2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

EC Number:

614-503-3

Cas Number:

68441-62-3

Molecular formula:

(C3H7O2)xC4H4O2Br2(C4H9O2)y with x + y = 2.5

IUPAC Name:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Polyol IXOL B 350
- Substance type: Halogenated polyetherpolyol
- Physical state: viscous, dark brown liquid
- Analytical purity: ≥99%
- Lot/batch No.: KB13017
- Expiration date of the lot/batch: 31 December 2013
- Storage condition of test material: ambient temperature, protected from light

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: 270 g (males) and 179 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 before blood withdrawal
- Water: domestic tap water suitable for human consumption, ad libitum, except during exposure and fasting period before blood withdrawal
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- 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: The average MMAD for the low, mid and high concentration groups were 2.3 µm (gsd of 2.5), 2.0 µm (gsd of 2.5) and 2.2 µm (gsd of 2.5) respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure units (a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom) consisting of a cylindrical polypropylene (group 1) or aluminium (groups 2-5) column, 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: heated air-driven atomizer (Schlick type 970/S, Coburg, Germany) placed at the top inlet of the exposure chamber
- Method of particle size determination: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra Instruments, Canne Valley, California, 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 be calculated
- Temperature and humidity in exposure chamber: 21.7 (± 0.4)°C, 22.9 (± 0.5)°C, 23.0 (± 0.4)°C and 23.1 (± 0.3) °C for the control, low, mid and high concentration groups, resp.; 40.1 (± 3.1)%, 48.5 (± 2.7)%, 47.9 (± 2.7)% and 48.1 (± 2.7) % 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 non-volatile fraction of Polyol IXOL B 350 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

VEHICLE (if applicable)
- Justification for use and choice of vehicle: To generate the test atmospheres, the test material was diluted with water (70% Polyol IXOL B 350 and 30% water, based on weight; solutions were prepared weekly)
- Concentration of test material in vehicle: 70%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration (by weight) of the non-volatile fraction of Polyol IXOL B 350 in the test atmospheres was determined at least three times per day during each exposure by means of gravimetric analysis. Representative test atmosphere samples were obtained by passing the test atmosphere at 4.6 Ln/min through fiber glass filters (Sartorius 13400-47) for groups 2, 3, 4 or respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere and weighed again. During preliminary measurements, it was established that drying of the loaded filters was not necessary, because the weight was constant.
To investigate whether filter weights should be corrected for dry matter content or possible hygroscopy of the test material, known amounts of undiluted Polyol IXOL B 350 were applied to glass fiber filters and the filters were dried with ambient air until a stable filter weight was reached. The percentage of captured weight on the filters was 102.8% for Polyol IXOL B 350, indicating slight hygroscopy of the test materials. This percentage was used to correct the weight of the filters after test atmosphere sampling for gravimetric analysis.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/m³ air (nominal)

Doses / concentrations 4

Dose / conc.:

30.14 mg/m³ air (analytical)

Remarks:

SD ± 3.21

Doses / concentrations 5

Dose / conc.:

100.1 mg/m³ air (analytical)

Remarks:

SD ± 4.54

Doses / concentrations 6

Dose / conc.:

300.7 mg/m³ air (analytical)

Remarks:

SD ± 25.46

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 two previously performed sub-acute (28-day) inhalation studies
- 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: daily in the morning hours; a group-wise observation was made halfway through each exposure day. During exposure, special
attention was paid to any breathing abnormalities and restlessness; observation of other abnormalities was limited due to the animals’ stay in restraining tubes.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded three (males) or four 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 4-day period for males; a 3-day period for females) and at the end of the exposure period (a 3-day period for males; a 4-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
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: Yes, overnight
- How many animals: all animals
- 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
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: yes, overnight
- How many animals: all animals
- 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.

Other examinations:

Functional Observational Battery (FAB) and measurement of motor activity were included in the study.
Urinalysis was performed. The following determination were carried out in individual samples:
- volume,
- density,
- appearance,
- dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen)
- microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).

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

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

The exposure to the test material up to a concentration of 300.70 mg/m3 was well tolerated as evidenced by the absence of any relevant toxicological effect. Clinical, neurobehavioural and ophthalmoscopic observations; growth and food consumption results; haematology, clinical chemistry and urinalysis parameters; organ weights; and necropsy and histopathology findings did not reveal any exposure-related changes.

Effect levels

Dose descriptor:

NOAEC

Remarks:

local and systemic effects

Effect level:

300 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects observed at the highest concentration tested.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Exposure to Polyol IXOL® B350 at concentrations up to 300 mg/m3 (the highest concentration tested) for 90 days, did not result in any treatment-related changes in the parameters tested. Therefore, the high concentration level of 300 mg/m3 was considered to be the No-Observed-Adverse-Effect-Level (NOAEL) for sub-chronic inhalation exposure to Polyol IXOL®B350 in male and female rats.

Executive summary:

The inhalation toxicity of Polyol IXOL® B350 was studied in a GLP compliant sub-chronic inhalation toxicity study in Wistar rats according to OECD Guideline 413. Four groups of 10 male and 10 female rats were exposed nose-only to target concentrations of 0 (control), 30, 100 or 300 mg/m3 Polyol IXOL® B350 for 6 hours/day, 5 days/week over a 14-week period, with a total of 65 exposure days. The animals were sacrificed on the day after the last exposure. To investigate the toxicity, data on clinical and neurobehavioural observations, ophthalmoscopy, body weight, food consumption, urinalysis haematology and clinical chemistry were collected. At necropsy, the animals were examined for gross macroscopic abnormalities, organs were weighed and a selection of organs and tissues (including the complete respiratory tract with nasal passages) was examined microscopically.

The mean actual concentrations (± standard deviation) as measured by gravimetric analysis were very close to the target concentrations, i.e. 30.14 (± 3.21), 100.06 (± 4.54) and 300.70 (± 25.46) mg/m3 Polyol IXOL® B350 for the low, mid and high concentration groups, respectively.

All animals survived until scheduled sacrifice. No treatment-related clinical or ophthalmoscopic abnormalities were observed in response to the exposure to Polyol IXOL® B350. Neurobehavioural observations (arena and Functional Observational Battery testing) and motor activity assessment did not indicate any neurotoxic potential of the test material. Treatment-related differences in growth or food consumption were not seen. Haematology, clinical chemistry and urinalysis conducted in all rats at the end of the exposure period, did not reveal any treatment-related abnormalities. No changes in absolute and relative (to body weight) organ weight were observed at the end of the exposure period. Furthermore, macroscopic examination at necropsy and histopathological examination of organs and tissues – including the full respiratory tract – did not reveal any treatment-related gross or microscopic changes

The high concentration level of 300 mg/m3 was considered to be the No-Observed- Adverse-Effect-Level (NOAEL) for sub-chronic inhalation exposure to Polyol IXOL® B350 in male and female rats.