Zinc sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2021 to June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested, which precludes an evaluation of dose-response relationships.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2021 to June 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested, which precludes an evaluation of dose-response relationships.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Expiry date: 30 June 2022

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Assigned to test groups randomly: yes: All animals were randomized before the start of the pre-exposure period (according to weight).
- Fasting period before study: No
- Housing: 5 rats per cage, Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding
- Diet: milled/ mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: +/- 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 45-65%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole-body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 40.7 and 50.8 %. Daily mean temperatures in the inhalation systems ranged between 20.4 and 23.0°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 20 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 6.9 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39).
To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

14 days

Frequency of treatment:

14 days, 6 h per day

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 - air control

Dose / conc.:

17.82 mg/m³ air (analytical)

Remarks:

SD: ± 0.90 mg/m³ ; target concentration: 18 mg/m³: Test group 8

No. of animals per sex per dose:

5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s):
- Route of administration: oral gavage which guarantees systemic distribution of the compound and thus exposure to all assessed tissues
- Doses / concentrations: 300mg/kg body weight

Tissues and cell types examined:

Comet assay:
1. Bone marrow
2. Liver
3. Lung
4. Nasal mucosa

Bronchoalveolar lavage fluid (BAL): for cytology and total protein and enzyme levels.

Details of tissue and slide preparation:

COMET ASSAY
Samples were minced or aspirated in cold mincing buffer to produce a cell suspension. Cell suspensions were diluted as necessary and kept cold until they were processed further. At least three comet slides were prepared per sample. An aliquot of cell suspension was mixed with low melting point agarose, layered onto microscope slides precoated with normal melting point agarose, and covered with an additional layer of low melting point agarose. After the agarose had solidified, slides were lysed in cold working high salt lysing solution and maintained cold for at least 1 hour. At least two comet slides were removed from lysis for electrophoresis. Slides were rinsed with 0.4M Tris buffer and submerged in alkaline electrophoresis buffer for 20 minutes at 1 to 10°C to unwind the DNA. After unwinding, slides were electrophoresed in the same buffer at 1 to 10°C for 40 minutes at a constant voltage of 0.7V/cm. The buffer level was adjusted as necessary at the start of electrophoresis to achieve a starting current of 300±10 mA. After electrophoresis, the slides were neutralized with 0.4M Tris buffer, rinsed in ethanol, and air dried. The air-dried comet slides were stored at room temperature at a RH of ≤60% until shipment with desiccant to Helix3 for analysis.

Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System (Andor Technology, Northern Ireland). The image analysis version and settings were documented in the raw data. For each sample ghosts defined as comets with heavily diffused tail and a non-discernable head that cannot be accurately measured by image analysis were counted in parallel with the image analysis scoring. Slides were scored without knowledge of the sample treatment group.

Evaluation criteria:

Criteria for a Valid Test
a. Where no statistically significant (p<0.05) response in DNA damage as measured by %Tail is detected at any test article dose concentration, the concurrent positive control must induce a statistically significant increase in the same genotoxic endpoint when compared to the concurrent vehicle control.

b. The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a statistically significant positive effect.

Criteria for a Positive Response
An experienced scientific investigator classifies a test article as positive, equivocal, or negative for inducing genotoxicity based on the results of the statistical analysis and the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data and the reproducibility of the results in any repeat experiments.

The test article may be classified as positive for inducing genotoxicity if the following criteria are met:

a. a statistically significant increase in DNA damage is detected at one or more dose concentrations and

b. a statistically significant dose dependent response is detected in the same tissue

If cytotoxicity is detected in the same tissue and dose concentration(s) at which a significant increase in DNA damage is detected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity. Where cytotoxicity may be a confounding factor and/or when cytotoxicity is present in all doses tested, a repeat study including lower non-cytotoxic doses may be conducted to evaluate for the presence of genotoxicity in the absence of cytotoxicity.

A test article may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If the results are equivocal and/or the biological relevance of the results are unclear, a repeat study at the same doses may be con

Statistics:

Mean values and standard deviations were calculated. In addition, the median of the values from each slide was determined and for each animal the mean of the median from the slides were calculated. To be consistent with historical control comet data generated at Helix3, the individual animal mean %Tail values was calculated as the mean of the total cells scored. The following statistical analyses were carried out, additionally. For each test, a 95% confidence Interval (P<0.05) was used to determine statistical significance:

Parameter: Statistical test
%Tail and %LMW data distribution: Shapiro-Wilk, test group 0 only

%Tail and %LMW equality of variances: Bartlett test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Normally distributed data with equal variances: Dunnett (2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Line fit trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Non-normally distributed data or unequal variances: Steel ((2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Kendall rank trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

%Tail and %LMW References and Positive control only: Fisher’s F-test (2-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0); student’s t-test for equal variances or Welch’s t-test for unequal variances (1-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0)

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMET ASSAY
- in the nasal epithelium tissue:
The assessment of the DNA damage in the nasal epithelium showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 14.6 – 15.9% (mean of mean values) and 8.3 - 9.3% (mean of medians) and for Zinc oxide T0421 the range was between 15.9 to 16.6% (mean of means) and 8.8 to 10.8% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (16.6 and 10.7% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a lower level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 13.4% (mean of means) and 7.8% (mean of medians). The difference between the mean of mean values was statistically significant. However, the difference between the mean of medians was not. Similarly, Zinc sulfate monohydrate also showed lower tail intensity values (15.4 and 7.6% mean of means and medians, respectively) as compared to the air control. However, in this case the drop in the mean of medians was statistically significant but not the mean of means. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (34.9 and 32.3% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (23.1%) of the historical control data range (mean of means).
- in the lung tissue:
The assessment of the DNA damage in the lung tissue also did not show a biologically relevant alteration of the level of observed DNA damage after treatment with either Zinc oxide T0420 or Zinc oxide T0421 as compared to the air control values (The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means) (see see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 9.9 – 11.5% (mean of mean values) and 4.9 - 6.9% (mean of medians) and for Zinc oxide T0421 the range was between 8.2 to 10.8% (mean of means) and 4.2 to 5.3% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (11.3 and 5.6% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test with T0420, however, a negative trend (dose related decrease in the % tail intensity values) was observed in the mean of mean values of the test groups treated with T0421. This effect was not, however, observed in the mean of median values. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 8.6% (mean of means) and 4.0% (mean of medians) and not statistically significant as compared to the air control values. Zinc sulfate monohydrate showed slightly lower tail intensity values (8.3 and 4.1% mean of means and medians, respectively) as compared to the air control. The drop in the mean of means was statistically significant but not the mean of medians. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (52.8 and 52.4% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means).
- in the liver:
The % tail DNA intensity in the liver tissue (see table in any other info on results) ranged between 6.9 – 8.7% (mean of means) and 2.7 to 3.8% (mean of medians) for test groups treated with various concentrations of T0420. The mean of mean value of the test group treated with 8 mg/m3 (8.7 ± 1.08%) was statistically higher than the respective air control value (6.6 ± 1.15%). Furthermore, a concentration related trend was observed in the groups treated with 0.5, 2.0 and 8.0 mg/m3 Zinc oxide T0420. However, the mean of median value at 8.0 mg/m3 (3.8 ± 1.19%) was not statistically higher than its respective air control value (2.8 ± 1.00) and the trend observed when using the mean of means was not observed when using the mean of medians. The mean % tail DNA of test groups treated with Zinc oxide T0421 ranged between 7.7 – 8.7% (mean of mean values) and 3.1-3.6% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.6 and 2.8% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 7.6% (mean of means) and 3.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also did not show significantly altered tail intensity values (7.5 and 3.1% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.1 and 30.7% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (16.9%) of the historical control data range (mean of means).
- in the bone marrow:
The assessment of the DNA damage in the bone showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 6.1 - 6.6% (mean of mean values) and 2.7 – 2.9% (mean of medians) and for Zinc oxide T0421 the range was between 5.2 to 6.6% (mean of means) and 2.6 to 2.9% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.2 and 2.9% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 5.4% (mean of means) and 2.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also showed similar and not statistically significant tail intensity values (6.0 and 2.5% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.4 and 30.6% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (11.0%) of the historical control data range (mean of means).

CYTOTOXICITY:
The assessment of the potential of the test and reference substances to induce cytotoxicity in the LMW DNA diffusion assay did not show any significant increases in the percentage of diffused low molecular weight DNA in any of the tested tissues and doses (see table in any other info on results), except for micro-scaled Zinc oxide T0242, which induced a statistically higher diffusible LMW DNA (12.6%) as compared to the corresponding air control (8.6%). The positive control (EMS), however, induced statistically significant increases in the amount of diffused LMW DNA in all examined organs.
The comparison of the obtained LMW DNA data with the historical control data showed that all values from the nasal epithelium (including the air control values; 26.8 - 36.8%) were above the maximum value obtained in the historical control data for this tissue (8.0%). In the lung all values (8.4 - 12.6%), except for the positive control group, were below the maximum value of the historical control data range for the lung (17.6%). In the liver the values were similarly below the maximum historical control data range (13.6%), except for the low dose group of T0420, which had a value of 15.2% and the positive control group (38.2%). All bone marrow values (12.0 – 16.8% for the test and reference substances and 34.6% for the positive control group) were also above the upper limit of the historical control data (6.8%).


TOXICITY
- General toxicity: No Signs of general toxicity. No impairment of body weight gain.
- Local effects: In lavage, the exposure of single concentration of reference substance 1 (T0242) and reference substance 2 (zinc sulfate monohydrate) caused significantly increases in most of the lavage parameters, indicating inflammation process in the lung (see table ' Changes in mean total protein and enzyme levels in BAL' in any other info on results)

Table. Results of the comet assay in the nasal epithelium

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	16.6 ± 2.64	10.7 ± 2.89
1	Zinc Oxide T0420	0.5	14.6 ± 3.62	9.3 ± 2.64
2		2.0	15.9 ± 3.60	9.0 ± 2.86
3		8.0	14.8 ± 3.00	8.3 ± 1.69
4	Zinc Oxide T0421	0.5	16.6 ± 5.28	10.8 ± 5.05
5		2.0	15.9 ± 4.73	9.9 ± 4.03
6		8.0	16.0 ± 3.34	8.8 ± 3.91
7	Zinc Oxide T0242	8.0	13.4 ± 2.67^#	7.8 ± 2.13
8	Zinc sulfate monohydrate	18.0	15.4 ± 1.89	7.6 ± 1.28^$
9	EMS	300 mg/kg b.w.	34.9 ± 3.63^*	32.3 ± 4.12^*

^#: statistically significant P=0.048 (decrease)
^$: statistically significant P=0.031 (decrease)
^*: statistically significant P<0.001 (increase)

 

Table. Results of the comet assay in the lung

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	11.3 ± 1.93	5.6 ± 1.50
1	Zinc Oxide T0420	0.5	11.5 ± 3.25	6.9 ± 4.15
2		2.0	9.9 ± 1.62	4.9 ± 1.19
3		8.0	10.1 ± 2.70	5.4 ± 2.89
4	Zinc Oxide T0421	0.5	10.8 ± 3.68^1	5.3 ± 2.67
5		2.0	9.0 ± 1.54^1	4.5 ± 1.34
6		8.0	8.2 ± 1.58^1	4.2 ± 1.60
7	Zinc Oxide T0242	8.0	8.6 ± 2.85	4.0 ± 1.60
8	Zinc sulfate monohydrate	18.0	8.3 ± 1.69^#	4.1 ± 1.51
9	EMS	300 mg/kg b.w.	52.8 ± 6.84^*	52.4 ± 7.84^*

^#: statistically significant P=0.015 (decrease)
^*: statistically significant P<0.001 (increase)
¹: negative trend statistically significant P=0.047

 

Table. Results of the comet assay in the liver

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.6 ± 1.15	2.8 ± 1.00
1	Zinc Oxide T0420	0.5	7.9 ± 0.49^1	3.2 ± 0.53
2		2.0	6.9 ± 1.30^1	2.7 ± 0.38
3		8.0	8.7 ± 1.08^#1	3.8 ± 1.19
4	Zinc Oxide T0421	0.5	8.7 ± 0.91	3.1 ± 0.80
5		2.0	7.8 ± 1.05	3.6 ± 0.73
6		8.0	7.7 ± 2.25	3.1 ± 0.83
7	Zinc Oxide T0242	8.0	7.6 ± 2.03	3.2 ± 1.11
8	Zinc sulfate monohydrate	18.0	7.5 ± 2.03	3.1 ± 1.26
9	EMS	300 mg/kg b.w.	32.1 ± 2.55^*	30.7 ± 3.07^*

^#: statistically significant P=0.016
^*: statistically significant P<0.001
¹: positive trend statistically significant P=0.032

 

Table. Results of the comet assay in the bone marrow

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.2 ± 1.00	2.9 ± 0.66
1	Zinc Oxide T0420	0.5	6.6 ± 1.02	2.9 ± 0.52
2		2.0	6.5 ± 0.78	2.8 ± 0.76
3		8.0	6.1 ± 0.30	2.7 ± 0.42
4	Zinc Oxide T0421	0.5	6.0 ± 0.60	2.9 ± 0.68
5		2.0	6.6 ± 1.63	2.9 ± 0.67
6		8.0	5.2 ± 0.80	2.6 ± 0.75
7	Zinc Oxide T0242	8.0	5.4 ± 0.55	2.2 ± 0.54
8	Zinc sulfate monohydrate	18.0	6.0 ± 0.83	2.5 ± 0.37
9	EMS	300 mg/kg b.w.	32.4 ± 4.84^*	30.6 ± 5.53^*

^*: statistically significant P<0.001

Table. Results of the LMW DNA diffusion assay

Test group	Test Substance	Concentration (mg/m³)	Group Mean ± SD (%)
			Nasal epithelium	Lung	Liver	Bone marrow
0	Air control	0	30.0 ± 9.19	8.6 ± 1.52	12.6 ± 5.37	14.8 ± 2.17
1	Zinc Oxide T0420	0.5	26.8 ± 6.76	11.6 ± 3.29	15.2 ± 6.02	13.6 ± 2.07
2		2.0	33.8 ± 8.98	11.4 ± 2.51	13.2 ± 3.35	14.0 ± 3.54
3		8.0	35.8 ± 16.40	10.6 ± 1.52	13.4 ± 6.95	13.8 ± 5.02
4	Zinc Oxide T0421	0.5	31.6 ± 8.62	11.8 ± 3.56	12.6 ± 6.80	12.0 ± 3.54
5		2.0	36.8 ± 15.67	8.4 ± 3.36	10.0 ± 4.30	16.8 ± 5.89
6		8.0	34.6 ± 14.24	10.4 ± 2.61	11.0 ± 3.74	15.2 ± 5.40
7	Zinc Oxide T0242	8.0	29.0 ± 11.05	12.6 ± 4.22^€	11.8 ± 4.09	15.6 ± 5.98
8	Zinc sulfate monohydrate	18.0	33.0 ± 8.80	12.0 ± 5.92	10.6 ± 7.64	14.6 ± 6.66
9	EMS	300 mg/kg b.w.	39.8 ± 4.49^*	26.8 ± 13.37^#	38.2 ± 7.05^$	34.6 ± 6.73^$

^*: statistically significant P=0.032
^€: statistically significant P=0.041
^#: statistically significant P=0.019
^$: statistically significant P=0.032

 

Table. Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats after inhalation exposure to Zinc oxide T0242 and zinc sulfate monohydrate on 14 consecutive days

Analyte	Gr. 7 8 mg/m^3	Gr8 18 mg/m^3
Total Protein	7.6**	2.4**
GGT	4.2**	3.9**
LDH	13.2**	4.6**
ALP	19.1**	4.3**
NAG	2.4**	1.5

GGT = γ-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

Conclusions:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 0.5, 2 and 8 mg/m³, as well as to 8 mg/m³ miconsize zinc oxide or 18 mg/m³ zinc sulfate monohydrate for 6 hours daily on 14 consecutive days. A concurrent control group was exposed to conditioned air.

The tested atmospheric were met and they were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes within the respirable range. There were no signs of toxicity, nor were there any impairment of body weight gain. In the range finding 14-day study (BASF project no.: 36I0050/20I005) animals of the mid dose groups were nominally exposed to 12 mg/m3. The actual assessment of test substance concentrations, however, revealed an exposure to 10.9 and 10.8 mg/m3 for T0420 and T0421, respectively. At this dose level significant histopathological alterations (grade 2 degeneration/regeneration, olfactory epithelium) as well as alterations indicative of inflammatory responses in the lung were observed, which could have had a confounding impact on the outcome of the comet assays results. Thus, the top exposure concentration was slightly reduced to 8 mg/m3. At this level in the lavage fluid, several parameters were increased in a concentration related manner in animals exposed to T0420 and T0421, indicating inflammation process in the lung. These findings were comparable with those observed previously in the range finding 14-day inhalation study with these two test materials. Similar findings were observed in animals exposed to the reference substances.


LMW DNA diffusion assay
In all test groups using the test or reference substances, the quantification of the diffused low molecular weight DNA as a measure for cytotoxicity did not show any indication of excessive cytotoxicity as compared to the respective air control value, which could have hampered the interpretation of the comet assay data. The significantly increased value observed for the lung using micro-scaled Zinc oxide T0242 was not high enough to interfere the comet data evaluation. The positive control group, however, showed significant increases in the LMW DNA value. This is, however, expected from the positive control considering the amount of DNA damage it has induced. The fact that all diffusible LMW DNA values from bone marrow and nasal epithelium samples were above the historical control data, does not have an impact on the data interpretation, since the air control values were also above the historical control data range and furthermore, the high LMW DNA values did not have an impact on the % tail intensities, which were all within the historical control data range for these two organs.

Comet assay
The study is considered as valid, since all concurrent negative control data are acceptable for addition to the historical data base. Furthermore, the positive control group showed that the test is able to detect positive response in all assessed tissues.
The assessment of the comet assay in the various tissues did not show and biologically relevant indication of a genotoxic potential in any of the test or reference substances. The significant concentration dependent increase observed in the mean of mean values obtained from the liver of animals treated with T0420 is not considered as relevant, since the same values were not statistically significant, when the mean of medians was used instead of mean of means. This indicates that the observed effect is more likely due to the greater impact of individual values on the group mean value when using the mean of means. The current OECD guideline, therefore, also recommends the use of mean of medians. The statistically significant decreases in the observed in the lung and nasal epithelium using Zinc oxide T0421, micro-scaled Zinc oxide T0242 and Zinc sulfate are not considered as biologically relevant effects, since once again the statistical significance is only observed in either mean of mean or mean of median calculations.

CONCLUSION
In conclusion it can be stated that under the described circumstances neither Zinc oxide T0420 nor Zinc oxide T0421 showed a genotoxic potential in the nasal epithelium, lung liver and bone marrow of rats exposed for 14 days via inhalation.

Executive summary:

In this study two nanosized test substances Zinc oxide T0420 and Zinc oxide T0421 were assessed for their genotoxic potential using the alkaline comet assay after a 14-day exposure period via inhalation. This was a multisite study, where the in-life phase, necropsy, as well as the examination of the lung lavage fluid was performed by the test facility. The processing of the isolated tissues and the comet assay was performed by the test site (Helix 3 Inc.). The target tissues addressed in this study were the nasal epithelium, the lung, the liver as well as the bone marrow.

The concentrations used in this study was based on a separately performed dose range finding assay (BASF Project no.: 36I0050/20I005). The three concentrations ,selected based on the dose range finding study, were 0.5, 2.0 and 8.0 mg/m³.  Wistar rats were exposed whole-body to the indicated concentrations of each test substance for a 6 h period per day for 14 days. In addition, for comparison, a micro-scaled Zinc oxide as well as well as a soluble Zinc salt (Zinc sulfate monohydrate) were tested in parallel under the same conditions at a single (equimolar) concentration. Ethylmethane sulfonate (EMS) was used as a positive control and applied orally at a single time point.

During the exposure period, the animals were observed for signs of toxicity before, during and after the exposure. Body weight was determined once weekly. The following mean concentrations and particle size distribution were determined.

In animals exposed to test item 1, concentration-related increases of lavage parameters were observed, as well as in animals exposed to test item 2. The exposure of single concentration of reference substance 1 and 2 caused significantly increases in most of the lavage parameters. The changes in lavage fluid demonstrate the toxicity in the lungs, which were comparable with the range finding study.

The assessment of the target tissues in the comet assay did not show any biologically relevant increases in the % tail intensity of the analyzed tissues under the indicated conditions. The positive control group showed a distinct and statistically significant increase in all analyzed tissues.

Thus, under the indicated circumstances, the two test substances as well as the reference substances (micro-scaled Zinc oxide and Zinc sulfate monohydrate) are considered as non-genotoxic in this assay.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Zinc oxide

EC Number:

215-222-5

EC Name:

Zinc oxide

Cas Number:

1314-13-2

Molecular formula:

ZnO

IUPAC Name:

oxozinc

Test material form:

solid

Remarks:

micro sized zinc oxide

Details on test material:

micro sized Zinc Oxide
Name of reference substance 1: Zinc oxide T0242
Reference substance No.: 20/0201-1
Batch identification: 56589
Appearance - physical state / color: Solid / white
Storage conditions: Room temperature
BET: 4.48 m2/g

Specific details on test material used for the study:

Expiry date: 01 Mar 2022

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld/Germany)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Assigned to test groups randomly: yes: All animals were randomized before the start of the pre-exposure period (according to weight).
- Fasting period before study: No
- Housing: 5 rats per cage, Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding
- Diet: milled/ mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water: tap water; ad libitum
- Acclimation period: +/- 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 45-65%
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole-body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ or 1.4 m³(BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 40.7 and 50.8 %. Daily mean temperatures in the inhalation systems ranged between 20.4 and 23.0°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 20 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 6.9 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39).
To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

14 days

Frequency of treatment:

14 days, 6 h per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 - air control

Doses / concentrations 2

Dose / conc.:

7.56 mg/m³ air (analytical)

Remarks:

SD: ± 1.88 mg/m³ ; target concentration: 8 mg/m³: Test group 7

No. of animals per sex per dose:

5 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s):
- Route of administration: oral gavage which guarantees systemic distribution of the compound and thus exposure to all assessed tissues
- Doses / concentrations: 300mg/kg body weight

Examinations

Tissues and cell types examined:

Comet assay:
1. Bone marrow
2. Liver
3. Lung
4. Nasal mucosa

Bronchoalveolar lavage fluid (BAL): for cytology and total protein and enzyme levels.

Details of tissue and slide preparation:

COMET ASSAY
Samples were minced or aspirated in cold mincing buffer to produce a cell suspension. Cell suspensions were diluted as necessary and kept cold until they were processed further. At least three comet slides were prepared per sample. An aliquot of cell suspension was mixed with low melting point agarose, layered onto microscope slides precoated with normal melting point agarose, and covered with an additional layer of low melting point agarose. After the agarose had solidified, slides were lysed in cold working high salt lysing solution and maintained cold for at least 1 hour. At least two comet slides were removed from lysis for electrophoresis. Slides were rinsed with 0.4M Tris buffer and submerged in alkaline electrophoresis buffer for 20 minutes at 1 to 10°C to unwind the DNA. After unwinding, slides were electrophoresed in the same buffer at 1 to 10°C for 40 minutes at a constant voltage of 0.7V/cm. The buffer level was adjusted as necessary at the start of electrophoresis to achieve a starting current of 300±10 mA. After electrophoresis, the slides were neutralized with 0.4M Tris buffer, rinsed in ethanol, and air dried. The air-dried comet slides were stored at room temperature at a RH of ≤60% until shipment with desiccant to Helix3 for analysis.

Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System (Andor Technology, Northern Ireland). The image analysis version and settings were documented in the raw data. For each sample ghosts defined as comets with heavily diffused tail and a non-discernable head that cannot be accurately measured by image analysis were counted in parallel with the image analysis scoring. Slides were scored without knowledge of the sample treatment group.

Evaluation criteria:

Criteria for a Valid Test
a. Where no statistically significant (p<0.05) response in DNA damage as measured by %Tail is detected at any test article dose concentration, the concurrent positive control must induce a statistically significant increase in the same genotoxic endpoint when compared to the concurrent vehicle control.

b. The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a statistically significant positive effect.

Criteria for a Positive Response
An experienced scientific investigator classifies a test article as positive, equivocal, or negative for inducing genotoxicity based on the results of the statistical analysis and the biological relevance of the results, taking into consideration the appropriateness of the concurrent control data and the reproducibility of the results in any repeat experiments.

The test article may be classified as positive for inducing genotoxicity if the following criteria are met:

a. a statistically significant increase in DNA damage is detected at one or more dose concentrations and

b. a statistically significant dose dependent response is detected in the same tissue

If cytotoxicity is detected in the same tissue and dose concentration(s) at which a significant increase in DNA damage is detected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity. Where cytotoxicity may be a confounding factor and/or when cytotoxicity is present in all doses tested, a repeat study including lower non-cytotoxic doses may be conducted to evaluate for the presence of genotoxicity in the absence of cytotoxicity.

A test article may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If the results are equivocal and/or the biological relevance of the results are unclear, a repeat study at the same doses may be con

Statistics:

Mean values and standard deviations were calculated. In addition, the median of the values from each slide was determined and for each animal the mean of the median from the slides were calculated. To be consistent with historical control comet data generated at Helix3, the individual animal mean %Tail values was calculated as the mean of the total cells scored. The following statistical analyses were carried out, additionally. For each test, a 95% confidence Interval (P<0.05) was used to determine statistical significance:

Parameter: Statistical test
%Tail and %LMW data distribution: Shapiro-Wilk, test group 0 only

%Tail and %LMW equality of variances: Bartlett test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Normally distributed data with equal variances: Dunnett (2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Line fit trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

Non-normally distributed data or unequal variances: Steel ((2-tailed; test groups 1 to 3 compared with test group 0; test groups 4 to 6 compared with test group 0); Kendall rank trend test (2-tailed; test groups 0 and 1 to 3; test groups 0 and 4 to 6)

%Tail and %LMW References and Positive control only: Fisher’s F-test (2-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0); student’s t-test for equal variances or Welch’s t-test for unequal variances (1-tailed; test group 7 compared with test group 0; test group 8 compared with test group 0; test group 9 compared with test group 0)

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMET ASSAY
- in the nasal epithelium tissue:
The assessment of the DNA damage in the nasal epithelium showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 14.6 – 15.9% (mean of mean values) and 8.3 - 9.3% (mean of medians) and for Zinc oxide T0421 the range was between 15.9 to 16.6% (mean of means) and 8.8 to 10.8% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (16.6 and 10.7% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a lower level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 13.4% (mean of means) and 7.8% (mean of medians). The difference between the mean of mean values was statistically significant. However, the difference between the mean of medians was not. Similarly, Zinc sulfate monohydrate also showed lower tail intensity values (15.4 and 7.6% mean of means and medians, respectively) as compared to the air control. However, in this case the drop in the mean of medians was statistically significant but not the mean of means. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (34.9 and 32.3% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (23.1%) of the historical control data range (mean of means).
- in the lung tissue:
The assessment of the DNA damage in the lung tissue also did not show a biologically relevant alteration of the level of observed DNA damage after treatment with either Zinc oxide T0420 or Zinc oxide T0421 as compared to the air control values (The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means) (see see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 9.9 – 11.5% (mean of mean values) and 4.9 - 6.9% (mean of medians) and for Zinc oxide T0421 the range was between 8.2 to 10.8% (mean of means) and 4.2 to 5.3% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (11.3 and 5.6% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test with T0420, however, a negative trend (dose related decrease in the % tail intensity values) was observed in the mean of mean values of the test groups treated with T0421. This effect was not, however, observed in the mean of median values. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 8.6% (mean of means) and 4.0% (mean of medians) and not statistically significant as compared to the air control values. Zinc sulfate monohydrate showed slightly lower tail intensity values (8.3 and 4.1% mean of means and medians, respectively) as compared to the air control. The drop in the mean of means was statistically significant but not the mean of medians. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (52.8 and 52.4% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (15.3%) of the historical control data range (mean of means).
- in the liver:
The % tail DNA intensity in the liver tissue (see table in any other info on results) ranged between 6.9 – 8.7% (mean of means) and 2.7 to 3.8% (mean of medians) for test groups treated with various concentrations of T0420. The mean of mean value of the test group treated with 8 mg/m3 (8.7 ± 1.08%) was statistically higher than the respective air control value (6.6 ± 1.15%). Furthermore, a concentration related trend was observed in the groups treated with 0.5, 2.0 and 8.0 mg/m3 Zinc oxide T0420. However, the mean of median value at 8.0 mg/m3 (3.8 ± 1.19%) was not statistically higher than its respective air control value (2.8 ± 1.00) and the trend observed when using the mean of means was not observed when using the mean of medians. The mean % tail DNA of test groups treated with Zinc oxide T0421 ranged between 7.7 – 8.7% (mean of mean values) and 3.1-3.6% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.6 and 2.8% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 7.6% (mean of means) and 3.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also did not show significantly altered tail intensity values (7.5 and 3.1% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.1 and 30.7% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (16.9%) of the historical control data range (mean of means).
- in the bone marrow:
The assessment of the DNA damage in the bone showed that neither Zinc oxide T0420 nor Zinc oxide T0421 showed a potential to induce DNA damage (see table in any other info on results). The mean % tail DNA of test groups treated with Zinc oxide T0420 ranged between 6.1 - 6.6% (mean of mean values) and 2.7 – 2.9% (mean of medians) and for Zinc oxide T0421 the range was between 5.2 to 6.6% (mean of means) and 2.6 to 2.9% (mean of medians). The values were not statistically significant as compared to the corresponding air control value (6.2 and 2.9% mean of mean and medians, respectively). A dose response was not observed as determined via the two-tailed trend test. Micro-scaled Zinc oxide T0242 showed a similar level of % tail DNA in the comet assay as compared to the air control values. The mean % tail DNA values were 5.4% (mean of means) and 2.2% (mean of medians) and not statistically significant as compared to the air control values. Similarly, Zinc sulfate monohydrate also showed similar and not statistically significant tail intensity values (6.0 and 2.5% mean of means and medians, respectively) as compared to the air control. The group treated with the positive control (EMS) showed a distinct, statistically significant and biologically relevant increase in the mean % DNA tail intensity values (32.4 and 30.6% mean of means and medians, respectively).
The % tail intensity values (mean of means) of all groups (except for the positive control group) were below the upper range (11.0%) of the historical control data range (mean of means).

CYTOTOXICITY:
The assessment of the potential of the test and reference substances to induce cytotoxicity in the LMW DNA diffusion assay did not show any significant increases in the percentage of diffused low molecular weight DNA in any of the tested tissues and doses (see table in any other info on results), except for micro-scaled Zinc oxide T0242, which induced a statistically higher diffusible LMW DNA (12.6%) as compared to the corresponding air control (8.6%). The positive control (EMS), however, induced statistically significant increases in the amount of diffused LMW DNA in all examined organs.
The comparison of the obtained LMW DNA data with the historical control data showed that all values from the nasal epithelium (including the air control values; 26.8 - 36.8%) were above the maximum value obtained in the historical control data for this tissue (8.0%). In the lung all values (8.4 - 12.6%), except for the positive control group, were below the maximum value of the historical control data range for the lung (17.6%). In the liver the values were similarly below the maximum historical control data range (13.6%), except for the low dose group of T0420, which had a value of 15.2% and the positive control group (38.2%). All bone marrow values (12.0 – 16.8% for the test and reference substances and 34.6% for the positive control group) were also above the upper limit of the historical control data (6.8%).


TOXICITY
- General toxicity: No Signs of general toxicity. No impairment of body weight gain.
- Local effects: In lavage, the exposure of single concentration of reference substance 1 (T0242) and reference substance 2 (zinc sulfate monohydrate) caused significantly increases in most of the lavage parameters, indicating inflammation process in the lung (see table ' Changes in mean total protein and enzyme levels in BAL' in any other info on results)

Any other information on results incl. tables

Table. Results of the comet assay in the nasal epithelium

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	16.6 ± 2.64	10.7 ± 2.89
1	Zinc Oxide T0420	0.5	14.6 ± 3.62	9.3 ± 2.64
2		2.0	15.9 ± 3.60	9.0 ± 2.86
3		8.0	14.8 ± 3.00	8.3 ± 1.69
4	Zinc Oxide T0421	0.5	16.6 ± 5.28	10.8 ± 5.05
5		2.0	15.9 ± 4.73	9.9 ± 4.03
6		8.0	16.0 ± 3.34	8.8 ± 3.91
7	Zinc Oxide T0242	8.0	13.4 ± 2.67^#	7.8 ± 2.13
8	Zinc sulfate monohydrate	18.0	15.4 ± 1.89	7.6 ± 1.28^$
9	EMS	300 mg/kg b.w.	34.9 ± 3.63^*	32.3 ± 4.12^*

^#: statistically significant P=0.048 (decrease)
^$: statistically significant P=0.031 (decrease)
^*: statistically significant P<0.001 (increase)

 

Table. Results of the comet assay in the lung

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	11.3 ± 1.93	5.6 ± 1.50
1	Zinc Oxide T0420	0.5	11.5 ± 3.25	6.9 ± 4.15
2		2.0	9.9 ± 1.62	4.9 ± 1.19
3		8.0	10.1 ± 2.70	5.4 ± 2.89
4	Zinc Oxide T0421	0.5	10.8 ± 3.68^1	5.3 ± 2.67
5		2.0	9.0 ± 1.54^1	4.5 ± 1.34
6		8.0	8.2 ± 1.58^1	4.2 ± 1.60
7	Zinc Oxide T0242	8.0	8.6 ± 2.85	4.0 ± 1.60
8	Zinc sulfate monohydrate	18.0	8.3 ± 1.69^#	4.1 ± 1.51
9	EMS	300 mg/kg b.w.	52.8 ± 6.84^*	52.4 ± 7.84^*

^#: statistically significant P=0.015 (decrease)
^*: statistically significant P<0.001 (increase)
¹: negative trend statistically significant P=0.047

 

Table. Results of the comet assay in the liver

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.6 ± 1.15	2.8 ± 1.00
1	Zinc Oxide T0420	0.5	7.9 ± 0.49^1	3.2 ± 0.53
2		2.0	6.9 ± 1.30^1	2.7 ± 0.38
3		8.0	8.7 ± 1.08^#1	3.8 ± 1.19
4	Zinc Oxide T0421	0.5	8.7 ± 0.91	3.1 ± 0.80
5		2.0	7.8 ± 1.05	3.6 ± 0.73
6		8.0	7.7 ± 2.25	3.1 ± 0.83
7	Zinc Oxide T0242	8.0	7.6 ± 2.03	3.2 ± 1.11
8	Zinc sulfate monohydrate	18.0	7.5 ± 2.03	3.1 ± 1.26
9	EMS	300 mg/kg b.w.	32.1 ± 2.55^*	30.7 ± 3.07^*

^#: statistically significant P=0.016
^*: statistically significant P<0.001
¹: positive trend statistically significant P=0.032

 

Table. Results of the comet assay in the bone marrow

Test group	Test Substance	Concentration (mg/m^3)	% Tail intensity ± SD
			Mean of means	Mean of medians
0	Air control	0	6.2 ± 1.00	2.9 ± 0.66
1	Zinc Oxide T0420	0.5	6.6 ± 1.02	2.9 ± 0.52
2		2.0	6.5 ± 0.78	2.8 ± 0.76
3		8.0	6.1 ± 0.30	2.7 ± 0.42
4	Zinc Oxide T0421	0.5	6.0 ± 0.60	2.9 ± 0.68
5		2.0	6.6 ± 1.63	2.9 ± 0.67
6		8.0	5.2 ± 0.80	2.6 ± 0.75
7	Zinc Oxide T0242	8.0	5.4 ± 0.55	2.2 ± 0.54
8	Zinc sulfate monohydrate	18.0	6.0 ± 0.83	2.5 ± 0.37
9	EMS	300 mg/kg b.w.	32.4 ± 4.84^*	30.6 ± 5.53^*

^*: statistically significant P<0.001

Table. Results of the LMW DNA diffusion assay

Test group	Test Substance	Concentration (mg/m³)	Group Mean ± SD (%)
			Nasal epithelium	Lung	Liver	Bone marrow
0	Air control	0	30.0 ± 9.19	8.6 ± 1.52	12.6 ± 5.37	14.8 ± 2.17
1	Zinc Oxide T0420	0.5	26.8 ± 6.76	11.6 ± 3.29	15.2 ± 6.02	13.6 ± 2.07
2		2.0	33.8 ± 8.98	11.4 ± 2.51	13.2 ± 3.35	14.0 ± 3.54
3		8.0	35.8 ± 16.40	10.6 ± 1.52	13.4 ± 6.95	13.8 ± 5.02
4	Zinc Oxide T0421	0.5	31.6 ± 8.62	11.8 ± 3.56	12.6 ± 6.80	12.0 ± 3.54
5		2.0	36.8 ± 15.67	8.4 ± 3.36	10.0 ± 4.30	16.8 ± 5.89
6		8.0	34.6 ± 14.24	10.4 ± 2.61	11.0 ± 3.74	15.2 ± 5.40
7	Zinc Oxide T0242	8.0	29.0 ± 11.05	12.6 ± 4.22^€	11.8 ± 4.09	15.6 ± 5.98
8	Zinc sulfate monohydrate	18.0	33.0 ± 8.80	12.0 ± 5.92	10.6 ± 7.64	14.6 ± 6.66
9	EMS	300 mg/kg b.w.	39.8 ± 4.49^*	26.8 ± 13.37^#	38.2 ± 7.05^$	34.6 ± 6.73^$

^*: statistically significant P=0.032
^€: statistically significant P=0.041
^#: statistically significant P=0.019
^$: statistically significant P=0.032

 

Table. Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats after inhalation exposure to Zinc oxide T0242 and zinc sulfate monohydrate on 14 consecutive days

Analyte	Gr. 7 8 mg/m^3	Gr8 18 mg/m^3
Total Protein	7.6**	2.4**
GGT	4.2**	3.9**
LDH	13.2**	4.6**
ALP	19.1**	4.3**
NAG	2.4**	1.5

GGT = γ-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = β-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

Applicant's summary and conclusion

Conclusions:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 0.5, 2 and 8 mg/m³, as well as to 8 mg/m³ miconsize zinc oxide or 18 mg/m³ zinc sulfate monohydrate for 6 hours daily on 14 consecutive days. A concurrent control group was exposed to conditioned air.

The tested atmospheric were met and they were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes within the respirable range. There were no signs of toxicity, nor were there any impairment of body weight gain. In the range finding 14-day study (BASF project no.: 36I0050/20I005) animals of the mid dose groups were nominally exposed to 12 mg/m3. The actual assessment of test substance concentrations, however, revealed an exposure to 10.9 and 10.8 mg/m3 for T0420 and T0421, respectively. At this dose level significant histopathological alterations (grade 2 degeneration/regeneration, olfactory epithelium) as well as alterations indicative of inflammatory responses in the lung were observed, which could have had a confounding impact on the outcome of the comet assays results. Thus, the top exposure concentration was slightly reduced to 8 mg/m3. At this level in the lavage fluid, several parameters were increased in a concentration related manner in animals exposed to T0420 and T0421, indicating inflammation process in the lung. These findings were comparable with those observed previously in the range finding 14-day inhalation study with these two test materials. Similar findings were observed in animals exposed to the reference substances.


LMW DNA diffusion assay
In all test groups using the test or reference substances, the quantification of the diffused low molecular weight DNA as a measure for cytotoxicity did not show any indication of excessive cytotoxicity as compared to the respective air control value, which could have hampered the interpretation of the comet assay data. The significantly increased value observed for the lung using micro-scaled Zinc oxide T0242 was not high enough to interfere the comet data evaluation. The positive control group, however, showed significant increases in the LMW DNA value. This is, however, expected from the positive control considering the amount of DNA damage it has induced. The fact that all diffusible LMW DNA values from bone marrow and nasal epithelium samples were above the historical control data, does not have an impact on the data interpretation, since the air control values were also above the historical control data range and furthermore, the high LMW DNA values did not have an impact on the % tail intensities, which were all within the historical control data range for these two organs.

Comet assay
The study is considered as valid, since all concurrent negative control data are acceptable for addition to the historical data base. Furthermore, the positive control group showed that the test is able to detect positive response in all assessed tissues.
The assessment of the comet assay in the various tissues did not show and biologically relevant indication of a genotoxic potential in any of the test or reference substances. The significant concentration dependent increase observed in the mean of mean values obtained from the liver of animals treated with T0420 is not considered as relevant, since the same values were not statistically significant, when the mean of medians was used instead of mean of means. This indicates that the observed effect is more likely due to the greater impact of individual values on the group mean value when using the mean of means. The current OECD guideline, therefore, also recommends the use of mean of medians. The statistically significant decreases in the observed in the lung and nasal epithelium using Zinc oxide T0421, micro-scaled Zinc oxide T0242 and Zinc sulfate are not considered as biologically relevant effects, since once again the statistical significance is only observed in either mean of mean or mean of median calculations.

CONCLUSION
In conclusion it can be stated that under the described circumstances neither Zinc oxide T0420 nor Zinc oxide T0421 showed a genotoxic potential in the nasal epithelium, lung liver and bone marrow of rats exposed for 14 days via inhalation.

Executive summary:

In this study two nanosized test substances Zinc oxide T0420 and Zinc oxide T0421 were assessed for their genotoxic potential using the alkaline comet assay after a 14-day exposure period via inhalation. This was a multisite study, where the in-life phase, necropsy, as well as the examination of the lung lavage fluid was performed by the test facility. The processing of the isolated tissues and the comet assay was performed by the test site (Helix 3 Inc.). The target tissues addressed in this study were the nasal epithelium, the lung, the liver as well as the bone marrow.

The concentrations used in this study was based on a separately performed dose range finding assay (BASF Project no.: 36I0050/20I005). The three concentrations ,selected based on the dose range finding study, were 0.5, 2.0 and 8.0 mg/m³.  Wistar rats were exposed whole-body to the indicated concentrations of each test substance for a 6 h period per day for 14 days. In addition, for comparison, a micro-scaled Zinc oxide as well as well as a soluble Zinc salt (Zinc sulfate monohydrate) were tested in parallel under the same conditions at a single (equimolar) concentration. Ethylmethane sulfonate (EMS) was used as a positive control and applied orally at a single time point.

During the exposure period, the animals were observed for signs of toxicity before, during and after the exposure. Body weight was determined once weekly. The following mean concentrations and particle size distribution were determined.

In animals exposed to test item 1, concentration-related increases of lavage parameters were observed, as well as in animals exposed to test item 2. The exposure of single concentration of reference substance 1 and 2 caused significantly increases in most of the lavage parameters. The changes in lavage fluid demonstrate the toxicity in the lungs, which were comparable with the range finding study.

The assessment of the target tissues in the comet assay did not show any biologically relevant increases in the % tail intensity of the analyzed tissues under the indicated conditions. The positive control group showed a distinct and statistically significant increase in all analyzed tissues.

Thus, under the indicated circumstances, the two test substances as well as the reference substances (micro-scaled Zinc oxide and Zinc sulfate monohydrate) are considered as non-genotoxic in this assay.