Potassium trifluorozincate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, the Netherlands

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Potassium trifluorozincate

EC Number:

237-537-7

EC Name:

Potassium trifluorozincate

Cas Number:

13827-02-6

Molecular formula:

F3Zn.K

IUPAC Name:

potassium trifluorozincuide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nocoloc Zn Flux
- Chemical name : Potassium trifluorozincate, KZnF3
- Appearance: white powder
- Batch number: BWF 91112
- Purity: ≥99%
- Storage conditions: ambient temperature
- Expiry date: 31 December 2012

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories
- Age at study initiation: 7 weeks
- Weight at study initiation: 242g
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and a wooden block and strips of paper (Enviro-dri) as environmental enrichment (3-5 animals per cage).
- Diet: ad libitum, a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diet Services, Whitham, England).
- Water:ad libitum, Tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Air changes (per hr): ca. 10
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%,
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

- Humidified compressed air.

Details on exposure:

The study comprised four groups, viz. two test groups exposed to Nocolok Zn flux at two concentrations, one negative control group (clean air) and one positive control group.

Exposure equipment:
The animals were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical polypropylene (group 1; P. Groenendijk Kunststoffen BV), aluminium (a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) or stainless steel ( a modification of the design of the chamber manufactured by ADG Developments Ltd.) column, surrounded by a transparent cylinder. The columns had a volume of approximately 40-50 litres and consisted of a top assembly with the entrance of the unit, one or two mixing chambers, one or two rodent tube sections and at the bottom the base assembly with the exhaust port. The rodent tube sections had 20 ports for animal exposure. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. Several empty ports were used for test atmosphere sampling, and measurement of temperature and relative humidity. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. It was noted that the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. Animals were rotated with respect to their position in the column, viz. they were moved 5 places each week. The units were illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the units was at least 1 litre/min for each rat. The air entering the units was controlled at 22 ± 2°C and the relative humidity was maintained between 30 and 70%.

Generation of the test atmosphere:
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. A test atmosphere was generated in a separate base exposure unit (in which no animals were exposed) at a concentration higher than the required concentrations by aerosolization of the test material using a turntable dust feeder (Reist and Taylor, 2000) and an eductor (Fox Valve Development Corp., Dover, NJ, USA; Cheng et al., 1989). The test material was aerosolized in the eductor, which was placed at the top inlet of the base exposure unit and was supplied with humidified compressed air. From the base exposure unit, parts of the test atmosphere were extracted using eductors (Fox Valve Development Corp., mounted in the rodent tube section of the base unit) to dilute and transport the test atmospheres towards the top inlet of the exposure units. In the exposure units, the test atmospheres were mixed with a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, The Netherlands) stream of humidified compressed air. The resulting aerosol was directed downward and led to the noses of the animals. At the bottom of the units, the test atmosphere was exhausted. The eductors were calibrated by measuring the total air flow, with and without the aspiration air flow, at a range of driving air pressures encompassing the driving pressures used. At the settings of the eductors and mass flow controllers – recorded hourly during expsosure – the total airflow through the exposure units was at least 17.7, 24.5, and 101.0 L/min for the 2, 8 mg/m3 and base exposure units, respectively. The exposure chamber for the control animals (group 1) was supplied with a stream of humified air only, which was controlled by a rotameter at a flow of approximately 26 L/min.

Since the aerodynamic particle size of the original test material (as delivered by the sponsor) was above the range of 1-3 μm recommended by OECD guideline 412, the test material was milled using a ball mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany) fitted with a zirconium beaker and 99 zirconium balls with a diameter of 10 mm. Among a few alternative milling scenarios, a three-fold repetition of milling during 5 minutes at a speed of 350 rotations per minute followed by a 1-minute pause with reversal of the direction of rotation after each pause resulted in a powder that could be aerosolized with a MMAD (mass median aerodynamic particle size) below 3 μm.

Time to attain chamber equilibration (T95):
The animals were placed in the exposure unit well after stabilization of the test atmosphere, at least 25 minutes after the start of atmosphere generation. The increase in concentration C (mg/L) in a perfectly stirred test atmosphere in a chamber with volume V (L) and flow F (L/min) is calculated by: C = C∞ * (1 – e-(F*T/V)), where T (min) is the time and C∞ is the steady state concentration. The time it takes to reach 95% of the steady state concentration (T95) follows from (1 – e-(F*T95/V)) = 0.95; thus T95 = 3V/F. Assuming the worst case scenario – high chamber volume (i.e. 50 L) and low flow (i.e. 17.7 L/min) – T95 was calculated to be 8.5 minutes. However, since the test atmospheres were extracted and diluted from a base exposure unit, T95 might have been slightly longer than this theoretical value.

Nominal concentration:
The nominal concentration of the base exposure unit was determined by dividing the total amount of test material used (by weighing) by the total volume of air passed through the exposure unit. The generation efficiency was calculated from the actual and the nominal concentration. Because of the chosen generation set-up (i.e. a base unit from which the test atmospheres for the individual exposure units is extracted and diluted), it was not possible to determine nominal concentrations for the individual exposure units.

Particle size measurement:
Particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA) once weekly and at least once during preliminary generation of the test atmosphere for each exposure condition. During preliminary test atmosphere generation the results of the APS were compared to particle size measurements obtained using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA). The cascade impactor was not used during exposure, since the relatively low target concentrations would require very long sampling periods. On 23 January 2012, the Mass Median Aerodynamic Diameter (MMAD) of the 8 mg/m3 concentration test atmosphere was determined to be 1.73 μm (with a geometric standard deviation – gsd – of 1.99) using the cascade impactor, compared to 1.88 μm (gsd of 1.62) using the APS. Similarly, on 24 January 2012, the MMAD of the 2 mg/m3 concentration test atmosphere was determined to be 1.65 μm (gsd of 2.34) using the cascade impactor, compared to 1.76 μm (gsd of 1.64) using the APS. Based on these results, the APS was considered to be suitable for particle size measurements during exposure. The MMAD and gsd were calculatedaccording to Lee (1972).

Total air flow, temperature and relative humidity:
The total airflow, temperature and relative humidity were recorded hourly during exposure. The airflow was monitored by recording the settings of the rotameter (group 1), mass flow controllers and the driving air pressure of the eductors (groups 2-4 and the base unit). Temperature and relative humidity were measured using an RH/T device (Testo 610 or 635, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany).

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week (i.e. 20 exposure days in total)

Post exposure period:

16-24 hr

Doses / concentrations

Remarks:

Doses / Concentrations:
2 and 8 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Six animals (including one reserve animal) were treated as a positive control group. The animals were dosed by gavage (12-16 hour prior to sacrifice) with the mutagen 2-acetamidofluorene (2-AAF) (50 mg/kg-bw, 2.5 mg/mL in corn oil).

Examinations

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

- Preparation of hepatocyte cultures: Within 16-24h after exposure, animals were sacrificed for isolation of hepatocytes. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a Ca2+- and Mg2+-free HEPES buffer (0.01 M) whilst under anaesthesia with sodium pentobarbital and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution. Directly after the start of the perfusion with the Ca2+ and Mg2+ free HEPES buffer to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10 % solution of formalin) for histopathological examination. After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking waterbath at ca 37 °C. Thereafter, they were filtered over a 200 mesh nylon filter, centrifuged and resuspended in WEC medium [= Williams medium E complete, which consisted of Williams medium E containing Glutamax supplemented with 10 % foetal calf serum and gentamycin (50 μg/mL)]. Cell counts were made. The viability of the hepatocytes was determined by trypan blue exclusion. The viability of the hepatocytes of the negative control animals was at least 85%, and therefore considered sufficient. Suspensions containing 5E5 cells/mL were prepared in WEC medium. Aliquots (1 mL) were seeded onto Thermanox 25 mm round plastic cover slips in 35 mm 6-well dishes, which already contained 1 mL of WEC medium. The cultures were then incubated at ca 37 ºC in a humidified incubator containing ca 5 % CO2 and 95 % air to allow cells to attach (2-4 h).
- Labelling of hepatocyte cultures: Two to four hours after seeding of the cells, the medium was removed and cells were washed twice with Williams E medium leaving only attached viable cells. Immediately after washing, 2 mL WEI [= Williams E medium Incomplete, which consisted of Williams medium E containing Glutamax, gentamycin (50 μg/mL), insulin (8 μg/mL), hydrocortisone (36 μg/mL) and ca 10 μCi [methyl-3H]thymidine (specific activity: 84.4 Ci.mmol-1 [3123 GBq/mmol, aqueous solution of 1.0 mCi/mL]) per mL was added to the cultures. The hepatocyte cultures were incubated for 16 to 20 hours at ca 37 ºC. Thereafter, the cover slips were rinsed three times with Williams E medium. The cover slips were then immersed in 2 mL of a 1 % sodium citrate solution for 10 min to allow cells to swell. Subsequently, cells were fixed in three 30 min changes of absolute ethanol-glacial acetic acid (3:1), air-dried, and mounted on glass slides.
- Autoradiography: Slides were processed for autoradiography using Ilford K5D emulsion. At two time points, 7 and 10 days of exposure at < –18 ºC, slides were developed, fixed and washed with water. Slides were stained with haematoxylin and eosin and coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. The slides that were developed after 7 days of exposure were chosen to be analyzed because these slides were considered of better quality than the slides that were developed 10 days after exposure.
- Grain counting and calculations: Sorcerer UDS software (Perceptive Instruments, UK) with microscopic attachment (Zeiss microscope connected to a high resolution camera) was used for grain counting of nuclei and cytoplasm. Fifty cells (randomly selected from each quadrant of the cover slip) per slide and 2 (out of 3) slides per animal were evaluated. The remaining slide of each animal was kept in reserve. Cells with abnormal morphology (pyknotic or lysed nuclei), or heavily-labelled S-phase cells were excluded from evaluation. Cytoplasmic labelling was determined by counting two nuclei-sized area of cytoplasm adjacent to the nucleus. The mean cytoplasmic count was subtracted from the nuclear count to give the net nuclear grains (NNG).

Evaluation criteria:

- The study is considered valid if the positive controls give a positive response and if the negative control is non-genotoxic.
- Cells are considered “in repair” if the NNG value is ≥ 5.
- A response at a data point is considered positive if at least 20 % of the cells are “in repair”.
- A response is considered weakly positive if the population average is between 0 to 5 NNG.
- A test substance is considered to cause DNA damage and induce DNA repair in liver cells if the dose levels result in a positive or weakly positive response.
- A test substance is considered non-genotoxic under the conditions of the test if the dose levels produce NNG ≤ 0.

Both numerical significance and biological relevance are considered in the evaluation ofthe results.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

systemic toxicity observed at the highest concentration tested.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The positive control substance 2-AAF induced the expected increase in unscheduled DNA synthesis in rat hepatocytes. The negative control group demonstrated the expected response. This demonstrates the validity of the study.
- The mean NNG determined for the mid and high dose group of the test substance (- 10.09 and -12.48, respectively) were comparable to the mean NNG determined for the negative control (-11.81) and clearly lower than the mean NNG found in the positive control group (+14.40). The percentage ‘cells in repair’ of the mid and high dose group of the test substance (0.40% and 0.20%) was comparable to the negative control group (0.20 % ‘cells in repair’) and clearly lower than the percentage found in the positive control group (89.60 %). This demonstrates that the test substance did not induce unscheduled DNA synthesis. Analysis of fluoride levels (as a marker for the test substance) in urine indicated systemic availability of the test compound upon inhalation exposure. Therefore, there is no reason to assume that the negative responses observed in the animals treated with the test substance were due to lack of systemic exposure.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that the test substance Nocolok Zn flux did not induce unscheduled DNA synthesis (UDS) in hepatocytes of male rats, exposed to the test substance by inhalation, under the conditions used in this study.

Executive summary:

In a GLP compliant unscheduled DNA synthesis-test performed according to OECD Guideline 486, Nocolok Zn Flux was examened for its portential to damage DNA as reflected by induced DNA synthesis to repair the damage using liver hepatocytes harvested from exposed male Wistar rats. The study comprised of four groups, viz. two test groups (2 and 8 mg/m3) exposed to Nocolok Zn flux at two concentrations, one negative control group (clean air) and one positive control group. The mean NNG determined for the mid and high dose group of the test substance (-10.09 and -12.48, respectively) were comparable to the mean NNG determined for the negative control (-11.81) and clearly lower than the mean NNG found in the positive control group (+14.40). The percentage ‘cells in repair’ of the mid and high dose group of the test substance (0.40% and 0.20%) was comparable to the negative control group (0.20 % ‘cells in repair’) and clearly lower than the percentage found in the positive control group (89.60 %). Analysis of fluoride levels (as a marker for the test substance) in urine indicated systemic availability of the test compound upon inhalation exposure. It is therefore concluded that the test substance Nocolok Zn flux did not induce unscheduled DNA synthesis (UDS) in hepatocytes of male rats, exposed to the test substance by inhalation, under the conditions used in this study.