Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-24 till 2016-09-21

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:

September, 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion BV, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Test type:

traditional method

Limit test:

yes

Test material

Test material information

1

Reference substance name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

EC Number:

944-405-9

Molecular formula:

C11H20O2

IUPAC Name:

Reaction mass of (4Z)-4-ethylidene-2-propoxycyclohexanol and (4E)-4-ethylidene-2-propoxycyclohexanol and (5Z)-5-ethylidene-2-propoxycyclohexanol and (5E)-5-ethylidene-2-propoxycyclohexanol

Test material form:

liquid

Details on test material:

- Substance name as cited in test report: FRET 13-0156
- Phystical state: clear, yellowish liquid
- Storage conditions: ambient temperature (15-25 °C), protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

outbred rats (Crl:WI(Han))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 9 weeks
- Weight at study initiation: mean weights of 261 g (males); 186 g (females)
- Fasting period before study: none
- Housing: except during exposure, 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. During exposure, the animals were housed individually in the exposure unit.
- Diet: cereal-based (closed formula) rodent diet (VRF1) from a commercial supplier (SDS Special Diets Services, Witham, England); available ad libitum
- Water: domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC); available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- 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:

air

Mass median aerodynamic diameter (MMAD):

3.83 µm

Geometric standard deviation (GSD):

1.86

Remark on MMAD/GSD:

- 3.83 ± 0.13 μm (MMAD)
- 1.86 ± 0.05 µm (GSD)

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 47.8 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. Unused 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 our experience, 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 animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow 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%.

- Generation of the test atmosphere
The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. The test atmosphere was generated by nebulization using an air-driven atomizer (Schlick type 970/S, Coburg, Germany) which was placed at the top inlet of the exposure chamber. The amount of test material delivered to the atomizer was controlled using a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France). The atomizer was supplied with a stream of dry compressed air, controlled at a pressure of 4 bar using a reducing valve. Air flow to the atomizer was measured using a mass view meter (Bronkhorst Hi Tec, Ruurlo, the Netherlands). 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. A schematic diagram of the generation and exposure system is presented in Figure 1. The animals were placed in the exposure unit after stabilization of the test atmosphere (T95 was about 3.8 minutes, see paragraph 5.1.2). The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 34 minutes. Test atmosphere generation was done in an illuminated laboratory at room temperature.

- Actual concentration
The actual concentration of the test substance in the test atmosphere was determined eight times during exposure by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone 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, 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 (9 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 0.7 g/m3 at 20ºC), which is not captured on the gravimetric filters. Therefore, potential evaporation of the test material was examined during preliminary experiments by applying known amounts (47.74 and 47.53 mg) of pure (unaerosolized) 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 7.5 hours after loading, and about 72-76% was still left after a day 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.

- 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
Particle size distribution measurements were carried out using an Aerodynamic Particle Sizer (APS; model 3321, TSI Incorporated, Shoreview, MN, USA). Eight samples were taken during exposure, which were diluted using an aerosol diluter (model 3302A, TSI Incorporated; dilution ratio of 2000:1) and subsequently measured by APS. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated (APS user’s manual, 2010). Given the nature of the test material (a slowly evaporating liquid), an APS was considered better suitable for particle size analysis than a cascade impactor – commonly used for aerodynamic particle size measurements – because the latter instrument relies on weighing of test material captured on substrates. Nevertheless, results of particle size measurements by APS were compared to data obtained using 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA) by simultaneous sampling during preliminary test atmosphere generation experiments performed on 11 August 2016. Cascade impactor measurements resulted in a slightly higher particle size when compared to results obtained by APS (4.05 μm for the cascade impactor vs 3.82 μm for the APS).

- 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

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 and weighing:
Observations: 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.
Weighing: once during the acclimatization period (on day -1), and on days 0 (just before exposure), 1, 3, 7 and on day 14 prior to necropsy.
- Necropsy of survivors performed: yes
- Other examinations performed: At the end of the 14-day observation period, the animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital). At necropsy, abdominal and thoracic organs were examined in situ for gross pathological changes. Carcasses were discarded after the post-mortem examination.

Statistics:

Not applicable

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.2 other: g/m3 air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: 5.20 ± 0.03, (N=8)

Remarks:

this is considered to be a slight underestimation of the total concentration of the test substance in the atmosphere, which will have been slightly higher due to potential evaporation of the test material.

Mortality:

No mortality occurred during the study.

Clinical signs:

other: During exposure, all animals displayed breathing abnormalities characterized by a decreased breathing rate and shallow breathing. The severity of these findings increased during the course of the exposure period, and males (showing slight to moderate abno

Body weight:

A slight loss of body weight was observed in most animals on the day after exposure (on average 5% in males and 1% in females). All animals recovered from the initial body weight loss within a week and showed normal growth during the second week of the observation period.

Gross pathology:

Scheduled necropsy at the end of the 14-day observation period, revealed red spots on one or more lung lobes – indicating pulmonary hemorrhages – of one female and four male animals. No macroscopic lesions were found in the five remaining animals

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 >5.20 g/m3.

Executive summary:

In this GLP-compliant acute toxicity inhalation study, performed according to OECD 403, toxicity of the test substance was examined. A group of five male and five female rats was exposed to a target concentration of 5 g/m³ for 4 hours. Thereafter, the animals were kept for an observation period of 14 days before necropsy. To detect adverse effects, clinical observations were made during and after exposure, body weight was determined before exposure (days -1 and 0) and on days 1, 3, 7 and 14, and a gross necropsy was performed on all animals.

 

The mean actual concentration (± standard deviation) during exposure, based on gravimetric analysis of test atmosphere samples, was 5.20 (± 0.03) g/m³. The average mass median aerodynamic diameter (MMAD) of the particles in the aerosol was 3.83 (± 0.13) μm and the distribution of particle sizes had an average geometric standard deviation (GSD) of 1.86 (± 0.05). Mortality did not occur during the study; all animals survived until scheduled sacrifice at the end of the 14-day observation period. During exposure, breathing abnormalities were observed in all animals, characterized by a decreased breathing rate and shallow respiration. Male animals were generally affected more severely than females. Shortly after exposure, animals displayed breathing abnormalities (dyspnoea, sniffing and/or shallow breathing) and general signs of discomfort (e.g. ataxia, tremors, hunched posture, hypoactive behavior, piloerection). At the end of the day, the condition of the animals had improved and clinical abnormalities were less apparent. Over the course of the following days, the animals recovered from these abnormalities. Most animals showed a slight loss of body weight on the day after exposure (on average 5% in males and 1% in females), from which they recovered within the first week. Normal growth was observed in the second week of the observation period. Macroscopic examination at scheduled necropsy revealed hemorrhages in one or more lung lobes of one female and four male animals. No macroscopic lesions were found in the five remaining animals.

 

Based on the results of this study, it was concluded that the 4-hour LC50 of the test substance in rats is above 5.20 g/m³.