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

Endpoint summary

Description of key information

Acute oral toxicity: The LD50 value of the test item was estimated to be between 300 mg/kg and 2000 mg/kg.

Acute inhalation toxicity: . The acute inhalation median lethal concentration (4 hr LC50) of the test material was considered to be greater than 10.66 mg/L.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes

Test type:

acute toxic class method

Species:

rat

Strain:

Crj: CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan (Hino Breeding Center)
- Age at study initiation: 8 weeks of age
- Weight at study initiation: 175-187 g.
- Fasting period before study: Animals were fasted from the evening of the day before administration (from 4:00-4:10 pm) for about 17.5-18.5 hours until administration, and then for about 6 hours after administration, after which feed was provided again
- Housing: The animals were housed individually in racks of 5 stainless steel cages (W: 755 × D: 210 × H: 170 mm) during both the quarantine/acclimation period and after grouping
- Diet: The animals had free access to pellet diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water: The animals were provided free access to tap water in water bottles. However, water was removed after grouping for about 6 hours until administration, after which it was provided again.
- Acclimation period: A five day quarantine period followed by a 2 day acclimation period was set for the animals before the first, second, third and fourth tests

ENVIRONMENTAL CONDITIONS
- Temperature: 23°C (measured values: 21-23°C),
- Humidity: nominal humidity of 55% (measured values: 40-61%)
- Air changes (per hr): 12 times/hour (filter-sterilized fresh air).
- Photoperiod: 12-hour light/dark cycle (illumination: 6:00 am to 6:00 pm)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED:
The dose volume was 10 mL/kg, calculated based on the body weight measured immediately before administration.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The LD50 value for oral administration of 2,4-diphenyl-4-methyl-1-pentene in rats is reported to be 3.8 mL/kg. 1) Therefore, the maximum dose of 2000 mg/kg according to the OECD Guideline for Testing of Chemical for Acute Oral Toxicity-Acute Toxic Class method (Revised Guideline 423) was selected for the first test.

Doses:

2000 and 300 mg/kg

No. of animals per sex per dose:

Three females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs and mortality for 6 hours after administration (immediately after to 30 minutes after administration and 2, 4 and 6 hours after administration) and once a day during the observation period from the day after administration. Body weight was measured on the day of administration (immediately before administration) and 1, 3, 7, 10 and 14 days after administration
- Necropsy of survivors performed: yes

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

300 - 2 000 mg/kg bw

Based on:

test mat.

Mortality:

One animal died on day 1 after administration in the first test at 2000 mg/kg, and 2 animals died on day 1 after administration in the second test at 2000 mg/kg. There were no deaths in the third and fourth tests at 300 mg/kg. From these results, the LD50 value of 2,4-diphenyl-4-methyl-1-pentene was estimated to be between 300 mg/kg and 2000 mg/kg.

Clinical signs:

other: Clinical signs observed in the first test at 2000 mg/kg were tremor in 1 animal at 4 hours after administration, tremor and clonic convulsions in 1 animal at 6 hours after administration, and soiled perineal region in 2 animals and clonic convulsions in 1

Gross pathology:

No abnormal necropsy findings were observed either in the animals that died in the first and second tests at 2000 mg/kg.

No abnormal necropsy findings were observed in the surviving animals in the first and second tests at 2000 mg/kg or in the surviving animals in the third and fourth tests at 300 mg/kg.

Results: (Test group 1: 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene group at 2000 mg/kg)

Concentration of 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene (mg/mL)	Measured concentrations			Recovery rate (%)
	1st	2nd	Mean
200	192.2	196.1	194.2	97.1

The concentration of the preparation was within the acceptable range (100 ± 10%)

Results: (Test group 2: 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene group at 2000 mg/kg)

Concentration of 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene (mg/mL)	Measured concentrations			Recovery rate (%)
	1st	2nd	Mean
200	194.6	186.4	190.5	95.3

The concentration of the preparation was within the acceptable range (100 ± 10%)

Results: (Test group 3: 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene group at 300 mg/kg)

Concentration of 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene (mg/mL)	Measured concentrations			Recovery rate (%)
	1st	2nd	Mean
30	28.77	28.78	28.78	95.9

The concentration of the preparation was within the acceptable range (100 ± 10%)

Results: (Test group 4: 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene group at 300 mg/kg)

Concentration of 1,1-(1,1-dimethyl-3-methylene-1,3-propanediyl) bisbenzene (mg/mL)	Measured concentrations			Recovery rate (%)
	1st	2nd	Mean
30	29.88	29.31	29.60	98.7

The concentration of the preparation was within the acceptable range (100 ± 10%)

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value of 2,4-diphenyl-4-methyl-1-pentene is estimated to be between 300 mg/kg and 2000 mg/kg. Based on the results the test substance should be classified as Acute Toxicity Category 4 according to CLP regulation 1272/2008

Respiratory paralysis or central nervous abnormality is thought to have led to deaths because tremor and clonic convulsions were observed following the administration of the test item at 2000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Quality of whole database:

Two separate acute toxicity studies via the oral route have been conducted on the test material, namely Anon (2007) entitled Single Dose Oral Toxicity Study of 2,4-Diphenyl-4-methyl-1-pentene in Rats and Baldrick (1991). The Anon (2007) study has been conducted according to OECD Guideline 423 and to GLP and is well documented and has been assigned reliability 1. The Baldrick (1991) study has been conducted according to OECD Guideline 401 and to GLP and is well documented and has been assigned reliability 1. The Anon (2007) study determined the LD50 value of test item to be between 300 mg/kg and 2000 mg/kg. Respiratory paralysis or central nervous abnormality is thought to have led to deaths because tremor and clonic convulsions were observed following the administration of the test item at 2000 mg/kg. The Baldrick (1991) study found that the acute lethal oral dose to rats of the test substance was found to be greater than 5.0 g/kg bodyweight. As such, the Baldrick (1991) study has been used as supporting study only.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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.

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

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

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.

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

10 660 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity via the oral route: An acute oral toxicity study was performed in the rat in accordance with GLP and OECD Guideline 423. Four groups of 3 female rats were given a single oral dose of the test item in corn oil; 2 groups received a dose level of 2000 mg/kg and 2 groups received a dose level of 300 mg/kg. One animal died on day 1 after administration in the first test at 2000 mg/kg, and 2 animals died on day 1 after administration in the second test at 2000 mg/kg. Clinical signs observed in the first and second tests at 2000 mg/kg were tremor, clonic convulsions, soiled perineal region and diarrhea. No abnormalities were observed in the tests at 300 mg/kg. Suppression of body weight gain was observed on day 1 after administration in the first and second tests at 2000 mg/kg. No abnormality in body weight change was observed in the tests at 300 mg/kg. No abnormal necropsy findings were observed either in the animals that died or those that survived in the first and second tests at 2000 mg/kg. No abnormal necropsy findings were observed in the tests at 300 mg/kg. Therefore, the LD₅₀ value of the test item is estimated to be between 300 mg/kg and 2000 mg/kg. Respiratory paralysis or central nervous abnormality is thought to have led to deaths because tremor and clonic convulsions were observed following the administration of the test item at 2000 mg/kg.

Acute toxicity via the inhalation route: An acute inhalation study was performed in the rat in accordance with GLP and OECD Guideline 436. A group of 6 RccHanTM : WIST strain rats (3 males and 3 females) was exposed to an aerosol atmosphere of the test item with a mean achieved atmosphere concentration of 10.66 mg/L (more than double the target “limit test” concentration of 5 mg/L) for 4 h using a nose only exposure system, followed by a 14 d observation period. Two deaths, 1 male and 1 female, occurred. Common abnormalities noted during the study included increased respiratory rate, decreased respiratory rate, laboured respiration, moisy respiration, ataxia, occasional body tremors, hunched posture, pilo-erection and wet fur, occasional instances of clonic convulsions ans sneezing were also noted. Surviving animals recovered to appear normal from Days 13 or 14 post-exposure. 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 one male animal which exhibited a bodyweight loss from Days 3 to 7 post-exposure. 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. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC₅₀) of the test item was greater than 10.66 mg/L.

Justification for classification or non-classification

Acute oral toxicity: The available study has been assigned a reliability 1 and is considered acceptable for classification. The acute oral median lethal dose (LD₅₀) of the test item in the female rat was estimated to be between 300 and 2000 mg/kg. Therefore, the test item should be classified as Category 4 for acute toxicity via the oral route in accordance with Regulation 1272/2008.

Acute inhalation toxicity: The available study has been assigned a reliability 1 and is considered acceptable for classification. The acute inhalation median lethal concentration (4 hr LC50) was considered to be greater than 10.66 mg/L. As the mean achieved atmosphere concentration was more that double the target "limit test" concentration of 5 mg/L and only 2 animals died the test item can be considered to be non-classified.