Summary of results from micronucleus test with 1,5-NDI

 Treatment group  Dose (mg/m3)  Dissection interval (h)  Number of evaluated PCE  Number of NCE per 2000 PCE  MNNCE per 2000 NCE  MNPCE per 2000 PCE
 Negative control 0 24 10,000

3946 ± 1300

1.5 ± 1.0

4.2 ± 1.3

  0 48  10,000

2749 ± 655

 2.5 ± 1.4

3.2 ± 1.5

1,5 -NDI 

5 24  10,000

2949 ± 691

1.2 ± 1.4

0.6 ± 0.9

  5 48 10,000 

3183 ± 1297

1.1 ± 1.0

1.2 ± 0.4

  25 24 10,000

2732 ± 770

 1.1 ± 1.1

1.2 ± 1.7

  25 48 10,000 

4040 ± 947

1.5 ± 1.1

2.2 ± 1.3

  50 24  10,000 

3086 ± 1032

2.5 ± 0.9

5.2 ± 1.9

  50 48  10,000 

3560 ± 930

2.2 ± 1.1

1.4 ± 0.9

  70 24  10,000 

 2528 ± 593

2.9 ± 0.7

3.8 ± 2.2

  70 48  10,000 

4778* ± 785

1.8 ± 0.9

3.2 ± 1.9

 Positive control

(Cyclophosphamide)

20 (mg/kg i.p.) 24 10,000 

3319 ± 962

1.7 ± 1.3

20.8* ± 6.5

* p<0.01 in non-parametric Wilcoxon ranking test

NCE = normochromatic erythrocytes

PCE = polychromatic erythrocytes

MNNCE = micronucleated NCE

MNPCE = micronucleated PCE


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

Male mice treated with 1,5‑Naphthylene diisocyanate received for 6 hours inhalative administrations of 5, 25, 50 and 70 mg/m³, respectively. The negative control animals received the air without 1,5‑Naphthylene diisocyanate using the same application regimen. Males of the positive control received a single intraperitoneal treatment with 20 mg/kg cyclophosphamide. The femoral marrow of all groups was prepared 24 hours after the last administration. Animals treated with 1,5‑Naphthylene diisocyanate and animals of the negative controls were sacrificed 24 and 48 hours after the mid of the inhalation period. Animals of the positive control were sacrificed 24 hours after treatment.  There was no altered ratio between polychromatic and normochromatic erythrocytes. After inhalative treatment for 6 hours of males with concentrations up to and including the maximum tolerated concentration of 70 mg/m³ no biologically relevant indications of a clastogenic effect of were found. Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

Thus, 1,5‑Naphthylene diisocyanate was concluded to be negative in the mouse micronucleus assay.