Polymer with 2-Butyne-1,4-Diol and (Chloromethyl-)Oxirane, Brominated, Dehydrochlorinated, Methoxylated

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion B.V.

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

(11E)-11,12-dibromo-7-(chloromethyl)-2,6,9,14,18-pentaoxanonadec-11-ene-4,16-diol; (12E)-12,13-dibromo-1,21-dichloro-8-(chloromethyl)-5,17-bis(methoxymethyl)-4,7,10,15,18-pentaoxahenicos-12-ene-2,20-diol; (8E)-8,9-dibromo-13-(hydroxymethyl)-2,6,11,15-tetraoxahexadec-8-en-4-ol; (8E)-8,9-dibromo-2,6,11,15-tetraoxahexadec-8-ene-4,13-diol

EC Number:

617-903-6

Cas Number:

86675-46-9

Molecular formula:

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

IUPAC Name:

(11E)-11,12-dibromo-7-(chloromethyl)-2,6,9,14,18-pentaoxanonadec-11-ene-4,16-diol; (12E)-12,13-dibromo-1,21-dichloro-8-(chloromethyl)-5,17-bis(methoxymethyl)-4,7,10,15,18-pentaoxahenicos-12-ene-2,20-diol; (8E)-8,9-dibromo-13-(hydroxymethyl)-2,6,11,15-tetraoxahexadec-8-en-4-ol; (8E)-8,9-dibromo-2,6,11,15-tetraoxahexadec-8-ene-4,13-diol

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Polyol IXOL M125
- Substance type: Halogenated polyetherpolyol
- Physical state: viscous, dark brown liquid
- Analytical purity: ≥99%
- Lot/batch No.: TAE6038
- Expiration date of the lot/batch: 31 December 2011
- Storage condition of test material: ambient temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7 weeks
- Weight at study initiation: 222 g (males)
- Housing: in macrolon cages with a bedding of wood shavings and strips of paper as environmental enrichment; 3 or 5 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: 6 days (dose-range finding study); 5 days (main study)

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

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

Vehicle: clean air

Details on 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, humidity, pressure in air chamber: 22.4 (± 0.7) °C (test group) and 22.1 (± 0.5) °C (control group) (main study); 45.4 (± 1.4)% (control group) and 36.3 (± 1.3) % (test group) (main study)


TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration (by weight) of the non-volatile fraction of Polyol IXOL M125 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 (880% Polyol IXOL M125 and 12% water, based on weight; solutions were prepared weekly)
- Concentration of test material in vehicle: 88%

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week ((i.e. 20 exposure days over a 28-day study period))

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/m3
Basis:
other: target conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 (±10), 290 (±30) and 950 (±70) mg/m3
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
380 (± 60), 770 (± 170), 3550 (± 370) mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

5 males

Control animals:

yes, sham-exposed

Positive control(s):

The rats of the positive control group were injected once intraperitoneally with the mutagen Mitomycin C at a dose level of 1.5 mg/kg body weight, using physiological saline as the vehicle (10 mL/kg body weight; body weights were recorded just before dosing).

Examinations

Tissues and cell types examined:

Bone marrow erythroblasts.

Details of tissue and slide preparation:

BONE MARROW COLLECTION AND PROCESSING
At necropsy, femoral bone marrow cells of one of the femurs were collected from each rat assigned to the micronucleus test. The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald-Giemsa solution. The other fixed unstained smear was kept in reserve and discarded after completion of analysis.

MICROSCOPIC EXAMINATION OF THE BONE MARROW SMEARS
The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.
The following criteria were used for the scoring of cells:
- A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
- A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
- A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

- The study was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were within the historical range.
- A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus (positive response) if the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control group.
- A test substance was considered to be negative in this micronucleus test if it did not result in a positive response at the dose level analysed.
- The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation if the test substance statistically significantly reduced the mean number of PE.

Statistics:

Data on MPE and PE were analysed by one-way Analysis of Variance (ANOVA) with treatment as factor. Data on MPE and PE were analysed by one-way Analysis of Variance (ANOVA) with treatment as factor. All statistical tests were performed using SAS V9.1 statistical software Copyright (c) 2002-2003 by SAS Institute Inc., Cary, NC, USA.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE FINDING STUDY
- See section 7.5.2

Any other information on results incl. tables

BROMIDE ANALYSIS

Analysis of blood sampled immediately after exposure on day 14/15 indicated highly increased levels of plasma bromide (37 to 44-fold) in animals exposed to the high concentration Polyol IXOL M 125, when compared to unexposed controls. Bromide levels were still highly increased in blood sampled the next day before the start of exposure. These results indicate the systemic availability of Polyol IXOL M125 – with plasma bromide as a marker – upon inhalation exposure, and therefore the results of the micronucleus test are considered valid.

Applicant's summary and conclusion

Conclusions:

Polyol IXOL M125 did not induce chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male rats.

Executive summary:

In a GLP compliant sub-acute (28-day) inhalation toxicity study a micronucleus test rats was performed according to OECD guideline 474. Four groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0, 0.1, 0.3 or 1.0 g/m3 Polyol IXOL M125 for 6 hours/day, 5 days/week over a 28-day period, with a total of 20 exposure days. Inhalation exposure to Polyol IXOL M125 did not induce chromosomal damage or damage to the mitotic apparatus of the bone marrow target cells.