Potassium trifluorozincate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP-compliant non-guideline study, 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:

no guideline followed

Principles of method if other than guideline:

Dose-range finding study for Sub-acute (28-day) inhalation study. Four groups of (3 male/3 female) wister rats were exposed to 0, 2, 10 and 50 mg/m3 Nocolok Zn Flux in a 7-day range finding study.

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48 3704 HE Zeist, the Netherlands

Test type:

other: Range-finding study

Limit test:

no

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): Nocolok 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/female

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: 256 g for males, 168 g for females
- 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 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, domestic mains tap-water suitable for human consumption
- Acclimation period: 7 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

Type of inhalation exposure:

nose only

Vehicle:

clean air

Details on inhalation exposure:

- Exposure equipment: The animals of groups 1-4 were exposed to the test atmosphere in nose-only inhalation units consisting of a cylindrical polypropylene (group 1; P. Groenendijk Kunststoffen BV), aluminium (groups 2 and 4; a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) or stainless steel (group 3; 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 previously noted that the animal's body often 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: 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 mid, and high concentration exposure units. For this range finding study, the low concentration test atmosphere was extracted from the mid concentration exposure unit, rather than from the base unit. In the low, mid, and high concentration 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 22.1, 46.7, 22.6, and 101.7 L/min. 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.
- Actual concentration: The actual concentration of Nocolok Zn Flux in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min through fiber glass filters (Sartorius, 13400- 47). Samples of 460, 92, 36.8 (or 18.4 during the first two days when the target concentration was 50 g/m3) and 5.0 Ln were taken for groups 2, 3, 4 and the base unit of the range finding study, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition, including the base exposure unit.
- 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 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 3 was not used during exposure, since the relatively low target concentrations would require very long sampling periods. The Mass Median Aerodynamic Diameter (MMAD) of the high 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, one day later, the MMAD of the mid 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 calculated according 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).

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetric analysis

Remarks on duration:

6 hrs/day 5 days/week

Concentrations:

- Nominal concentrations: 0, 2, 10 and 50 mg/m3. Due to unexpected exposure-related mortality in the 50 mg/m3 group after the first exposure day, the target concentration was reduced to 25 mg/m3 as from the third exposure day. On the second exposure day, instead of reducing the target concentration, the exposure period for group 4 was reduced from 6 to approximately 3 hours.
- Actual concentrations: The mean actual concentrations (± standard deviation) as determined by gravimetric analysis were 2.02 (± 0.14) and 10.18 (± 0.94) mg/m3 for the low and mid concentration groups, respectively. Due to unexpected mortality in the high concentration group, the target concentration was reduced from 50 to 25 mg/m3 as from the third exposure day. The mean actual concentrations were 56.43 (± 8.57) and 23.51 (± 3.95) mg/m3 during the first two and the last three exposure days of the high concentration group, respectively. The mean actual concentration (± standard deviation) in the base exposure unit was 287.32 (± 33.18) mg/m3.

No. of animals per sex per dose:

3 males, 3 females

Control animals:

yes

Details on study design:

The 7 day range finding study, as part of a sub-acute (28-day) inhalation toxicity study, comprised four test groups of 3 male and 3 female rats each, viz. one negative control group and three groups exposed to different concentrations of Nocolok Zn Flux. The negative control group was exposed to clean air, but otherwise treated in the same manner as the exposed groups. The animals were exposed for 6 hours per day, 5 days per week, over a 7-day period (5 exposure days in total). Animals were sacrificed on the day after the last exposure.

OBSERVATIONS
- Clinical signs: Animals of groups 1-4 were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway through each exposure day. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. The observations included, but were not restricted to, the signs listed in Annex 6 of the studie report. During exposure however, observation was limited due to the animals’ stay in restraining tubes and attention was directed towards any breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.
- Body weights: The body weight of each animal was recorded one or two (days before the start of exposure and prior to exposure on the first day (day 0). Subsequently, animals were weighed on days 3 and 6. All animals were also weighed on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.
- Food consumption: Food consumption of animals of groups 1-4 was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured for a single 6-day period, starting on day 0.
- Fluoride analysis in urine: To investigate the systemic availability of the test material, urine was collected and analyzed for fluoride content. On the day before necropsy, all animals were transferred to stainless-steel metabolism cages (one animal per cage) for overnight urine collection. Prior to overnight collection of urine samples, the individual metabolism cages were washed with a solution of nitric acid in deionised water (1:20 v/v dilution), rinsed with deionised water and air dried. In addition, the urine separators were treated similarly. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. Urine samples were stored at <-18ºC until analysis. The concentration of fluoride was determined in urine samples of animals of the control and high concentration group by a gas chromatographic method based on Fresen et al. (1968). In short, after acidification of the samples with concentrated sulphuric acid, fluoride was selectively liberated using a solution of trimethylchlorosilane (TMCS) in toluene. The concentration of trimethylfluorosilane formed was measured by GC analysis with head space injection.
- Haematology: Blood samples were collected of all surviving animals of the range finding study for possible future analysis, in case systemic availability of the test material could not be determined by fluoride analysis in urine. To this end, blood was sampled from the tail tips of all animals of the range finding study just after exposure on the day of the last exposure. Blood samples were collected in heparinized tubes (one sample per animal) and stored at <-18°C. Since fluoride analysis in urine proved to be suitable for analysis of systemic availability, these blood samples were discarded without further analysis.
- Pathology: Gross examination: At the end of the exposure period, surviving animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes. A thorough autopsy was also performed on the animals that died intercurrently. The following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying: adrenals, heart, kidneys, liver, spleen, testes, lungs with trachea and larynx. The tissues and organs were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin).
- Histopathological examination: The nose, larynx and lungs of all animals of the control group and the high concentration group (groups 1 and 4) were examined histopathologically (by light microscopy). The tissues were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin. The nasopharyngeal tissues were examined at 6 levels (Woutersen et al., 1994) with 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT). The larynx was examined at 3 levels (1 level to include the base of the epiglottis), and each lung lobe at 1 level. Since treatment-related changes were observed in animals of the high concentration group, examination of the nose, larynx and lungs was extended to animals of the intermediate concentration groups.

Statistics:

- Body weight data: Ancova & Dunnet`s Test. (See any other information on materials and methods)
- Organ weight data: Generalised Anova/Ancova Test. (See any other information on materials and methods)

Fluoride data in urine were checked for homogeneity of variances (Levene test) and subsequently evaluated by Anova after log-transformation.
Tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01. Because numerous variables are subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results is based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results
are significant in the light of other biological and pathological findings.

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

other: LC33

Effect level:

50 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

6 h

Effect levels 2

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 75 mg/m³ air (nominal)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Applying Haber's rule on the 6 hour LC33

Mortality:

One female and one male animal of the high concentration group were found dead on days 2 and 3, respectively.

Clinical signs:

other: Surviving animals of the high concentration group showed restlessness during exposure, which was reduced to from 6 to approximately 3 hours in response to the observed mortality. Shortly after exposure, these animals demonstrated blepharospasm, piloerecti

Body weight:

Substantial body weight loss was observed in the high concentration group after the first exposure. Upon reducing the target concentration, body weights slightly recovered, but were still decreased relative to controls at the end of the exposure, reaching statistical significance in males only. No exposure-related effects on growth were observed in the low and mid concentration groups.

Gross pathology:

Macroscopic examination: No macroscopic abnormalities were observed in the female animal of the high concentration group which was found dead on day 2 . Macroscopic examination of the male animal of the high concentration group which was found dead on day 3 revealed dilated atria, red lungs and a nasal haemorrhagic discharge. At the end of the exposure period, the remaining animals of the high concentration group showed spongy lungs. The other macroscopic observations were unremarkable.
Organ weights: Absolute lung weights were statistically significantly increased in males and females of the high concentration group, and in females of the mid concentration group. Relative lung weights were also clearly elevated in animals of the high concentration group. This was, however, not confirmed by statistical analysis, probably because of the low number of animals in the high concentration group and the choice of rank transformation by the decision tree. No significant differences in weights of the other organs were found between exposed rats and controls.

Other findings:

Food consumption: Decreased food consumption was observed in males and females of the high concentration group.
Microscopic examination: The animal found dead on day 2 showed hyperaemia, focal haemorrhages, oedema and slight interstitial pneumonia in the lungs. In the nasal cavity, slight olfactory epithelium degeneration and slight rhinitis was observed. The animal found dead on day 3 showed hyperaemia, multifocal haemorrhages and moderate interstitial pneumonia in the lungs. In the nasal cavity, slight olfactory epithelial degeneration and slight rhinitis was observed. In agreement with the observed haemorrhagic discharge, blood was found in the nasal cavity. The remaining animals of the high concentration group showed slight to moderate interstitial pneumonia. Focal alveolitis was found in one animal of the mid concentration group and two animals of the low concentration group. Slight degeneration of the olfactory epithelium was found in the nasal cavity of the remaining animals of the high concentration group. All animals of the midconcentration group, showed olfactory epithelial degeneration as well, albeit to a lesser extent. Olfactory epithelial degeneration was not observed in the control or low concentration groups. The other histological observations were unremarkable.

Applicant's summary and conclusion

Interpretation of results:

very toxic

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

A single 6-hour exposure to a target concentration of 50 mg/m3 Nocolok Zn Flux resulted in mortality in 2/6 animals. Using Haber's rule, the LC50 is therefore estimated to be greater then 75 mg/m3 using Haber's rule.

Executive summary:

In a GLP-compliant 7-day range finding study groups of 3 animals/sex were exposed to target concentrations of 0 (control), 2, 10 or 50 mg/m3 Nocolok Zn Flux. In the weekend following the first 6-hour exposure to 50 mg/m3, 2/6 of the animals of the high concentration group were found dead. Histopathological examination revealed hyperaemia, haemorrhages and interstitial pneumonia in the lungs, and olfactory epithelial degeneration and rhinitis in the nasal tissues of these animals. After a reduced 3-hour exposure period to 50 mg/m3 Nocolok Zn Flux on the second exposure day, the target concentration for the high concentration group was reduced to 25 mg/m3 for the remainder of the study. The surviving animals of the high concentration group showed decreased body weight gain and food consumption; increased lung weights and spongy lungs at necropsy; and histopathological examination revealed interstitial pneumonia and degeneration of olfactory epithelium in the nasal cavity. At the mid concentration level, increased lung weights were observed in females, all animals showed olfactory epithelial degeneration (though less severe than at the high concentration), and one female also showed focal alveolitis. The focal alveolitis was also found in the lungs of two animals of the low concentration group, exposed to a target concentration of 2 mg/m3 Nocolok Zn Flux. The LC33 is therfore 50 mg/m3. If Haber's rule is applied, the 4 -hour LC33 would be 75 mg/m3. The LC50 will be above this concentration.