Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-12 till 2016-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
7 September 2009
GLP compliance:
yes (incl. QA statement)
Remarks:
Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
EC Number:
944-553-4
Cas Number:
1632042-40-0
Molecular formula:
C11H20O
IUPAC Name:
1-(2,4-dimethylcyclohex-3-en-1-yl)propan-1-ol
Test material form:
solid
Details on test material:
- Substance name as cited in test report: FRET 11-0571
- Phystical state: white solid
- Storage conditions: ambient temperature (15-25 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 12 weeks
- Weight at study initiation: mean males 346 g, mean females 202 g
- Fasting period before study: no access to feed or water during exposure
- Housing: animals were housed in groups of five, separated by sex, in macrolon cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) and a piece of gnaw wood (from ABEDD, Austria) and shreds of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) as environmental enrichment.
- Diet: ad libitum, cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England)
- Water: ad libitum, domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC).
- Acclimation period: 6 weeks

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
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.49 - <= 2.59 µm
Geometric standard deviation (GSD):
2.25
Details on inhalation exposure:
EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The inhalation chamber consisted of a cylindrical stainless steel column, surrounded by a transparent cylinder. The column had a volume of 47.6 liters and consisted of a top assembly with the entrance of the unit, two mixing sections, a rodent tube section and at the bottom the base assembly with the exhaust port. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling (for analysis of the actual concentration and particle size) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012). In the experience of the test laboratory, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animal’s thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 liter/min for each rat. The air entering the unit was maintained between 22 ± 3˚C and the relative humidity between 30% and 70%, as much as possible.

GENERATION OF TEST ATMOSPHERE
The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, the test material was diluted in ethanol (50% test material and 50% ethanol [VWR Chemicals, productno. 20823.293], based on weight) and subsequently nebulized using an air-driven atomizer (type 970/S, Düsen-Schlick GmbH, Untersiemau, Germany), located at the top of the exposure chamber. The test material solution was continuously stirred on a magnetic stirring plate, and the amount of material delivered to the atomizer was controlled using a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France). The atomizer was supplied with humidified compressed air, the flow of which was measured using a mass view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands) and controlled by a reducing valve. The aerosol was mixed with a mass flow controlled (Bronkhorst Hi Tec) stream of humidified compressed air in the top of the exposure chamber, and the resulting test atmosphere was directed downward and led to the noses of the animals. At the bottom of the unit, the test atmosphere was exhausted. The animals were placed in the exposure unit after stabilization of the test atmosphere (T95 was about 5 minutes). The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 17 minutes. Exposure was shortly intermitted twice to clean the atomizer; lost time (6 minutes) was compensated at the end of the exposure period. During extensive preliminary experiments, it appeared that test atmosphere generation was not possible without the addition of a vehicle. The neat test material, a waxy solid with a melting point just above ambient temperature, had to be heated for nebulization; all attempts to aerosolize this material (using various nebulizers, tested and heated at several settings) resulted in clogging of the equipment. Various vehicles were subsequently tested. Water could not be used given the poor solubility of the test material. Dilution in acetone did not prevent the clogging problems. After testing dilutions in isopropanol and ethanol which both resulted in stable test atmospheres, ethanol was selected because of its slightly lower toxicity.

ACTUAL CONCENTRATION
The actual concentration of the test material in the test atmosphere was determined thirteen times during exposure by means of gravimetric analysis. Representative test atmosphere samples were obtained by passing approximately 5 L test atmosphere at 5 L/min through fibre glass filters (Sartorius 13400-47, Ø 47 mm). Filters were weighed before sampling, loaded with a sample of test atmosphere, dried for 10 minutes at ambient conditions (which was determined to be sufficient to eliminate the ethanol), and were then weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken. Given the available vapor pressure of the test substance (16.84 Pa at 20ºC), some of the material in the test atmosphere could have been present in the vapor phase (in theory, up to the saturated vapor concentration of about 1.2 g/m3 at 20 ºC), which is not captured on the gravimetric filters. Therefore, potential evaporation of the test material was examined during preliminary experiments by applying known amounts (26.69 and 26.78 mg) of pure (unaerosolized and undiluted) test material onto fibre glass filters, which were weighed after prolonged periods of drying at ambient conditions. Evaporation was determined to be a relatively slow process: only 5% of the test material had evaporated 6 hours after loading; about 50% was still left after 2 days, and over 10% was left after 10 days of drying. Thus, the potential effect of evaporation on the results of the gravimetric analysis was considered to be small, as long as filters were weighed shortly after loading.

TIME TO ATTAIN CHAMBER EQUILIBRATION (T95)
The concentration C in a perfectly stirred test atmosphere in a chamber with volume V (L) and flow F (L/min) increases according to C = C∞ * (1 – e-(F*T/V)), in which T (min) is the time and C∞ is the steady state concentration. The time it takes to reach 95% of the steady state concentration (T95) was calculated from the above formula as: T95 = (-V * Ln 0.05) / F = 3V/F.

NOMINAL CONCENTRATION AND GENERATION EFFICIENCY
The nominal concentration was determined by dividing the total amount of test material used (by weighing) by the total volume of air passed through the exposure unit. The generation efficiency was calculated from the actual concentration (determined by gravimetric analysis) and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

PARTICLE SIZE MEASUREMENT
Three particle size distribution measurements were carried out: one during preliminary generation of the test atmosphere (at the settings used during exposure) and two during exposure of the animals. A 10-stage cascade impactor was used (2110k cascade impactor, Sierra instruments, Carmel Valley, California, USA). The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated (Lee, 1972).

TOTAL AIR FLOW, TEMPERATURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The chamber air flow, temperature and relative humidity of the test atmosphere were recorded eight times during exposure. Air flow was measured by recording the readings of the mass view meter and mass flow controller. The temperature and relative humidity were measured using an RH/T device (TESTO type 0636 9735 probe with 635-1 read-out unit, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany). The oxygen (Oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) concentrations were measured once during exposure.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5 g/m3 (target concentration); 4.94 g/m3 (± 0.43) (actual concentration)
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: On the exposure day, the animals were observed for clinical signs just before exposure, four times during exposure (about once per hour), and twice after exposure. During exposure, when observation was limited due to the animals’ stay in restraining tubes, attention was directed to breathing abnormalities and restlessness. During the observation period, each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were checked again in the afternoon.
- Frequency of weighing: The body weight of each animal was recorded once during the acclimatization period (on day -1) and just prior to exposure on day 0. Surviving animals were also weighed on days 1, 3, 7 and on day 14 prior to necropsy. Body weights were also recorded at the time of discovery after intercurrent death.
- Necropsy of survivors performed: yes

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4 940 mg/m³ air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Two female animals died during the last hour of exposure and one male was found dead shortly afterwards; the remaining 7 animals survived until scheduled sacrifice.
Clinical signs:
other: During exposure, all animals showed a decreased breathing rate, dyspnea and lethargy, which increased in severity – from slight or moderate to severe – during the course of the 4-hour exposure period. Shortly after exposure, surviving animals displayed br
Body weight:
All surviving animals showed a slight exposure-related loss of body weight (3-8%) on the day after exposure. Most animals – except one female – had gained weight again on day 3. Normal growth was observed in all surviving animals during the second week of the observation period.
Gross pathology:
Necropsy of the three animals which were found dead revealed pulmonary hemorrhages and/or incompletely collapse of the lungs, large amounts of air in the gastrointestinal tract (probably the result of gasping in response to the respiratory distress), and all animals had a wet fur. In addition, deposition of test material was found in the nose and mouth of one female and in the pharynx of the male animal; the latter animal also had a fully filled urinary bladder. Exposure-related gross macroscopic changes observed at scheduled necropsy of surviving animals were limited to red spots (indicating small hemorrhages) on one or more lung lobes of two males and a female animal. Other changes (swollen uterus, skin wound) are commonly observed as background findings and were not considered to be related to the exposure.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions (OECD 403, GLP) the LC50 of the test substance is >4.94 g/m3.
Executive summary:

In a GLP compliant acute inhalation study, performed according to OECD guideline 403, five Wistar rats per sex were exposed to the test substance. Animals were exposed to 4.94 g/m3 of the aerosolized test material for 4 hours. After an observation period of 14 days animals were necropsied. Two female animals died during the last hour of exposure and one male was found dead shortly afterwards. The remaining four males and three females survived until scheduled sacrifice. During exposure, all animals showed a decreased breathing rate, dyspnea and lethargy, which increased in severity during the course of the 4-hour exposure. Shortly after exposure, surviving animals displayed breathing abnormalities (dyspnea, sniffing, mouth breathing, decreased breathing rate) and general signs of discomfort (e.g. ataxia, hypoactivity, hunched posture, muscle weakness, vocalization, piloerection). The incidence of these abnormalities gradually decreased over the course of the following days. Generally, exposure-related clinical abnormalities were no longer observed by the end of the first week of the observation period, with the exception of one male which displayed piloerection up to day 7 and dyspneic breathing until scheduled sacrifice. All surviving animals showed a slight (3-8%) exposure-related body weight loss on the day after exposure. Recovery of the loss of body weight was observed during the first week, followed by normal growth during the second week of the observation period. Necropsy of the three animals which were found dead revealed pulmonary hemorrhages and/or incompletely collapse of the lungs, large amounts of air in the gastrointestinal tract (a sign of gasping in response to respiratory distress), and all animals had a wet fur. In addition, deposition of test material was found in the nose and mouth of one female and in the pharynx of the male animal; the latter animal also had a fully filled urinary bladder. At scheduled necropsy of the surviving animals, exposure-related macroscopic observations were limited to red spots on one or more lung lobes of two males and a female animal. Based on the results of this study, it was concluded that the 4-hour LC50of the test material in rats is above 4.94 g/m3.