Potassium trifluorozincate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment. For KZnF3 a new chemical substance notification in China is ongoing under the Regulation ‘Measures for the Environmental Management of New Chemical Substances' (Ministery of Environment Order No. 7 also known as ‘China REACH’). Under this regulation a 90-day study is part of the data requirements for substances that are produced or imported in volumes > 10 t/y. For this reason a 90-day inhalation study with KZnF3 was performed in 2013 at TNO. The results of the study were included in the dossier and serve as the valid testing related to this endpoint. For this reason no testing proposal for the 90-day study was included in the orginally submitted dossier.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes (incl. QA statement)

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

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 2013

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: 8 weeks
- Weight at study initiation: main study: 274 g (males) and 181 g (females),
- Housing: in macrolon cages with a bedding of wood shavings and strips of paper as environmental enrichment; 5 animals of the same sex per cage
- Diet: Rat & Mouse No. 3 Breeding Diet, RM3, ad libitum, except during exposure and fasting period prior to scheduled sacrifice
- Water: domestic tap water suitable for human consumption, ad libitum, except during exposure and fasting period prior to scheduled sacrifice
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: - Particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS, model 3321, TSI Incorporated, Shoreview, MN, USA) 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. The average particle size (Mass Median Aerodynamic Diameter; MMAD) was 1.43 µm (gsd of 1.52), 1.60 µm (gsd of 1.57) and 1.82 µm (gsd of 1.67) for the low, mid and high concentration test atmospheres, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure units consisting of a cylindrical polypropylene (group 1; P. Groenendijk Kunststoffen BV) or a stainless steel column (groups 2, 3 and 4; a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) surrounded by a transparent cylinder
- Method of holding animals in test chamber: plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column
- Source of air: humidified compressed air
- System of generating particulates/aerosols: 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 low, mid, and high concentration exposure units. 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 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 45 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 413, 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.
- Method of particle size determination: 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. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated
- Temperature and humidity in exposure chamber: 23.7 (± 0.6)°C, 21.9 (± 0.3)°C, 21.9 (± 0.3)°C and 22.8 (± 0.5) °C for the control, low, mid and high concentration groups, resp.; 37.5 (± 2.2)%, 38.3 (± 2.5)%, 40.8 (± 1.4)% and 41.1 (± 3.8) % for the control, low, mid and high concentration groups, resp.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration (by weight) of the Nocolok Zn Flux in the test atmospheres was determined at least three times per day during each exposure by means of gravimetric analysis.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 1380, 460, 276 (or 138 for measurement on 9 and 10 January 2013) and 46 (23 for all but the first measurement on 9 and 10 January 2013) Ln test atmosphere were obtained for groups 2, 3, 4 and the base exposure unit, 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 except for group 2. Since a minimum amount of test material should be present on each filter to allow accurate weighing, the low target concentration for this group meant that only one sample – encompassing almost the complete exposure period – could be taken during each exposure.
- Results: The mean actual concentrations (± standard deviation) as determined by gravimetric analysis were 1.09 (± 0.39), 3.05 (± 0.47) and 10.48 (± 2.75) mg/m3 for the low, mid and high concentration groups, respectively. These mean concentrations were relatively close to the respective target concentrations of 1, 3 and 10 mg/m3 Nocolok Zn Flux. The mean actual concentration (± standard deviation) in the base exposure unit was 161.97 (± 20.38) mg/m3.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1, 3 and 10 mg/m3
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.09 (± 0.39), 3.05 (± 0.47), and 10.48 (± 2.75) mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: based on the results of a previous sub-acute (28-day) inhalation study
- Rationale for animal assignment (if not random): computer randomization proportionally to body weight (males and females separately)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On exposure days, each animal was observed daily in the morning, prior to exposure, by cage-side observation and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure, a group-wise observation was made about half-way through the 6-hour exposure period. In weekends and on public holidays only one check per day was carried out. During exposure,
attention was paid to any breathing abnormalities and restlessness.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ophthalmoscopic observations were made prior to the start of treatment in all animals (on day -2) and towards the end of the exposure period in the animals of the control and high concentration main groups (males on day 83; females on day 84). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Because treatment-related ocular changes were not observed in the high concentration main group, the eye examinations were not extended to the animals of the intermediate concentration groups or to the recovery groups

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded one day (males) or two days (females) before the start of exposure and prior to exposure on the first day (day 0). Subsequently, animals of the range finding study were weighed twice weekly for the first four weeks (Mondays and Fridays). Thereafter, the frequency was reduced to once weekly (Fridays), because there were no statistically significant effects on body weight in the first four weeks. At the end of the in-life phase, the animals were weighed on the day before overnight fasting prior to their scheduled sacrifice, and on the day of sacrifice in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
- Food consumption was measured per cage by weighing the feeders. The consumption was measured over 7-day periods, except at the start (a 5-day period followed by a 4-day period for males; two 4-day periods for females) and at the end of the exposure period (a 5-day period for males; a 6-day period for females) and at the end of the recovery period (a 3-day period for males and a 2-day period for females). The results were expressed in g per animal per day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy from the abdominal aorta of overnight fasted rats. Since no treatment-related changes in haematology parameters were observed in animals of the main study, haematology was not extended to animals of the recovery groups.
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: Yes, overnight
- How many animals: all animals of the main study
- Parameters examined: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, trombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy from the abdominal aorta of overnight fasted rats. Since no treatment-related changes in clinical chemistry parameters were observed in animals of the main study, clinical chemistry was not extended to animals of the recovery groups.
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: yes, overnight
- How many animals: all animals of the main study
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, the weights of the following organs were determined: adrenals, brain, epididymides, heart, kidneys, liver, lungs with trachea and larynx, spleen, testes, thymus, thyroid, ovaries, uterus
HISTOPATHOLOGY:
For histopathological examination, samples of the following tissues and organs of all animals of groups 1-4 were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation.
- adrenals,
- aorta,
- axillary lymph nodes,
- brain (brain stem, cerebrum, cerebellum),
- caecum,
- colon,
- epididymides,
- eyes (with optic nerve),
- exorbital lachrymal glands,
- femur with joint,
- heart,
- kidneys,
- liver,
- lungs/trachea/larynx,
- mammary glands (females),
- cervical lymph nodes,
- nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth),
- nerve peripheral,
- oesophagus,
- olfactory bulb,
- ovaries,
- pancreas,
- parathyroids,
- pharynx,
- parotid salivary glands,
- pituitary,
- prostate,
- rectum,
- seminal vesicles,
- skeletal muscle,
- skin (flank),
- small intestines,
- spinal cord (cervical, mid-thoracic, and lumbar),
- spleen,
- sternum with bone marrow,
- stomach,
- sublingual salivary glands and submaxillary salivary glands,
- testes,
- thymus,
- thyroid,
- tongue,
- tracheobroncial (mediastinal) lymph nodes,
- ureter,
- urethra,
- urinary bladder,
- uterus (with cervix).

Slide preparation
Tissues to be examined were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.
 
Histopathological examination
All preserved tissues of all animals of the control and high concentration groups were examined histopathologically (by light microscopy). In addition, all relevant gross lesions observed in rats of the intermediate concentration groups were examined microscopically. The nasopharyngeal tissues were examined at 6 levels with 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at 3 levels (1 level to include the base of the epiglottis), the trachea at 3 levels (including the bifurcation, and 1 longitudinal section through the carina), and each lung lobe at 1 level.
Since treatment-related changes were observed in the respiratory tract (nasal tissues and lungs) and initially apparently also in the testes of animals of the high concentration main group, histopathological examination of these tissues was extended to animals of groups 2 and 3 of the main study and to the recovery groups.

Statistics:

Body weight data collected after initiation of treatment: ‘Ancova & Dunnett’s Test’ with ‘Automatic’ as data transformation method.
Pretreatment body weight, Haematology, clinical chemistry, quantitative urinalysis and organ weight data: ‘Generalised Anova/Ancova Test’ with ‘Automatic’ as data transformation method.
Food consumption data: Dunnett’s multiple comparison test.
Incidences of histopathological changes: Fisher’s exact probability test.
Tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Results and discussion

Results of examinations

Organ weight findings including organ / body weight ratios:

no effects observed

Details on results:

- Mortality and clinical signs: No treatment-related clinical abnormalities or mortality was observed in response to the exposure to KZnF3.
- Mean body weight, mean body weight change and mean food consumption: Treatment-related differences in growth or food consumption were not seen.
Ophthalmological examination: Ophthalmoscopic examination did not reveal any treatment-related abnormalities.
Hematology and clinical chemistry: Haematology and clinical chemistry, conducted in the rats of the main study groups at the end of the exposure period, did not reveal any treatment-related abnormalities.
Pathology: Absolute and relative (to body weight) weights of the lungs were statistically significantly increased in males and females of the high concentration main group and in males of the mid concentration main group. These changes were no longer observed at the end of the recovery period. Other treatment-related changes in organ weights were not found. Macroscopic examination at necropsy at the end of the exposure period (main study animals) revealed white spots on the lungs in most high concentration animals. These or other treatment-related macroscopic abnormalities were not seen at necropsy at the end of the recovery period. Microscopic examination of organs and tissues at the end of the exposure period (main study) revealed treatment-related histopathological changes in the nasal tissues and in the lungs. The changes in the nasal tissues consisted of degeneration of the olfactory epithelium at levels 3-6. These were seen in all animals of the high concentration group, in 2 male and 6 female animals of the mid concentration group and in 1 male and 4 female animals of the low concentration group. The changes in lungs consisted of multifocal accumulations of alveolar macrophages seen in all high concentration animals and in one mid-concentration male animal. At the end of the recovery period, changes in the nasal tissues were no longer seen and changes in the lungs had recovered to a large extent.

Effect levels

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

1.09 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Exposure to Nocolok Zn Flux resulted in treatment-related changes in the nose and lungs. Since the changes in the nose were also observed – although at a lower incidence – at the low concentration level of 1.09 mg/m3, this concentration is considered to be the Lowest-Observed-Adverse-Effect-Level (LOAEL) in rats after sub-chronic (90-day) exposure by inhalation to Nocolok Zn Flux. A two-month recovery period after the last exposure resulted in complete (for changes in the nose) or almost complete (for changes in the lungs) recovery.

Executive summary:

The sub-chronic toxicity of potassium trifluorozincate was studied in a 90-day inhalation toxicity study in Wistar rats. Four groups of 10 male and 10 female rats were exposed nose-only to target concentrations of 0 (control), 1, 3 or 10 mg/m3 Nocoloc Zn Flux for 6 hours/day, 5 days/week over a 14-week period, with a total of 65 exposure days.The mean actual concentrations (± standard deviation) of Nocolok Zn Flux in the various test atmospheres – based on gravimetric analysis – were 1.09 (±0.39), 3.05 (±0.47) and 10.48 (±2.75) mg/m3 for the low, mid and high concentrationgroups, respectively. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main study animals of the control and high concentration groups, and were sacrificed after a two-month recovery period following the 13-week exposure period.

All animals survived until scheduled sacrifice. No treatment-related changes in growth, food consumption, haematology, clinical chemistry or ophthalmoscopic or clinical abnormalities were observed during the study. Absolute and relative weights of the lungs were statistically significantly increased in males of the mid and high concentration main groups and in females of the high concentration main group. These changes in lung weight were no longer observed at the end of the 2-month recovery period. No further treatment-related changes in organ weights were observed.

Microscopic examination of organs and tissues at the end of the exposure period (main study) revealed treatment-related histopathological changes in the nasal tissues and in the lungs. The changes in the nasal tissues consisted of degeneration of the olfactory epithelium at levels 3-6. These were seen in all animals of the high concentration group, in 2 male and 6 female animals of the mid concentration group and in 1 male and 4 female animals of the low concentration group. The changes in lungs consisted of multifocal accumulations of alveolar macrophages seen in all high concentration animals and in one mid-concentration male animal. At the end of the recovery period, changes in the nasal tissues were no longer seen and changes in the lungs had recovered to a large extent. 

Since the changes in the nose were also observed – although at a lower incidence – at the low concentration level of 1.09 mg/m3, this concentration is considered to be the Lowest-Observed-Adverse-Effect-Level (LOAEL) in rats after sub-chronic (90‑day) exposure by inhalation to Nocolok Zn Flux. A two-month recovery period after the last exposure resulted in complete (for changes in the nose) or almost complete (for changes in the lungs) recovery.