Ziram

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun - 13 Sep 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted in 1981

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

No ophthalmological examination, body weight only recorded weekly, not all organs histopathologically examined

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ziram

EC Number:

205-288-3

EC Name:

Ziram

Cas Number:

137-30-4

Molecular formula:

C6H12N2S4Zn

IUPAC Name:

ziram

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kent, UK
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 312 - 402 g (males) and 200 - 252 g (females)
- Housing: Groups of 5 animals of the same sex were housed in suspended stainless steel cages fitted with mesh tops and floors.
- Diet: Rat and Mouse No. 1 (E) SQC maintenance diet, ad libitum (no access to food during each 6-h inhalation exposure)
- Water: Tap water, ad libitum (no access to food during each 6-h inhalation exposure)
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 21.5
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.8 - <= 2 µm

Remarks on MMAD:

MMAD / GSD: 1.8 - 2.0 / 2.76 - 2.87

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each exposure system comprised a snout-only inhalation chamber, rat restraining tubes, a Wright Dust Feed mechanism, an electrical power interrupter unit, polymethyl methacrylate pre-chamber, a diluent air control system and an exhaust system incorporating in-line particulate filters.
- Method of holding animals in test chamber: rat restraining tubes
- Source and rate of air: filtered air, aerosol was generated into a calibrated airflow of 7 L/min, which was diluted by an additional airflow of 50 L/min
- System of generating particulates/aerosols: Wight Dust Feed (WDF) particulate aerosol generator
- Temperature, humidity, pressure in air chamber: 20.1 - 22.3 °C, humidity and pressure were not specified
- Method of particle size determination: Size distribution in each test chamber was determined twice during each week of treatment using a cascade impactor operated at an airflow of 3 L/min. The collection substrates for the device were stored pending extraction of the test substance collected and spectrophotometric analysis of the mass of test substance on each stage of the device.
- Treatment of exhaust air: The extract air passed through a trapping system compromising polypropylene fiber depth filters followed by a glass filter.

TEST ATMOSPHERE
- Brief description of analytical method used: Three samples were taken daily per 6 h exposure after approximately 1, 3, 5 h. The samples were collected onto previously weighed glass fiber filters in an open face holder. The atmosphere samples were taken by drawing a previously selected volume of chamber air through the filters at a calibrated flow of 4 L/min. The air volume of each sample collected was measured (in-line wet type gas meter). The test substance collected on each filter was extracted and the extract was analysed to determine the mass of the test substance collected (spectrophotometric assessment)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The achieved chamber concentrations are presented in terms of µg/L air and were derived from the analytically determined mass of test material collected on each filter and the measured volume of chamber air sampled. Acetone was used to extract the test material from the collection substrate. the resulting solutions of approximate concentrations of 10 to 0.5 µg/mL were quantified by spectrophotometry.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 days/week for 6 h/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

corresponding to 0.095 mg/m³ air (achieved concentration)

Doses / concentrations 2

Dose / conc.:

0.3 mg/m³ air (nominal)

Remarks:

corresponding to 0.318 mg/m³ air (achieved concentration)

Doses / concentrations 3

Dose / conc.:

1 mg/m³ air (nominal)

Remarks:

corresponding to 1.153 mg/m³ air (achieved concentration)

Doses / concentrations 4

Dose / conc.:

3 mg/m³ air (nominal)

Remarks:

corresponding to 2.898 mg/m³ air (achieved concentration)

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

The study consists of two study groups, one main group and one recovery group.
Main group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at target concentrations of 0.1,0.3, 1.0 and 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks.
Recovery group: Groups of 5 male and 5 female rats, which were exposed to either the negative control (air only) or to the test substance at the target concentration of 3.0 µg/L air for 6 h/day, 5 days a week for a period of 4 weeks. The recovery group was retained untreated for a further period of 4 weeks, thus serving to assess reversibility of any effects.
For details on test concentrations, please refer to attachment 1 under "Overall remarks, attachments".

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day (mortality) and once per day (clinical signs)
- Cage side observations checked: mortality and signs of ill-health and behavioural changes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly interval with special attention on audible respiratory noise

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting 7 days before exposures

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was calculated as g food per rat per week.
Food consumed by each cage of rats was recorded daily, commencing 7 d before the start of exposures. Food intake was determined based on total amounts of food given to and left by each cage in each group and the number of rats surviving in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 4
- Anaesthetic used for blood collection: Yes (identity not indicated)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: Haematocrit (HCT), haemoglobin (HB), red cell count (RBC), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean cell haemoglobin (MCH), total white cell count (WBC), differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells (LUC)) were determined as well as cell morphology, common morphological changes (anisocytosis, micro/macrocytosis, hypo/hyperchromasia), platelet count (Pit), reticulocyte count (Retic), prothrombin time (PT), and activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 4
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked; Total protein (Total Prot), Albumin (Alb), globulin (Glob Total), urea, creatinine (Creat), sodium (Na), potassium (K), calcium (Ca Total), inorganic phosphorus (Phos), chloride (CI), total cholesterol (Choi Total), alkaline phosphatase (Alk. Phos), total bilirubin (Bili. Total), glucose (Gluc) hexokinase mediated, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma Glutamyl transferase (gGT) and creatinine phosphokinase (CPK Total) [creatinine kinase].

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Following 4 weeks of exposure all rats were sacrificed on the first two days of Week 5. Exposures were continued until the day prior sacrifice. The recovery groups were sacrificed following 4 weeks of exposure and a 4 week withdrawal period on the first day of Week 9. All rats were sacrificed by intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial blood vessels.
At scheduled necropsy, adrenals, kidneys, liver, lungs, spleen, testes and epididymides of all animals (males, females) were dissected and weighed. Adrenals and kidneys were weighed together and testes and epididymides separated.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart. Any abnormalities in the appearance and size of the thoracic viscera were recorded. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. Gastrointestinal tract (GIT) was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver was sectioned at intervals of a few millimetres; the kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Gross lesions were examined from all animals and the following organs were collected: adrenals, alimentary tract (oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum), aorta, eyes, epididymides, femur (with joint), heart, kidney, larynx, liver, lungs (all lobes, mainstream bronchi), lymph nodes (cervical, mesenteric, tracheobronchial), mammary gland, nasal passages, optic nerve, ovaries, oviduct, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal column, spinal cord (cervical, thoracic, lumbar), spleen, sternum (bone and marrow), testes, thymus, thyroid (with parathyroids), tongue, trachea (including bifurcation), ureter, urinary bladder, uterus (corpus and cervix) and vagina.

HISTOPATHOLOGY: Yes
Larynx, lungs (all lobes, mainstream bronchi), trachea (including bifurcation), and any macroscopic abnormalities were examined in all animals (males, females) using a light microscope. In the animals (males, females) of control groups and high-dose groups also adrenals, epididymides, heart, kidneys and liver were examined using a light microscope.

Statistics:

Statistical analyses were carried out separately for each sex. Data relating to food consumption were analysed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit. Bw data were analysed using weight gain. Food consumption data to Week 4 were analysed using cumulative cage totals.
Statistical tests used for body weight, food consumption, organ weight and clinical pathology data were frequency, parametric and/or non-parametric analysis. For details, please refer to "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations that were considered to be as a result of treatment with the test substance. The type and frequency of clinical signs observed during the exposure and recovery were those routinely seen in this laboratory in this age and strain of animals.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no conclusive evidence of a reaction to treatment, with body weight gains being essentially comparable in all groups including the control group.
Body weight gain was statistically significantly higher in the high dose group than concurrent controls during the 4 week reversibility period. It was a minor change, which was not considered to be of toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Values for all treated group animals were similar to that of the control animals during the exposure and recovery periods.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the haematological parameters. Occasional differences from control values attained statistical significance, but in the absence of any correlation across the sexes these are all considered to be of no toxicological importance.
For details, please refer to attachment 2 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on the biochemical parameters. Occasional differences from control values attained statistical significance, most notably group mean glucose values of high and high intermediate dose males, but in the absence of any correlation across the sexes, and the absence of effects on any other parameters, these are all considered to be of no toxicological importance.
For details, please refer to attachment 3 under "Overall remarks, attachments".

Endocrine findings:

not specified

Description (incidence and severity):

For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An increase in lung weights (body weight adjusted) was observed in animals (males, females) of the high dose groups compared to the control animals. For both sexes this was a reversible finding (as evident by the recovery groups). Increased lung weights (body weight adjusted) were also noted for females of the high intermediate dose group compared to the control animals. Females of the recovery high dose group (previously exposed to 3.0 µg/L) showed slightly but statistically significantly lower kidney weights. Since no similar finding was noted at termination of animals following 4 weeks exposure, this change is of no biological significance.
No other test substance-related effects on organ weights were noted.
For details, please refer to attachment 4 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no lesions evident at either the terminal kills or recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes were observed for larynx, lungs and trachea and tracheal bifurcation.

Larynx:
Main group:
Changes of the larynx were seen at all dose levels except the low-dose groups. In the high dose and high intermediate dose groups those changes included squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage, subepithelial inflammatory infiltration and epithelial hyperplasia (ventral pouch, arytenoid cartilage). The changes seen were similar in character and degree. In the low intermediate dose groups squamous metaplasia of the ventrolateral epithelium, hyperplasia of the ventral epithelium, necrosis of the ventral cartilage and subepithelial inflammatory infiltration were observed.
Recovery group:
Following the 28 day reversibility period, histopathology revealed changes of the larynx in the high-dose groups. There was some evidence of recovery. No lesions were observed in the ventral pouch and arytenoids and no evidence of vesiculation in the ventrolateral epithelium. Squamous metaplasia was still present in ventrolateral epithelium of males and females accompanied by ventral epithelial hyperplasia in affected males. The severity of these changes was reduced. Subepithelial inflammatory infiltration was decreased when compared with animals killed at the end of the treatment period. There was no evidence of recovery in the ventral cartilage in investigated animals; necrosis was still present in all animals.

Lungs:
Main group:
In rats from high dose and high intermediate dose groups also changes of the lungs were observed when compared with the controls. Those changes were fibrosis, granulomatous inflammation, bronchiolar metaplasia in the alveolar ducts, prominent goblet cells in the bronchioles and terminal bronchioles, mucous/cellular debris in the airways, epithelial hyperplasia in the bronchioles and terminal bronchioles, bronchiolitis and an increased incidence of foamy alveolar macrophages and extravasation of eosinophils. In the low intermediate and low dose groups the changes of the lungs were similar to the controls.
Recovery group:
In lungs, there was evidence of a degree of recovery. No granulomatous inflammation, bronchiolar metaplasia in alveolar duct and changes in bronchioles were observed. No treatment-related effect on the incidence or severity of foamy alveolar macrophages or in the extravasation of eosinophils occurred. Minimal degree of fibrosis was still present in alveolar ducts. All other findings in the respiratory tracts of rats killed after the reversibility period were considered to be incidental.

Trachea and tracheal bifurcation:
Main group:
In the high-dose and the high intermediate dose-groups changes in trachea and tracheal bifurcation were present. Epithelial hyperplasia was seen at the tracheal bifurcation. The incidence of this finding was dose-related. Treatment-related epithelial hyperplasia was also observed in trachea of 2 females from the high dose group.
Recovery groups:
A complete recovery of the trachea and tracheal bifurcation lesions was observed in the high-dose recovery groups.

Other findings:
Main group:
In the nasal turbinates, there was low incidence of changes in the respiratory and olfactory epithelium throughout all main dose groups. The nature of these findings was inconsistent and they were considered to be of no toxicological significance.
Recovery group:
There were no lesions evident at the recovery kills that were considered to be attributable to treatment with the test substance. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic findings.

For details, please refer to attachment 5 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

general systemic toxicity

Effect level:

3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic effects observed at the highest concentration tested.

2

Key result

Dose descriptor:

NOAEC

Remarks:

respiratory system

Effect level:

0.1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at the respiratory system at this concentration.

3

Key result

Dose descriptor:

LOAEC

Remarks:

respiratory system

Effect level:

0.3 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.3 mg/m³ air (nominal)

System:

other: respiratory system

Organ:

larynx

lungs

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 and GLP conditions. Male and female rats were exposed to the test item as aerosol at nominal concentrations of 0.1, 0.3, 1.0 and 2.0 mg/m³ air via snout-only exposure conditions. The aerosol was highly respirable to rats, with mass median aerodynamic diameter (MMAD) of 1.8 - 2.0 µm. There was no clear evidence for general toxicity in male and female rats. Histological changes included inflammatory and proliferative lesions in the respiratory tract of rats from the high, high intermediate and low intermediate dose groups sacrificed after 28 d of exposure. Noted increase in lung weight in high-dose group animals and females of the high intermediate dose group was regarded as attributable to these lesions. A complete recovery of the trachea and tracheal bifurcation lesions, partial recovery of the lung lesions; and some evidence of recovery of the laryngeal lesions was observed in the high-dose recovery groups. No treatment-related histological changes were noted in all low-dose group rats. Therefore, the NOAEC regarding the respiratory system was set to 0.1 mg/m³ air. As no clear evidence for systemic effects was observed, the NOAEC for general toxicity was set to 3 mg/m³ air.