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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: The study was conducted according to the OECD Guideline and in compliance with GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: nanoform
Details on test material:
TEST ITEM
- Name of test material (as cited in study report): Z-COTE HP1
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: Solid
- Composition of test material, percentage of components: Z-COTE HP1 (98%), coated with triethoxycaprylylsilane (CAS # 2943-75-1; 2%)
- Lot/batch No.: NPL Ref#: ZB250#65
- Expiration date of the lot/batch: June 2014
- Storage condition of test material: Room temperature, dry, exclusion of light
- Particle size: <200nm
- Particle surface area: 12-24 m2/g
- Other: MMAD of the aerosol < 3.0 µm, GSD about 1.5


REFERENCE ITEMS
- Name of test material (as cited in study report): Zinc Oxide 205532, Micron Size Powder
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: Solid
- Composition of test material, percentage of components: Non-coated microscaled ZnO
- Lot/batch No.: NPL Ref#: ZrA250#60
- Expiration date of the lot/batch: May 2014
- Storage condition of test material: Room temperature, dry, exclusion of light
- Other: MMAD of the aerosol < 3.0 µm, GSD about 1.5

- Name of test material (as cited in study report): Z-COTE
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: Solid
- Composition of test material, percentage of components: Non-coated nanoscaled ZnO; purity ZnO: 99%
- Lot/batch No.: NPL Ref#: ZC250#32
- Expiration date of the lot/batch: June 2014
- Particle size: <200nm
- Particle surface area: 12-24 m2/g
- Storage condition of test material: Room temperature, dry, exclusion of light
- Other: MMAD of the aerosol < 3.0 µm, GSD about 1.5

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld/Germany
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: Approx. 230g (males), approx. 165g (females)
- Fasting period before study: No
- Housing: 2 rats per cage, absorbing softwood bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 1 d followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 55 +/- 15°C
- Air changes (per hr): Fully airconditioned
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

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

GENERATION OF TEST ATMOSPHERE CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 +/- 2°C, 55 +/- 15%,
- Air flow rate: 1L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically by filter samples, feed back loop to actual aerosol concentrations measured by an aerosol photometer
- Samples taken from breathing zone: Yes
Duration of treatment / exposure:
2 weeks, 5 consecutive days per week, 6 h per day
Frequency of treatment:
5 consecutive days per week, 6 h per day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.5, 2, and 8 mg/m3
Basis:
other: target aerosol concentration of test substance Z-COTE HP1
Remarks:
Doses / Concentrations:
8 mg/m3
Basis:
other: target aerosol concentration of reference substance Z-COTE
Remarks:
Doses / Concentrations:
8 mg/m3
Basis:
other: target aerosol concentration of reference substance ZnO
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent no treatment
Positive control(s):
5 additional rats per sex were dosed once orally with 20 mg/kg cyclophosphamide (CP) monohydrate 24 h pior to sacrifice

Examinations

Tissues and cell types examined:
bone marrow tissue- polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
From suspensions of bone marrow tissue, cleaned of nucleated cells by a cellulose column procedure, smears were prepared on slides, fixed for 10 minutes in absolute methanol and stained with May-Grünwald- and Giemsa-solution. Microscopic analysis was conducted on a blind basis. Micronuclei were counted in 2000 polychromatic erythrocytes (PCE) per animal. The ratio of polychromatic to normochromatic erythrocytes (NCE) was determined in 500 red blood cells.
Evaluation criteria:
Slide Reading and Measurements
The slides were analyzed microscopically under 630-1000 x magnification. For each animal, the incidence of micronucleated cells per 2.000 PCE of the bone marrow was determined. The ratio of PCE to NCE was calculated by counting the number of PCE per 500 red blood cells (RBC). For both endpoints one of the two existing (A and B) bone marrow smears was used.

Data Evaluation
The micronucleus assay is judged as valid if the clean air controls demonstrate low spontaneous frequencies of micronucleus induction and if the positive controls demonstrate significantly higher frequencies of micronucleus induction as compared to the clean air controls. In addition, in test item treated animals the PCE ratio should not fall below 20% of the clean air control values to avoid unspecific effects due to excessive cytotoxicity in the bone marrow.
In the mammalian erythrocyte micronucleus test a genotoxic effect is claimed if a dose-related increase in the number of polychromatic erythrocytes or a statistically significant increase in the number of micronucleated cells in a single dose group at a single sampling time is observed, but biological relevance of the results is considered first.
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's test.
The statistical evaluation of the histopathological findings was done with the two-tailed Fisher test by the P.L.A.C.E.S. system.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

As assessed by differential cell counting of bone marrow smears, Z‑COTE® HP1 and the particulate reference items Z-COTE®and micron-scaled ZnO in the given doses (0.5, 2.0, and 8.0 mg/m3) didn’t significantly influence red blood cell formation in male Wistar rats (strain: Crl:WU) after 14 days of inhalation exposure. However, there was a high fluctuation rate in PCE numbers, in part, perhaps based on the combination of the micronucleus test with the hOGG1-modified comet assay ex vivo in the male animals. In female Wistars rats (strain: Crl:WU), number of PCE/500 RBC was significantly reduced to 143±23.0 (p 0.05) by Z‑COTE®and to 124±23.1 (p 0.01) by the positive control CP, as compared to 179±18.0 for the clean air control. In addition, a significant decrease (p 0.05, only by using the Student’s t-Test) was ob­served for the highest Z‑COTE® HP1 dose group (8 mg/m3), indicating some systemic availabilty of, in particular, the nano-scaled particles.

Under the conditions of this assays, Z-COTE® HP1, Z-COTE®, and microscaled ZnO didn’t significantly en­hance the number of micronuclei in polychro­matic erythro­cytes of the bone marrow (after 14 days of inhalation exposure) of both male and female Wistar rats (strain: Crl:WU). In contrast, as expected, the positive control CP significantly induced micronucleus formation in PCE of the bone marrow, with higher rates in male than in female animals.

Table Red blood cell formation and micronucleus induction in bone marrow of rats, after 14 days of exposure to clean air, Z-COTE®HP1, Z-COTE®, or microscaled ZnO

Treatment group

Concentration, sampling time

PCE/500 RBC

PCE:NCE

MN/2000 PCE

% MN PCE

Negative control,
clean air

24 h

♂ 86  
 ♀ 179

 ♂ 0.22  
 ♀ 0.56

  ♂ 5.8 
  ♀ 5.4

♂ 0.29  
♀ 0.27

Positive control,
CP

20 mg/kg b.w.,
p.o., 24 h

♂ 67  
    ♀ 124**

 ♂ 0.16  
    ♀ 0.33**

  ♂ 55.4**  
  ♀ 22.0**

   ♂ 2.77**
  ♀ 1.10**

Test item,

Z-COTE®HP1

0.5 mg/m3, 24 h

 ♂ 127  
 ♀ 185

 ♂ 0.36  
 ♀ 0.59

  ♂ 5.0  
  ♀ 4.0

♂ 0.25  
♀ 0.20

Test item,

Z-COTE®HP1

2.0 mg/m3, 24 h

♂ 79  
 ♀ 170

 ♂ 0.19 
 ♀ 0.52

  ♂ 5.4  
  ♀ 3.8

 ♂ 0.27  
♀ 0.19

Test item,

Z-COTE®HP1

8.0 mg/m3, 24 h

♂ 84  
   ♀ 125(*)

 ♂ 0.21  
    ♀ 0.34(*)

  ♂ 2.6  
  ♀ 4.2

♂ 0.13  
♀ 0.21

Reference item,

Z-COTE®

8.0 mg/m3, 24 h

♂ 86  
 ♀ 143*

 ♂ 0.21  
  ♀ 0.41*

  ♂ 4.2  
  ♀ 5.4

♂ 0.21  
♀ 0.27

Reference item,

Microscaled ZnO

8.0 mg/m3,24 h

 ♂ 109  
 ♀ 173

 ♂ 0.29  
 ♀ 0.53

  ♂ 3.2  
  ♀ 2.0

♂ 0.16  
♀ 0.10

 

 PCE: Polychromatic erythrocytes; NCE: Normochromatic erythrocytes; RBC: Red blood cells; MN: Micronuclei; % MN PCE: Percent micronucleated PCE; Significantly different from negative controls:*:P 0.05, **:P 0.01, Mann-Whitney Rank Sum Test; (*): P  0.05, Student’s t-Test.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this assay, no systemic chromosome mutagenic activity was detected in the rat bone marrow micronucleus assay after inhalation exposure to Z-COTE HP1 at dose levels inducing local effects in the lung. Inhaled Z‑COTE® HP1 is considered non-mutagenic in immature bone marrow erythro­cytes (PCE) of Wistar rats (strain: Crl:WU).
Executive summary:

A 14-day repeated dose inhalation toxicity study was conducted to establish exposure dose-response relationships of the nanoscaled ZnO (with functionalized surface) in rats after subacute exposure and to compare the effects observed with two reference substances, i.e., nanoscaled ZnO (uncoated) and a microscaled ZnOin rats using nose-only exposure according to the OECD Guideline 412 in compliance with GLP.

In the framework of this study also systemic clastogenic and aneugenic effects were investigated in male and female rats using the bone marrow micronucleus assay according to OECD Guideline 474.

In this study, male rats were exposed (nose only) atconcentration levels of 0, 0.5, 2, or 8 mg/m³ with nanoscaled ZnO (with functionalized surface)and at 8 mg/m3 with reference test substances. Fresh air treated animals served as concurrent control.

There was no treatment-related reduction of body weights in any test group; no clinical signs were detected. In male rats the test item and the reference substances did not mediate significant repression of red blood cell formation. In females the PCE/NCE was significantly reduced in the positive control as well as in females exposed to 8 mg/m³ Z-COTE® HP1 or uncoated Z-COTE®. This effect might indicate that these test itemsreached the bone marrow as target organ.
There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes due to Z‑COTE® HP1, Z‑COTE®or microscaled ZnO exposure in males or females, as compared to the vehicle control groups (clean air) at any dose level. The positive and vehicle controls gave valid results.

Conclusion: No systemic chromosome mutagenic activity was detected in the rat bone marrow micronucleus assay after inhalation exposure to Z-COTE HP1 at dose levels inducing local effects in the lung.