1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 05 September 2012 and 18 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

EC Number:

228-846-8

EC Name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Cas Number:

6362-80-7

Molecular formula:

C18H20

IUPAC Name:

(4-methyl-4-phenylpent-1-en-2-yl)benzene

Details on test material:

- Name of test material (as cited in study report): 1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene
- Physical state: Clear colourless liquid
- Analytical purity: 95 %
- Lot/batch No.: 2554* and 2X55**
- Expiration date of the lot/batch: 28 February 2013
- Storage condition of test material: Room temperature, in the dark

* utilised during the sighting exposure and the first formal exposure
** utilised during the second formal exposure

Test animals

Species:

rat

Strain:

other: RccHan™: WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8- 12 weeks
- Weight at study initiation: 200 g- 350 g
- Fasting period before study:
- Housing: The animals were housed in groups of three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 25
- Humidity (%): 30- 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The test item was aerosolised using a glass concentric jet nebuliser located at the top of the exposure chamber. The nebuliser was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply. Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebuliser. The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The extract from the exposurechamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatised (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the day of exposure, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Temperature, humidity, pressure in air chamber: 21- 22 °C, 78- 83 %. The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty
min.
- Oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser located in a port in the
animals breathing zone during the 4 h exposure period. The test atmosphere was generated to contain at least 19% oxygen.

TEST ATMOSPHERE
- The test atmosphere was sampled 5 times during the exposure period. The sampling procedure involved pumping 2 L of the chamber atmosphere through a glass impinger containing 40 mL of methanol. After sampling the dreschel head was flushed through with a further 40mL of methanol to remove any deposits. This gave an 80mL sample (per impinger) to be submitted for chemical analysis.
- Partical size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined 3 times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of 6 impactor stages (8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 μm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 μm was calculated. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

The actual concentration of the test item was measured off-line by Gas Chromatography. The test atmospheres were sampled after theoretical chamber equilibration and then at approximately hourly intervals during the exposure period.

Duration of exposure:

4 h

Concentrations:

Mean achieved concentraiton: 10.66 mg/L

No. of animals per sex per dose:

3/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Necropsy of survivors performed: yes

Other examinations performed:
- Clinical signs: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 h after termination of exposure and subsequently once daily for up to 14 d. Any deaths or evidence of overt toxicity were recorded at each observation.
- Bodyweight: Individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.
- Necropsy: At the end of the 14 d observation period, the surviving animals were killed by intravenous overdose of sodium pentobarbitone. All animals, including the2 that died during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Results and discussion

Preliminary study:

Sighting exposure: No significant observations were noted during the exposure period, on removal from the chamber and 1 h post-exposure significant observations noted in both animals included increased respiratory rate, laboured respiration and ataxia. On Day 1 both animals exhibited increased respiratory rate, hunched posture and piloerection, the female also exhibited laboured respiration. Both animals recovered to appear normal on Day 5 post-exposure. No macroscopic abnormalities were detected amongst these 2 animals at necropsy.

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 10.66 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

At a mean achieved atmosphere concentration of 10.66 mg/L, the following deaths occured:
Male deaths: 1/3
Female deaths: 1/3
Total deaths: 2/6

Clinical signs:

other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a

Body weight:

All surviving animals exhibited slight bodyweight losses or showed no bodyweight gain on the first day post-exposure. Bodyweight gains were noted in all surviving animals during the remainder of the recovery period, with the exception of 1 male animal which exhibited a bodyweight loss from Days 3 to 7 post-exposure.

Gross pathology:

No macroscopic abnormalities were detected amongst animals that survived until the end of the recovery period at necropsy.

Pale or abnormally dark lungs were detected in the male or female animal respectively that died during the course of the study.

Due to the observations noted it is considered that the deaths noted during this study may have been mainly attributable to Systemic Toxicity.

Any other information on results incl. tables

Exposure chamber concentration

Atmospheric Concentration
Mean achieved (mg/L)	Standard deviation	Nominal (mg/L)
10.66	0.52	76.2

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour. The theoretical chamber equilibration time (T₉₉) was 3 min* (*The test atmosphere was generated for a total of 15 minutes prior to animal insertion to ensure test item concentration was being achieved).

Particle size distribution

The particle size analysis of the atmosphere drawn form the animals' breathing zone, was as follows:

Mean achieved atmosphere concentration (mg/L)	Mean mass median aerodynamic diameter (µm)	Inhalable fraction (% <4 µm)	Geometric standard deviation
10.66	2.69	67.4	2.42

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute toxicity of the test material via the inhalation route was assessed according to OECD guideline 436. Two deaths (1 male and 1 female) occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 10.66 mg/L for 4 h. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of the test material, in the RccHanTM : WIST strain rat, was greater than 10.66 mg/L. As the mean achieved atmosphere concentration was more than double the target “limit test” concentration of 5mg/L and only two animals died, it is considered that the test item should not be classified for inhalation toxicity under the conditions of this study.