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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014 - June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered as reliable without restriction as the study has being conducted under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes (incl. certificate)
Remarks:
42/2014. (VIII. 19.)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: vapour (from nebulized liquid)
Details on test material:
- The analysis was performed for the reference sample "Sample 2014" as defined in Section 1.4
- Name: reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA)
- Batch/Lot Number: 14547370
- CAS number: 6863-58-7; 108-20-3; 78-92-2; 75-65-0
- Appearance: Colourless liquid
- Expiry date: 09 September 2015
- Purity: 100% mixture
- Substance classification: Industrial chemical
- Storage conditions: Controlled room temperature (15-25oC, below 70 RH%) in tightly closed vessel in a dry, cool and well ventilated place
- Safety Precautions: Routine safety precautions (nitrile gloves, goggles, lab coat) will be applied to assure personnel health and safety. For respiratory protection, 3M EN140 face mask with ABEK1 filter (combination organic vapours, inorganic gases, acid gases and ammonia) will be used. Precautions required in handling are outlined in the Material Safety Data Sheet.
- Generation of the teste item atmospheres: Before animals were exposed, test item atmospheres were generated within the exposure chamber. For the test atmosphere generation, TSE jet nebulizers (TSE Systems GmbH, Bad Homburg, Germany) were used, connected to the infusion pump (TSE) and to pressurized air supply.
Two segments of the inhalation system were used in each exposure unit. For vaporization of the test item, the air flow rate was set at 30 L/min. The test item was supplied by a constant rate (approximately 2.7, 11 and 30 mL/hour, for low, mid and high concentrations, respectively).

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Wistar Crl:WI (Han)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, from SPF colony
Justification of strain: Rats are the preferred species of choice as historically they have been used for safety evaluation studies and they are specified by the appropriate regulatory authorities.
Number of animals: 40 male and 40 female toxicity rats (10 rats/sex/ group in 4 groups, plus a sufficient number of spare animals
Sex: Male and nulliparous, non pregnant female
Age of animals: Young adult rats, less than 9 weeks old at the initiation of treatment
Body weight: Not exceeded ± 20% of the mean weight for each sex at onset of treatment and were in range:Males: 193-214 g; females: 162-192 g
Acclimatisation time:At least 5 days

Husbandry:

Animal health: Prior to acceptance into the study, the health status of the animals was certified by the staff Veterinarian. Only healthy animals were used for the test
Housing: Group caging (in groups of 3 or 2 per cage, by sex)
Cage type: Polycarbonate solid floor cages (type II or III) with stainless steel mesh lids.
Light: 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
Temperature: 20.0-24.8°C
Relative humidity: 30 - 68%*
Ventilation: At least 15 air exchanges per hour.
The relative humidity in the animal room was below the target 30% on few occasion on 4 days toward the end of the study.
The temperature and relative humidity were checked and recorded twice daily during the study.

Diet and Water:

Animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottle ad libitum.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The standard content of the diet as provided by the supplier and a copy of the certificates of analysis (Batch numbers: 190 1786 and 6802237, Expiry dates: January and March 2015, respectively) are retained in the archives of CiToxLAB Hungary Ltd.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives of CiToxLAB Hungary Ltd.

Bedding:

Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG, Holzmühle 1, D-73494 Rosenberg, Germany (Batch numbers: 03018140226 and 03018140228; Expiry: February 2017) was used up to 27 November 2014. Grade 5 Fibre Litter Animal Bedding, manufactured by Johannes Brandenburg GmbH & Co. KG Arkeburger Str. 31 DE-49424 Goldenstedt, Germany, (Batch number: 171014; Expiry: 2017), was used from 27 November 2014 to the end of the study on 02 February 2015. The quality of the bedding material was guaranteed by the supplier. A copy of the certificates are retained in the archives of CiToxLAB Hungary Ltd. Nest building material (Cotton Rolls, DC GmbH, Germany, batch: F37640) were also added to the cages. A copy of the certificate is retained in the archives of CiToxLAB Hungary Ltd.
The bedding and nest building material was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: - Concentration of 1.00mg/L: MMAD 0.99 ± 0.04 µm /GSD: 1.80 ± 0.22/inhalable fraction (%< 3 um): 97.0%
- Concentration of 3.00mg/L: MMAD 0.68 ± 0.07 µm /GSD: 1.85 ± 0.34 /inhalable fraction (%< 3 um): 99.0%
- Concentration of 12.00mg/L: MMAD 0.54 ± 0.14 µm /GSD: 2.01 ± 0.79 /inhalable fraction (%< 3 um): 99.3%
Detailed results are provided in the attached document Interim_Data_Week_7_13_353_212P.pdf
Details on inhalation exposure:
Animals were acclimated to laboratory conditions for at least five days prior to involvement in the study. Animals were also acclimated to the test apparatus (restrain procedures) for a short period prior to start of the testing (5 daily periods of 1 h, 2 h, 4 h and 6 h , respectively) in order to reduce the stress during exposure.

The animals were treated by the inhalation route using a nose only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminum cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.

The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors.

These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn.

The exposure units were placed in closed hoods in order to avoid cross-contamination and contamination of the laboratory environment.

The animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.

Atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, the aerosol was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.

Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.5 L/min (in the range of 0.7-0.8 L/min). This flow rate is considered adequate to minimise re-breathing of the test atmosphere and to maintain oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.

To avoid any possible differences in exposure, the position of the animals was rotated on an exposure session basis.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual (achieved) concentration of generated atmospheres was measured analytically and gravimetrically. Samples were obtained at regular intervals during each exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through sorbent tubes filled with charcoal (Charcoal 20/40, manufactured by SKC inc. 863 Valley View Road Eighty Four, PA 15330 U.S.A., batch 11509, expiry 2018). Sampling was performed each day shortly after chamber equilibration and then approximately hourly during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). Approximately 6 samples/day were obtained at each concentration level.
The actual concentration was measured by a GC method validated under Study code:13/353-316AN. Samples of the test atmosphere were collected through sorbent tubes filled with charcoal, used for gravimetry as described above and were analysed once a week in each dose group. The amount of 2,2’-oxybisbutane (DSBE) the main component (43% according to the Analytical Certificate) of the test item deposited in the sorbent tube was measured and the test atmosphere calculated. Samples were collected from a vacant animal exposure port (animals breathing zone) and 6 samples at each concentration level were analysed at each occasion. Samples were analysed on the day of the sampling.
The following variables were in addtion monitored during each exposure period by the TSE-DACO monitoring system integrated into the exposure system:
- Chamber airflow rates,
- Test atmosphere temperature,
- Test atmosphere relative humidity,
- Test atmosphere carbon dioxide concentration,
- Test atmosphere oxygen concentration.
The relative humidity values during exposure were lower than the required range due to use of the filtered, dry air for the dispersion of the test item. The air was not humidified in order to avoid modification in the test atmosphere behavior. This deviation had no effect on the purpose and integrity of the study.
Summarizing, the test atmosphere temperature, relative humidity, oxygen and carbon dioxide concentration were considered to be satisfactory for this type of study.
Duration of treatment / exposure:
13 weeks (total study duration of 90 days)
Frequency of treatment:
6 hours per day on a 5 day per week basis
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.02±0.07 mg/L
Basis:
other: GC analysis
Remarks:
Doses / Concentrations:
3.01±0.23 mg/L
Basis:
other: GC analysis
Remarks:
Doses / Concentrations:
11.35±0.40 mg/L
Basis:
other: GC analysis
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
other: yes, concurrent vehicle (same airflow as treatment groups)

Examinations

Observations and examinations performed and frequency:
IN-LIFE PROCEDURES
MORBIDITY/MORTALITY:
Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day.

CLINICAL SIGNS:
Individual clinical observations were performed prior to exposure and twice during exposure whilst the animals were still restrained. Following exposure, clinical observations were performed twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure).
Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
The animals were observed for changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical sings were noted.

NEUROLOGICAL ASSESSMENT
Towards the end of the treatment period, during Week 12/13 as, each animal was subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.
To measure the landing foot splay, the hind paws of the rat was painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they release the bar; the device measured the maximum grip strength. The procedure was repeated with the hind limbs with the appropriate grip support.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), the general physical condition and behaviour of animals were tested. A modified Irwin test will be performed (ref.3).
Parameters such as, but not limited to, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software.Data of travelled distance, recorded in 5 minute segments were evaluated.

EXAMINATION OF VAGINAL SMEARS
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

OPHTALMOLOGY EVALUATION
Ophthalmoscopy examination was conducted in all animals before initiation of the treatment and in the control group 1 and high dose group 4 animals, during Week 13 (Day 86). No treatment related alterations are found at Week 13, therefore no examination of the lower dose group was necessary.
Mydriasis was produced after instillation of eye drops "Humapent", 5mg/mL Cyclopentolat-hydrochloride, (TEVA Pharmaceutical Works Inc. Hungary, Batch no: 5810513 and 2730614, expiry May 2015 and June 2016, respectively) into the conjunctival sac. The evaluation was performed by external examination and using a Gowlland® ophthalmoscope.

BODY WEIGHT:
Body weights were recorded with a precision of 1 g at randomisation, on Day 0, prior to start of exposure, twice a week thereafter, including on Day 90, and prior to necropsy (Day 91).

FOOD CONSUMPTION MEASUREMENTS:
Food consumption was recorded with precision of 1 g twice weekly. The weekly and mean daily food consumption were calculated.

CLINICAL PATHOLOGY:
At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals.
After an overnight period of food deprivation, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.
Urine collection was conducted, during an overnight period of food but not water deprivation, from each animal by placing the animals in metabolic cages.
Regarding the haematology and blood clotting times, the parameters provided in table 1 and 2 were evaluated in all animals euthanized at termination.
Blood smears were prepared (fixed, then stained) for all animals; the smears were not examined, as no adverse effects were observed at the standard haematology evaluation. The smears are stored/archived at CiToxLAB Hungary Ltd.
Regarding the clinical chemistry, the parameters presented in table 3 were evaluated in all animals at termination.
The evaluation of the urine samples was performed by observation (colour/appearance) or test strips as applicable. The Parameters provided in table 4 were investigated during urinalysis.
Sacrifice and pathology:
PATHOLOGY EVALUATION
TERMINAL PROCEDURES
Necropsy and macroscopic examination was performed on every experimental animal at the end of treatment period on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia (RELEASE 300 mg/ml pentobarbital injection A.U.V; Wirtschaftsgenossenschaft Deutscher Tierärzte, Siemensstr. 14, 30827 Garbsen, Germany; Lot no.: 085093, Retest/Expiry date: September 2016).

MACROSCOPIC EVALUATION
After exsanguination the external appearance of each rat was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality were recorded with details of the location, colour, shape and size.
In addition, bone marrow smears from the femur of each animal was prepared at necropsy. The smears were fixed, then stained with May Grünwald - Giemsa but not analysed. They are stored/archived at CiToxLAB Hungary Ltd.
The organs listed in Table 5 below were weighed in surviving animals. Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated.
On completion of the macroscopic examination the tissues and organs were retained from all animals. The list of tissues and organs which were preserved for histopathology investigation is presented in Table 6 below.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Full histopathology was performed in control and high dose groups. In addition, lungs were investigated in Mid and Low dose groups.
Organs or tissues with macroscopic abnormalities were also subjected to histological examination as follows: thymus in 3 and 2 females at 1 and 3 mg/L, respectively, stomach in one low dose female, kidney in one mid dose male and skin in one male at 1 and 3 mg/L.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Statistics:
Data were collected using the software PROVANTIS v.9 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means and standard deviations.
The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was no mortality at any of the exposure level. Slight and transient ataxia was observed in all males and females at 12 mg/L concentration level at the end of the exposure following the removal from the restrain. All males and females were symptom free from Days 21 and 14, respectively. Thin fur (focal) or focal alopecia and/or small skin lesions (scar) were occasionally observed in few males in all test item treated groups and in females at 3 mg/L and control groups. These observations were regarded as incidental and not related to the test item. Additionally, wet fur or red-brown staining of the fur around eyes or nose were occasionally observed in single males at all exposure levels including control and in one female at 1 mg/L concentration level. These observations were considered to be procedure related and not related to the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no mortality at any of the exposure level. Slight and transient ataxia was observed in all males and females at 12 mg/L concentration level at the end of the exposure following the removal from the restrain. All males and females were symptom free from Days 21 and 14, respectively. Thin fur (focal) or focal alopecia and/or small skin lesions (scar) were occasionally observed in few males in all test item treated groups and in females at 3 mg/L and control groups. These observations were regarded as incidental and not related to the test item. Additionally, wet fur or red-brown staining of the fur around eyes or nose were occasionally observed in single males at all exposure levels including control and in one female at 1 mg/L concentration level. These observations were considered to be procedure related and not related to the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain of high dose females did not differ significantly from control. Males at 12 mg/L had slightly lower bodyweights than control, related to a decreased food consumption. The difference was not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased food consumption (overall value lower than control by 4%) was observed at the highest dose. The differences in food consumption noted at 1 and 3 mg/L were not regarded as toxicologically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse effects at any dose level, all parameters falling with the normal physiological ranges for the age, strain and sex of rats on test. There was no effect of treatment on blood clotting parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Although cholesterol was slightly higher at 12 mg/L in both males and females and the differences in total bilirubin and albumin/protein concentrations attained statistical significance, the values were not considered to reflect an adverse effect.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No effects were noted, which could be related to the test item.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment related effects noted during neurological assessment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increase in liver and adrenal weights, slight increase in kidney weights and decrease in Thymus weights were noted in both sexes at 12 mg/L. None of these effects was considered as adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No effects were noted, which could be related to the test item.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal centrilobular hypertrophy of the hepatocytes were noted at 12 mg/l. In the lungs, focal/multifocal accumulation of foamy alveolar macrophages was observed with low incidence in all treated groups. These effects not considered as adverse.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No adverse effects observed.
Details on results:
TEST ATMOSPHERE DATA

Actual and nominal concentrations:
The mean achieved actual and nominal test atmosphere concentrations are provided in Table 7 below.
The exposure concentrations were measured by gravimetrical analysis of the test item captured in a charcoal filled sorbent tubes and measured by a validated analytical method (gas chromatography) once a week, based on DSBE concentration. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis are presented in the Table 7. The nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) is also presented in table 7.
During the gravimetry analysis, small differences in sample masses and consequently in the calculated test atmosphere concentrations occurred on a daily basis because, in order to achieve the target concentration, the feed rate of the test item was adjusted according to the result of the actual measurement. In result, the stability of the concentration on a day-by day basis was sufficient, below ±10% of the mean concentration, with the exception of the first week of males including Day 7 and Day 0 of females at mid dose level, when the achieved gravimetric concentration was lower, by approximately 11-15% of the mean concentration. The average concentration achieved was equal to the target concentration at low and at mid dose level and within slightly lower at high dose level, but within acceptance range of 10%.
Results of the analytical measurements (based on DSBE concentration) were in agreement with the gravimetrical analysis.
Nominal concentration was higher than actual concentration measured gravimetrically by approximately 30%, 60% and 15% at the low, mid and high dose level, respectively

Particle size analysis:
Although the test atmosphere appeared as vapour, a very small amounts of dispersed test item condensed to form aerosol, which was characterized by particle size analysis.
According to the results, the Mass Median Aerodynamic Diameter of the small fraction in the test atmospheres of all groups was in the range of 0.54-0.99 µm with Geometric Standard Deviation of 1.80-2.01.

Exposure conditions:
The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres. Temperature of the test atmospheres and air used for the control group exposure was increased over the limit of 25ºC (up to 27.5ºC) due to temporary malfunctioning of the cooling system on few occasions. At low dose level, temperature of the test atmosphere was lower than 19ºC (minimum value: 17.4ºC) on few occasion. The relative humidity was lower than the optimal range of 30-70% due to the use of filtered, dry air for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study (Ref.7).
The oxygen and carbon dioxide concentrations were considered to be satisfactory for this type of study.
In conclusion, the results of the atmosphere characterization exhibited that the test atmospheres were suitable for the purposes of the study.

MORTALITY AND CLINICAL OBSERVATION

There was no mortality at any of the exposure level.
Clinical signs: Slight and transient ataxia was observed in all males and females at 12 mg/L concentration level at the end of the exposure following the removal from the restrain. One hour after the exposure all animals were generally symptom free. A ceasing tendency was observed, resulting in decreasing incidence by the exposure days. All males and females were symptom free from Days 21 and 14, respectively.
Thin fur (focal) or focal alopecia and/or small skin lesions (scar) were occasionally observed in few males in all test item treated groups and in females at 3 mg/L and control groups. See overview table 8 . These observations were regarded as incidental and not related to the test item.
Additionally, wet fur or red-brown staining of the fur around eyes or nose were occasionally observed in single males at all exposure levels including control and in one female at 1 mg/L concentration level. These observations were considered to be procedure related and not related to the test item.

NEUROLOGICAL ASSESSMENT

There were no treatment related effects noted during neurological assessment.
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, in the control or treated groups, when subjected to the modified Irwin test (functional observation battery).
No test item related effects were observed in the grip strength tests for both fore and hind limbs in any of the experimental groups.
At evaluation of landing foot splay results of the hind limbs, slightly lower mean values were recorded for females at 12 mg/L. The differences attained statistical significance (p<0.01). In males, the mean values of all treatment groups were comparable with the control means. Therefore, this isolated finding in only one sex was considered to be incidental.
During evaluation of motor activity, no statistical or toxicologically significant differences to control were noted at any of the test item treated groups.
The total travelled distance was comparable with the controls in both males and females groups. The patterns of activity were similar in all experimental groups including controls.

OPHTHALMOSCOPY

No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination.

TERMINAL EXAMINATION OF OESTRUS CYCLE

Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats

BODY WEIGHT MEASUREMENTS

Males at 12 mg/L had slightly and consistently lower bodyweights than control, from Week 1 to termination. Compared to control mean, the values were approximately 6% lower at the termination of the treatment on Day 90. The difference was not statistically significant. See Table 9 below.
Compared to the control, slightly lower body weight gains were noted for high dose males at the end of exposure intervals. The differences attained statistical significance between Days 11-14 and 32-35 (p<0.01 and p<0.05, respectively). Reduced body weight gain compared to controls resulted in slightly lower overall gain value (by 12%, not statistically significant). The lower body weight values were in agreement with decreased food consumption.
Body weight and body weight gain values of high dose females did not differ significantly from the control mean.
At 3 mg/L, the body weight and body weight gain values of both males and females were comparable to the control throughout the study.
At 1 mg/L, slightly lower body weight and body weight gain values were noted for males (not statistically significant differences) and the lower mean overall body weight gain was mostly attributed to the low individual values of males 2001, 2004 and 2005. This observation was regarded as incidental.
The mean body weight gain of females at 1 mg/L was higher than control (p<0.05) and was regarded as incidental finding of no toxicological significance.

FOOD CONSUMPTION

The mean food consumption of males at 12 mg/L was lower than control. The difference were no more than 15%, and attained statistical significance between Days 4-7, 11-21, 25-28, 32-35, 39-42, 53-56, 67-70, 81-84 and 88-90 (p<0.01) and Days 21-25 and 74-81 (p<0.05). Compared to the control mean, the overall value for Days 0-90 was decreased by approximately 4% and attained statistical significance (p<0.01).
For females at 12 mg/L, reduced food consumption compared to controls was measured between Days 4-14, 18-21, 25-28, 39-42, 46-49, 53-56, 60-63, 67-70, 74-77 and 81-88 (p<0.01) and between Days 77-81 (p<0.05), resulting in an overall value for Days 0-90 approximately 5% (p<0.05) lower than for controls.
Slightly decreased food consumption was occasionally noted for males at 3 mg/L (between Days 11-14, 25-28 and 32-35) with the overall value for Days 0-90 comparable with the control mean. The average food consumption of females at this dose level was comparable to the control group.
At 1 mg/L, slightly lower food consumption was noted for males (between Days 4-7, 11-28 and 77-84) and the overall value for Days 0-90 was lower by approximately 2% (p<0.05). The food consumption of females was at control level.
The differences in food consumption noted at 1 and 3 mg/L were of low magnitude and were not regarded as toxicologically significant.

CLINICAL PATHOLOGY

Haematology:
There were no test item related adverse effects at any dose level, all parameters falling with the normal physiological ranges for the age, strain and sex of rats on test.
Variations, attaining statistical significance were noted in higher mean platelet volume (MPV) in males in the high dose group (p<0.01).
Statistical differences (p<0.05) were noted for relative counts of lymphocyte and neutrophil granulocyte (%) in females in Mid dose, with no differences in absolute counts.
As the differences were of low magnitude, were regarded as incidental or individual findings, unrelated to treatment and/or with no toxicological significance.

Blood Clotting Parameters:
There was no effect of treatment on blood clotting parameters Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PTT).
Compared to controls, slightly lower prothrombin time (PTT) was measured in all test item exposed groups of males and in females at 12 mg/L (p<0.01) and was regarded as incidental finding with no toxicological significance..

Clinical chemistry:
At 12 mg/L, cholesterol was slightly higher in both males and females. This finding was consistent with results of the 28-day DRF study.
It should be noted, that control values were in a very low range (i.e. approximately half of individual control values were below or at the LOQ, which was 1.16 mmol/L).
Albumin concentration in high dose males was slightly higher than in controls and in all treated females. The differences attained statistical significance. Consequently Total protein and the albumin to globulin ratio were also higher.
Total bilirubin concentration was slightly higher in males at 12 mg/L, while in females slightly increased values were measured in 3 of 10 females.
Although the differences in total bilirubin and albumin/protein concentrations attained statistical significance, the values remained within the normal ranges and were not considered to reflect an adverse effect.
Other differences observed between control and treated groups, occasionally attaining statistical significance, were regarded as incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.

Urinalysis:
No effects were noted, which could be related to the test item.
Statistical differences were recorded in the urine pH values in males at 12 mg/L (p<0.05) and in the volume of collected urine in males at 3 and 12 mg/L. These findings were regarded as off toxicological significance.
Results of sediment analysis revealed no significant differences between control and treated animals.

PATHOLOGY

Macroscopic findings:
No test item-related macroscopic changes were detected.
Incidental gross observations
The following observations were made:
Diffuse dark red discoloration of the thymus in females with incidence of 6/10, 3/10, 2/10 and 2/10, in control, low, mid and high dose respectively, was regarded as terminal procedure related.
Raised area on epididymides (bilateral) in 1/10 control male, dilated renal pelvis in 1/10 mid dose male, few black foci in glandular mucosa of the stomach in 1/10 control and 1/10 low dose females and dilated uterus in 1/10 high dose female, were regarded as incidental.
In addition, at external examination thin fur on different body region (forelimb /cheek) were observed with low incidence in control (2/10) and mid dose (2/10) females, or isolated, small skin lesions (scab) were noted in single males in low and mid dose. These observations were regarded as incidental finding.

Organ weight data:
Exposure to the test item was associated with an increase in liver weights in both sexes at 12 mg/L. The increase was approximately 28-30% in both sexes (for values relative to body weight) and the differences attained statistical significance. See Table 10 below.
The increase in liver weights was in line with findings in the 28-day DRF study.
The increased liver weight was in agreement with hypertrophy of centrilobular hepatocytes revealed during microscopic examination.
Slightly increased liver weights compared to controls were noted in both males and females at 3 mg/L and in females at 1 mg/L. The differences were in the range of 9-13% (for values relative to body weight) and attained statistical significance. These slightly increased weights were not associated with any microscopic finding.
Slightly increased kidneys weights were noted in both males and females at 12 mg/L and at 3 mg/L. The increases compared to controls were in general similar at both dose level and no more than 8.1%, except for the value relative to body weight of high dose males (13.5%). This finding was accompanied by minimal tubular basophilia, which is a common observation in rats but was noted in 4/10 males and 2/10 females at 12 mg/L. (Mid dose was not examined). Although the increase was of a minimal degree, it was in line with the results in the 28-day DRF study, where slightly increased kidney weights were noted in high dose males.
Thymus weights in both sexes at 12 mg/L were slightly lower than in controls (in the range of approximately 11-20%), and attained statistical significance for both absolute and relative values only in males. No microscopic change correlated with decreased thymus weights, however this finding was in line with results in the 28-day DRF study.
Compared to the control mean, weight of adrenals was higher in both males and females at 12 mg/L and in males at 3 mg/L. The differences were in the range of 20-30% in males and 10-12% in females. No microscopic changes were detected in the adrenals (only high dose was examined).
Although a causative role of the test item on the changes in the thymus and adrenals weight cannot be excluded, the changes were mild and were not correlated with any pathological findings. Therefore they were considered rather to be stress related than to reflect an adverse effect of the test item.
Other differences observed between the control and treated groups, occasionally attaining statistical significance were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance

Histopathology:
Test item-related hypertrophy of centrilobular hepatocytes was observed in the liver of both males and females at 12 mg/L. This hypertrophy was of minimal grade and was observed in 4/10 males and 5 of 10 females and corresponded with the increase of liver weights. See Table 11 below.
The centrilobular hypertrophy of the hepatocytes in the high dose males and females appeared as the enlargement of the cytoplasm with homogeneous structure. This change was not associated with cellular necrosis/degeneration and was considered to be a non-adverse adaptive response.
In the lungs, focal/multifocal accumulation of the foamy alveolar macrophages was observed with a low incidence in all treated groups. See table below. In addition, mild mixed cellular, multifocal, peribronchial infiltration was noted in 1/10 female at 12 mg/L (Animal 4509).
The findings in the lungs were not regarded to be adverse.
Minimal tubular basophilia , was noted in 4/10 males and 2/10 females at 12 mg/L. This finding is a common observation in rats. Nevertheless, a relation to treatment cannot be excluded.
Incidental findings: In addition, spermatocele in the epididymis, cortical cyst, or pelvic dilatation of the kidneys, perivascular, multifocal, mononuclear cell infiltrate in the prostate, multifocal, congestion/hemorrhage in the thymus, focal hyperkeratosis in the skin, were considered as incidental or background.

Effect levels

Dose descriptor:
NOAEC
Effect level:
11.35 mg/L air

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 7: Mean achieved actual and nominal test atmosphere concentrations

Group No.

Group Designation

Target Concentration (mg/L)

Achieved Concentration(mg/L)

Nominal

Concentration
(mg/L)

Gravimetry

GC analysis

Mean of all samples

Mean of daily exposures

Mean of all investigated samples

2

Low

1

1.02

(SD: 0.05)

1.02

(SD: 0.03)

1.02

(SD: 0.07)

1.30

(SD: 0.07)

3

Mid

3

2.91

(SD: 0.14)

2.91

(SD: 0.13)

3.01

(SD: 0.23)

4.70

(SD: 0.20)

4

High

12

11.12

(SD: 0.46)

11.12

(SD: 0.44)

11.35

(SD: 0.40)

12.85

(SD: 0.50)

 

Table 8: Incidence of skin lesions through experimental groups

 

 

Dose level/Concentration 

Air control

Low dose

1mg/L

Mid dose

3 mg/L

High dose

12 mg/L

Males

Thin fur/alopecia (focal)

0/10

0/10

0/10

2/10

Isolated scar/crust

0/10

1/10

1/10

1/10*

Females

Thin fur/alopecia (focal)

4/10

0/10

5/10

0/10

Isolated scar/crust

0/10

0/10

1/10

0/10

*Isolated scar was observed in one of 2 males with thin fur (4008 and 4006)

Table 9: Mean body weight values on Day 90 and overallbody weight gainsbetween Days 0 and 90 of males and females.

 

 

 

Dose level/Concentration 

 

Air control

Low dose

1mg/L

Mid dose

3 mg/L

High dose

12 mg/L

 

Males

 

Terminal Body weight (g)

361.5

346.0

364.7

340.4

NS

difference %

-4.3

0.9

-5.8

 

Overall body weight gain (%)

154.5

141.9

159.2

136.3

NS

difference %

-8.2

3.0

-11.8

 

Females

 

Terminal Body weight (g)

226.3

234.1

231.7

224.2

NS

difference %

3.4

2.4

-0.9

 

Cumulative body weight gain (%)

53.0

60.9*

56.9

52.2

DN

difference %

14.9

7.4

-1.5

 

* =p<0.05; DN = Duncan's Multiple Range Test; NS = not significant

difference % =percentage differences versus control mean

Table 10: Mean weights of selected organs

 

 

Groups/Concentration

 

Air control

Low dose

1 mg/L

Mid dose

3 mg/L

High dose

12 mg/L

 

Males

 

  Liver weight

Absolute (g)

8.53

8.30

9.73**

10.36**

DN

differences %

-2.7

14.1

21.5

 

Body weight relative (%)

2.51

2.57

2.85**

3.27**

DN

differences %

2.4

13.5

30.3

 

Brain relative (%)

395

398

455**

488**

DN

differences %

0.8

15.2

23.5

 

  Kidney weight

Absolute (g)

2.18

2.06

2.34

2.31

NS

differences %

-5.5

7.3

6.0

 

Body weight relative (%)

0.643

0.637

0.685

0.730**

DN

differences %

-1.4

6.5

13.5

 

Brain relative (%)

101

99

109

109

NS

differences %

-2

7.9

7.9

 

Females

 

   Liver weight

Absolute (g)

5.75

6.48**

6.53**

7.26**

U

differences %

12.7

13.6

26.3

 

Body weight relative (%)

2.76

3.00*

3.05**

3.52**

DN

differences %

8.7

10.5

27.5

 

Brain relative (%)

289

327**

331**

368**

DN

differences %

13.1

14.5

27.3

 

  Kidney weight

Absolute (g)

1.48

1.50

1.58*

1.58*

DN

differences %

1.4

6.8

6.8

 

Body weight relative (%)

0.707

0.694

0.740

0.764**

DN

differences %

-1.8

4.7

8.1

 

Brain relative (%)

74

76

80*

80*

DN

differences %

2.7

8.1

8.1

 

 * = p<0.05,   ** = p<0.01 , DN = Duncan's Multiple Range Test; U = Mann-Whitney U-Test, NS = Non Significant

difference % =percentage differences versus control mean

Table 11: Main microscopic findings

 

Microscopic findings

Concentration (mg/L)

Male

Female

A.C. 0

Low

1

Mid

3

High

12

A.C. 0

Low

1

Mid

3

High

12

Liver

Hypertrophy, minimal

0/10

-

-

4/10

0/10

-

-

5/10

Lungs

Accumulation of alveolar macrophages

0/10

2/10

4/10

2/10

0/10

2/10

3/10

4/10

focal

0/10

2/10

2/10

1/10

0/10

0/10

1/10

1/10

multifocal

0/10

0/10

2/10

1/10

0/10

2/10

2/10

3/10

Infiltration peribronchial mild, multifocal

0/10

0/10

0/10

0/10

0/10

0/10

0/10

1/10

 

Applicant's summary and conclusion

Conclusions:
The exposure to the test item reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) in the form of a vapour to Hannover Wistar rats for 91 days for 6 hour/day on a 5 day per week basis at analytical concentrations of 1.02, 3.01 and 11.35 mg/L was associated with the following effects:
At the high dose, transient and slight ataxia was noted following the end of the exposure in both sexes and slight body weight gain suppression was found in males associated with decreased food consumption. A slight increase in liver weights in both sexes consistent with slightly increased serum cholesterol concentration and minimal centrilobular hypertrophy of the hepatocytes were also noted. The weight of adrenals was slightly increased, while thymus weight decreased without any microscopic finding. In the lungs, focal/multifocal accumulation of foamy alveolar macrophages was observed with low incidence in all treated groups.
None of these findings was considered to be adverse.
In conclusion, under the conditions of this study, a no observed adverse effect-level (NOAEL) of 11.35 mg/L was established for reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) administered to Hannover Wistar rats as a vapour by the inhalation route, for for 91 days 6 hour/day on a 5 day per week basis.
In accordance with Annex VIII, section 8.6.1, column 2 and Annex IX, section 8.6.1, column 1, no testing of oral or dermal repeated dose toxicity is required as inhalation is the most probable exposure pathway.
According to the results of the peformed 90-day repeated dose toxicity inhalation study, reaction mass of 2,2'-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) does not need to be classified for Specific target organ toxicity – repeated exposure.
Executive summary:

Repeated inhalation toxicity of test item reaction mass of 2,2'-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) was tested according to OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day). Wistar Crl:WI (Han) rats were exposed 6 hours/day on a 5 day per week basis to the test atmosphere at concentrations of 10, 3 and 0.8 mg/L, as the High, Mid and Low Concentration, respectively. Analytical concentrations of 1.02, 3.01 and 11.35 mg/L were achieved in the respective groups. The control animals were exposed in similar way to filtered air. Forty Hannover Wistar rats of both sexes (twenty males and twenty females) were involved in the study, 10 males and 10 females in each experimental groups. Parameters monitored during the study included mortality, clinical observations, neurological investigations, ophthalmoscopy, terminal examination of oestrus cycle, body weight, food consumption and clinical pathology evaluation (haematology, coagulation, clinical chemistry and urinalysis). Gross macroscopic examination was performed at necropsy and selected organs were weighed.

Full histopathology was performed in control and high dose groups. In addition, lungs were investigated in Mid and Low dose groups.

The test atmosphere concentration was monitored based on the gravimetric analysis and by a validated GC method. The results of the test atmosphere characterization were considered suitable for the study purposes.

 Under the experimental conditions of this study, there was no mortality. There were no clinical signs related to treatment at the low and mid concentration levels. At the high dose (12 mg/L), slight and transient ataxia was observed in all animals at following the end of the exposure. There were no treatment related effects noted during neurological assessment. No test item related changes compared to pre-treatment were noted at ophthalmoscopy examination. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats. Body weights and body weight gains were slightly suppressed in males at 12 mg/L. The mean body weight 6% lower compared to controls and the overall body weight gain (D0-90) was decreased by 12% (not statistically significant).The lower body weight values were in agreement with decreased food consumption (overall value lower than control by 4%). Body weight and body weight gain values of high dose females did not differ significantly from the control mean, however the food consumption was slightly lower than in controls . No significant differences were noted in body weight, body weight gain and food consumption at 3 or 1 mg/L. There were no test item related adverse effects in haematology, blood coagulation or urinalysis parameters at any dose level. At 12 mg/L, cholesterol was slightly higher in both males and females. Albumin concentration, total protein and albumin to globulin ratio were also slightly higher than in control for both sexes and total bilirubin concentration was slightly higher in males. There were no test item-related macroscopic findings noted at necropsy. Liver weights were increased in both sexes at 12 mg/L (by approximately 28-30% for values relative to body weight). The increased liver weight was in agreement with hypertrophy of centrilobular hepatocytes revealed during microscopic examination and increased serum cholesterol concentration. This hypertrophy was of minimal grade and was observed in 4/10 males and 5 of 10 females. It was not associated with cellular necrosis/degeneration and was considered to be an adaptive response. Slightly higher liver weights compared to controls were noted in both males and females at 3 mg/L and in females at 1 mg/L. Slightly increased kidneys weights were noted in both males and females at 12 mg/L and at 3 mg/L. This finding was accompanied by minimal tubular basophilia , in 4/10 males and 2/10 females at 12 mg/L. This finding is a common observation in rats and therefore considered to be not adverse. Nevertheless, a relation to treatment cannot be excluded (only high dose was examined). Thymus weights were slightly lower than control in both sexes at 12 mg/L but they were not correlated with any microscopic change. Weight of adrenals was higher in both males and females at 12 mg/L and in males at 3 mg/L without any microscopic changes (only high dose was examined). In lungs, focal/multifocal accumulation of the foamy alveolar macrophages was observed with low incidence in all treated groups and was regarded as no adverse effect.

 

In conclusion, the exposure to the test item reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) in the form of a vapour to Hannover Wistar rats for 91 days for 6 hour/day on a 5 day per week basis at analytical concentrations of 1.02, 3.01 and 11.35 mg/L was associated with the following effects:

At the high dose, transient and slight ataxia was noted following the end of the exposure in both sexes and slight body weight gain suppression was found in males associated with decreased food consumption. A slight increase in liver weights in both sexes consistent with slightly increased serum cholesterol concentration and minimal centrilobular hypertrophy of the hepatocytes were also noted. The weight of adrenals was slightly increased, while thymus weight decreased without any microscopic finding. In the lungs, focal/multifocal accumulation of foamy alveolar macrophages was observed with low incidence in all treated groups.

None of these findings was considered to be adverse.

In conclusion, under the conditions of this study, a no observed adverse effect-level (NOAEL) of 11.35 mg/L was established for reaction mass of 2,2’-oxybisbutane (DSBE), DIPE, SBA and 2-methylpropan-2-ol (TBA) administered to Hannover Wistar rats as a vapour by the inhalation route, for for 91 days 6 hour/day on a 5 day per week basis.