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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (OECD 471) and a mammalian gene mutation assay (OECD 478) performed with a structural analogue of Polyol IXOL M125, Polyol IXOL B350, genotoxicity in the presence of S9-mix was observed.

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test (OECD 474) and an in vivo unscheduled DNA Synthesis test (OECD 486) were performed with polyol IXOL M125. Both in vivo tests indicate that Polyol IXOL M125 is not considered mutagenic in vivo.

Link to relevant study records

Reference

Reference 1

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

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

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

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))

Remarks:

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

Remarks:

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

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).

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.

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

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.

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.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion B.V.

Type of assay:

unscheduled DNA synthesis

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

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))

Remarks:

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

Remarks:

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

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):

Six animals (including one reserve animal) were treated as positive control group. The animals were dosed by gavage (12-16 hour prior to sacrifice) with the mutagen 2-AAF (50 mg/kg-bw, 2.5 mg/mL in corn oil). Body weights were measured just prior to dosing. The concentration of the test solution was not determined analytically; the dose quoted in this final report is therefore a nominal one.

Tissues and cell types examined:

Liver hepatocytes

Details of tissue and slide preparation:

PREPARATION OF HEPATOCYTE CULTURES
Within 16-24h after exposure, animals were sacrificed for isolation of hepatocytes. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a Ca2+- and Mg2+-free HEPES buffer (0.01 M) whilst under anaesthesia with sodium pentobarbital and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution.
Directly after the start of the perfusion with the Ca2+- and Mg2+-free HEPES buffer to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10 % solution of formalin) for histopathological examination.
After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at ca 37 °C. Thereafter, they were filtered over a 200 mesh nylon filter, centrifuged and resuspended in WEC medium [= Williams medium E complete, which consisted of Williams medium E containing Glutamax supplemented with 10 % foetal calf serum and gentamycin (50 μg/mL)]. Cell counts were made. The viability of the hepatocytes was determined by trypan blue exclusion. The viability of the hepatocytes of the negative control animals was at least 50%, and therefore considered sufficient. Suspensions containing 5E+05 cells/mL were prepared in WEC medium. Aliquots (1 mL) were seeded onto Thermanox 25 mm round plastic cover slips in 35 mm 6-well dishes, which already contained 1 mL of WEC medium. The cultures were then incubated at ca 37 ºC in a humidified incubator containing ca 5% CO2 and 95% air to allow cells to attach (2-4 h).

LABELLING OF HEPATOCYTES CULTURES
Two to four hours after seeding of the cells, the medium was removed and cells were washed twice with Williams E medium leaving only attached viable cells. Immediately after washing, 2 mL WEI [= Williams E medium Incomplete, which consisted of Williams medium E containing Glutamax, gentamycin (50 μg/mL), insulin (8 μg/mL), hydrocortisone (36 μg/mL) and ca 10 μCi [methyl-3H]thymidine (specific activity: 84 Ci/mmol [3.108 TBq/mmol]) per mL was added to the cultures.
The hepatocyte cultures were incubated for 16 to 20 hours at ca 37ºC. Thereafter, the cover slips were rinsed in three successive washes with Williams E medium. The cover slips were then immersed in 2 mL of a 1% sodium citrate solution for 10 min to allow cells to swell. Subsequently, cells were fixed in three 30 min changes of absolute ethanol-glacial acetic acid (3:1), air-dried, and mounted on glass slides.

AUTORADIOGRAPHY
Slides were processed for autoradiography using Ilford K5D emulsion. At two time points, 7 and 10 days of exposure at < –18ºC, slides were developed, fixed and washed with water. Slides were stained with haematoxylin and eosin and coded by a qualified person not involved in analysing the slides to enable ‘blind’ scoring. The slides that were developed after 7 days of exposure were chosen to be analyzed.

GRAIN COUNTING AND CALCULATIONS
Sorcerer UDS software (Perceptive Instruments, UK) with microscopic attachment (Zeiss microscope connected to a high resolution camera) was used for grain counting of nuclei and cytoplasm. Fifty cells (randomly selected from each quadrant of the cover slip) per slide and 2 (out of 3) slides per animal were counted. The remaining slide of each animal was kept in reserve. Cells with abnormal morphology (pyknotic or lysed nuclei), or heavily-labelled S-phase cells were not counted. Two slides of animal 68 could not be analyzed and 11 cells were analyzed of the third slide, because too few cells were present on the slides. Cytoplasmic labelling was determined by counting two nuclei-sized area of cytoplasm adjacent to the nucleus.
The mean cytoplasmic count was subtracted from the nuclear count to give the net nuclear grains (NNG).

The following calculations were made for each slide:
a) the population average NNG ± SD (cell to cell)
b) the percent of cells in repair
c) the population average NNG ± SD for the subpopulation of the cells in repair

The following calculations were made for each animal:
a) the population average NNG ± SD (slide to slide)
b) the percent of cells in repair (slide to slide)

The following calculations were made for each data point:
a) the population average NNG ± SD (animal to animal)
b) the percent of cells in repair ± SD (animal to animal)

Evaluation criteria:

- The study is considered valid if the positive controls give a positive response and if the negative control is non-genotoxic.
- Cells are considered “in repair” if the NNG value is ≥ 5 NNG.
- A response at a data point is considered positive if at least 20% of the cells are “in repair”.
- A response is considered weakly positive if the population average is between 0 to 5 NNG.
- A test substance is considered to cause DNA damage and induce DNA repair in liver cells if the dose levels result in a positive or weakly positive response.
- A test substance is considered non-genotoxic under the conditions of the test if the dose levels produce NNG ≤ 0.

Both numerical significance and biological relevance are considered in the evaluation of the 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

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 UDS test are considered valid.

Conclusions:

Polyol IXOL M125 did not induce unscheduled DNA synthesis in hepatocytes of male rats.

Executive summary:

In a GLP compliant sub-acute (28-day) inhalation toxicity study, unscheduled DNA Synthesis in rats was determined according to OECD guideline 486. 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 unscheduled DNA synthesis in hepatocytes, under the conditions used in this study.

Additional information

No in vitro genetic toxicity studies on Polyol IXOL M125 were available. Therefore, information from the structural analogue Polyol IXOL B350 was used to evaluate the possible in vitro genotoxic effects of Polyol IXOL M125.

In vitro: Bacterial reverse mutation assay

Polyol IXOL B350 was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiringSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and the tryptophan-requiringEscherichia colistrain WP2uvrA, in the absence and presence of S9 -mix. All strains were used, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance. The mean number of his+and trp+revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants or a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In the presence of S9-mix, in strains TA1535, TA98, TA100 and WP2uvrA, Polyol IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA1535, at and above 185 µg/plate; in strain TA98, at 5000 µg/plate; in strain TA100, at and above 556 µg/plate and in strain WP2uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA1535 at 5000 µg/plate. In the absence of S9-mix in all strains, Polyol IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained with the test substance inSalmonella typhimuriumstrains TA1535, TA98 and TA100 and in theEscherichia colistrain WP2uvrA, indicate that Polyol IXOL B350 is mutagenic in the presence of the S9-mix and all strains used indicate that Polyol IXOL B350 is not mutagenic in the absence of S9-mix under the conditions employed in this study.

In vitro: mammalian gene mutation assay 

Polyol IXOL B350 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence S9-mix (GLP study according to OECD guideline 478). One assay was conducted in which single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively.The highest concentrations of the substance evaluated for mutagenicity were 0.37 and 0.7 mmol/L in the absence and presence of S9-mix, respectively. The maximum concentrations were limited by cytotoxicity. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix a dose related increase in mutant frequency was observed at and above 0.5 mmol/L. In presence of S9-mix at the concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. The mean percentage of small colonies formed was 64%. Based on these results it cannot be excluded that Polyol IXOL B350 is clastogenic. The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.It is concluded that under the conditions used in this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.

As indicated in the REACH Regulation (Annex VIII), an in vivo mutagenicity test shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. In addition, in the REACH guidance (Chapter R.7A – Endpoint Specific Guidance version 2.0, November 2012 page 345) it is stated that an in vivo test should be initiated as soon as possible following a positive result in an in vitro mammalian cell mutagenicity test. For the substance Polyol IXOL M125 no in-vitro genetic toxicity studies are available. However, for the structural analogue Polyol IXOL B350, in vitro, positive results were observed in the mammalian gene mutation assay and the bacterial reverse mutation assay in the presence of S9 mix.Based on these positive results, it was decided to perform the in vivo unscheduled DNA synthesis (UDS) test and the in vivo micronucleus test as part of a repeated dose toxicity test (28 days) of Polyol IXOL M125.

In vivo: Micronucleus test

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. The systemic availability of the test material was demonstrated by the highly increased concentration of bromide in blood of animals of the high concentration Polyol IXOL M 125 group, when compared to control levels.

In vivo: Unscheduled DNA Synthesis

In a GLP compliant sub-acute (28-day) inhalation toxicity study, unscheduled DNA Synthesis in rats was determined according to OECD guideline 486. 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 unscheduled DNA synthesis in hepatocytes, under the conditions used in this study. The systemic availability of the test material was demonstrated by the highly increased concentration of bromide in blood of animals of the high concentration Polyol IXOL M 125 group, when compared to control levels.

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.