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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 November 2020 – 9 September 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
GLP compliance:
yes (incl. QA statement)
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylstyrene
EC Number:
202-889-2
EC Name:
3-methylstyrene
Cas Number:
100-80-1
Molecular formula:
C9H10
IUPAC Name:
1-methyl-3-vinylbenzene
Constituent 2
Chemical structure
Reference substance name:
4-methylstyrene
EC Number:
210-762-8
EC Name:
4-methylstyrene
Cas Number:
622-97-9
Molecular formula:
C9H10
IUPAC Name:
1-methyl-4-vinylbenzene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by Sponsor, Batch no.: 20200608
- Expiration: 07 August 2021
- Purity, including information on contaminants, isomers, etc.: 99.66% (3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at +2 °C to +8 °C in a tightly closed container

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories,Research Models and Services, Germany GmbH, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: 49 days; Females: 63 days
- Housing: During the 14-day observation period the animals are caged by sex in groups of 2 or 3 by sex in MAKROLON cages (type III plus). Granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany) is used as bedding material for the cages. The cages are changed and cleaned twice a week.
- Diet: Commercial diet ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany). This food was offered ad libitum. Food residue was removed.
- Water: Tap water was offered ad libitum.
Acclimitisation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 10%
- Air changes (per hr): air changes/h (≥12)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle


Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 9.205 µm
Geometric standard deviation (GSD):
ca. 4.913
Remark on MMAD/GSD:
Feasibility tests:

1st: 5.20 mg/L air MMAD = 9.124 µm GSD = 5.443;

2nd: 2.06 mg/L air MMAD = 9.768 µm GSD = 2.057.




The feasibility tests revealed that the MMAD at a concentration of 2 mg/L air was even slightly worse compared to a concentration at 5 mg/L air. The cumulative mass of particles less than 4 µm was approximately 31% at a concentration at 5 mg/L air and only 11% at a concentration at 2 mg/L air. Hence, the use of 5 mg/L air is from a toxicological point of view more suitable than employing 2 mg/L air, as at a concentration of 5 mg/L air the absolute concentration of smaller particles reaching the alveoli is considered to be larger than at 2 mg/L air.



Limit test:
Nominal concentration: 49.88 mg/L air;
Gravimetric (actual) concentration: 5.02 ± 0.02 mg/L air;
Mean MMAD: 9.205 µm;
Mean GSD: 4.913.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Feasibility test:
Before initiating the study with the animals, a feasibility test was carried out with the exposure system in order to verify that under the experimental settings chosen, the limit concentration of 5 mg/L air could be achieved by gravimetric analysis. The assay confirmed the functionality of the inhalation system and a limit concentration of 5.20 mg/L air was achieved. The determination of the aerosol particles at a concentration of 5 mg/L air revealed a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) out of the recommended range of 1 - 4 µm for MMAD and 1.5 - 3.0 for GSD as recommended in the OECD guideline 403. Hence, a further feasibility test was carried out with a limit concentration of 2 mg/L air.

The feasibility tests revealed that the MMAD at a concentration of 2 mg/L air (9.768 µm) was even slightly worse compared to a concentration at 5 mg/L air (9.124 µm). The cumulative mass of particles less than 4 µm was approximately 31% at a concentration at 5 mg/L air and only 11% at a concentration at 2 mg/L air. Hence, the use of 5 mg/L air is from a toxicological point of view more suitable than employing 2 mg/L air, as at a concentration of 5 mg/L air the absolute concentration of smaller particles reaching the alveoli is considered to be larger than at 2 mg/L air. For results of the feasibility tests, see Appendix 3.

- Exposure apparatus: The study was carried out using a dynamic inhalation apparatus (RHEMA-LABORTECHNIK, 65719 Hofheim/Taunus, Germany; air changes/hour approximately 30 times). The apparatus consists of a cylindrical exposure chamber which holds the animals in pyrex tubes at the edge of the chamber in a radial position.

- Exposure chamber volume: 28.5 L (Inner Diameter: 23.9 cm; Height: 63.0 cm)

- Method of holding animals in test chamber: The study animals were acclimatised to the test apparatus for approximately 5 minutes on 3 days prior to testing. The restraining tubes did not impose undue physical, thermal or immobilisation stress on the animals. Exposition started by locating the animals into the exposure chamber after equilibration of the chamber concentration for at least 15 minutes. Nose-only exposure was carried out according to KIMMERLE & TEPPER.
- Source and rate of air (airflow): A manometer and an air-flow meter (ROTA Yokogawa GmbH & Co. KG, 79664 Wehr/Baden, Germany) were used to control the constant supply of compressed air (inflow 900 L/h) and the exhaust (outflow 800 L/h), respectively. Flow rates were checked hourly and corrected if necessary.

Method of conditioning air: The oxygen content in the inhalation chamber was 21%. It was determined at the beginning and at the end of the exposure with a DRÄGER Oxygen-analysis test set . No determination of the carbon dioxide concentration was carried out as there was no evidence that the CO2 concentration exceeded 1% during the exposure.

- System of generating particulates/aerosols: The aerosol of the test item was obtained using a spray-jet (ALUP Kompressorenfabrik, 73257 Köngen, Germany). The spray-jet was fed with compressed air (5.0 bar) from a compressor and with the test item using an infusion pump (TSE infustion pump no. 37, jet model no. 970, TSE Systems). At the bottom of the exposure chamber, the air was sucked off at a lower flow rate than it was created by the spray-jet in order to produce a homogenous distribution and a positive pressure in the exposure chamber.

- Method of particle size determination: An analysis of the particle size distribution was carried out twice during the exposure period (80 minutes and 160 minutes after start of exposure) using a cascade impactor according to May.

The impactor is a device that classifies particles present in a sample of air or gas into known size ranges by drawing the air sample through a cascade of progressively finer nozzles. The air jets from this impact on pre-weighed plane sampling surfaces (slides). Each stage represents an aerodynamic size range and collects finer particles than its predecessor. Each successive stage represents a special aerodynamic cut-off diameter.

The aerosol from the exposure chamber is sucked through the cascade impactor for a specified period of time at a constant flow rate which depends on the physical properties of the test item. Aerosols with mass median aerodynamic diameter (MMAD) ranging from 1 to 4 µm with a geometric standard deviation (σg) in the range of 1.5 to 3.0 are recommended to allow the exposure of all relevant regions of the respiratory tract.

In the present study, the aerosol from the exposure chamber was drawn through the cascade impactor for 5 minutes at a constant flow rate of 5 L/min. The slides were removed from the impactor and weighed on an analytical balance with a precision of 0.1 mg. Deltas of slides’ weight were determined. The mass median aerodynamic diameter (MMAD) was estimated by means of non-linear regression analysis. The 32 µm particle size range and the filter (particle size range < 0.5 µm) were not included in the determination of the MMAD in order not to give undue weight to these values. The Geometric Standard Deviation (GSD) of the MMAD was calculated from the quotient of the 84.1%- and the 50%-mass fractions, both obtained from the above-mentioned non-linear regression analysis.

- Temperature, humidity in air chamber: The correct temperature (22°C ± 3°C) and humidity (55% ± 15%) in the inhalation chamber were checked once every hour with a climate control monitor.

TEST ATMOSPHERE
The actual aerosol concentration in the inhalation chamber was measured gravimetrically with
an air sample filter (0.45 µm; Minisart SM 17598) and pump (Membrane Pump,Vacuubrand GmbH
+ Co. KG, 97877 Wertheim/Main, Germany) controlled by a rotameter (Rota Yokogawa Deutschland GmbH, 40880 Rattingen, Germany). Aerosol samples were taken once every hour during the exposure. For that purpose, a probe was placed close to the animals' noses and air was drawn through the air sample filter at a constant flow of air of 5 L/min for 60 seconds. The filters were weighed before and after sampling (accuracy 0.1 mg). Individual chamber concentration samples did not deviate from the mean chamber concentration by more than 1%. In the report, both the nominal and the gravimetric concentrations are given, the inhalation chamber was equilibrated for at least 15 minutes.

TEST ATMOSPHERE (if not tabulated)
Limit test:
Nominal concentration: 49.88 mg/L air;
Gravimetric (actual) concentration: 5.02 ± 0.02 mg/L air;
Mean MMAD: 9.205 µm;
Mean GSD: 4.913.



Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Main test:
Target concentration = 5 mg/L air;
Gravimetric (actual) concentration = 5.02 ± 0.02 mg/L air

No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days: yes

- Frequency of observations and weighing: A careful clinical observation of each animal was made on the day of exposure (i.e. test day 1) and frequently during the treatment period, at least twice thereafter. Further observations were made at least once daily for 14 days or longer, if necessary. Records were maintained for each animal and any occurrence of findings. Observations on mortality were made at least once daily (in the morning starting on test day 2) to minimize loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.

Individual body weights of animals were determined once during the adaptation period on test day -1, before the exposure on test day 1, and on test days 4, 8, 15 and at time of death. Changes in weight were calculated and recorded in case of survival exceeded one day.

- Necropsy of survivors performed: Necropsy of all animals was carried out and all gross pathological changes were recorded. The lung weights of all animals were determined.

- Clinical signs including body weight : Cageside observations included, but were not limited to: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system as well as somatomotor activity and behaviour pattern. Particular attention was directed to observations in form of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The animals were also observed for possible indications of respiratory irritation such as dyspnoea (shortness of breath, observed in form of a reduced respiratory rate).

Results and discussion

Preliminary study:
The feasibility tests revealed that the MMAD at a concentration of 2 mg/L air (9.768 µm) was even slightly worse compared to a concentration at 5 mg/L air (9.124 µm). The cumulative mass of particles less than 4 µm was approximately 31% at a concentration at 5 mg/L air and only 11% at a concentration at 2 mg/L air. Hence, the use of 5 mg/L air is from a toxicological point of view more suitable than employing 2 mg/L air, as at a concentration of 5 mg/L air the absolute concentration of smaller particles reaching the alveoli is considered to be larger than at 2 mg/L air. For results of the feasibility tests, see Appendix 3.
Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.02 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 5.02 ± 0.02 mg/L air
Mortality:
The test item led to mortality/morbidity in only 1 of the 6 animals. One female rat revealed a pronounced sedation until 60 minutes p.a. and was sacrificed prematurely 2 hours after end of exposure due to animal welfare reasons.
Clinical signs:
other: Refer to Any other information on results incl. tables
Body weight:
The body weight did not reveal test item-related changes in any of the rats at the end of the study.
Gross pathology:
The macroscopic examination at necropsy did not reveal any changes. Hence, no histopathological examination was performed. No influence on the lung weights was observed.

Any other information on results incl. tables

Clinical signs


The following signs of systemic toxicity occurred in all 3 male and 3 female animals immediately after end of the 4-hour exposure and lasted for up to test day 3:


Moderately to severely reduced motility and ataxia were noted in all male and female rats until 60 minutes p.a., and slightly reduced motility and ataxia until 3 hours p.a.


Slightly reduced respiratory rates, tonic and clonic convulsions and slight tremor were observed in all 3 male and 2 of 3 female animals until 3 hours p.a. Slight lacrimation was observed for all animals up to 3 hours p.a. One female rat (no. 5 f) revealed a pronounced sedation until 60 minutes p.a. and was sacrificed prematurely 2 hours after end of exposure due to animal welfare reasons.


Slight pilo-erection was noted up to test day 2 (all 3 females) or test day 3 (all 3 males) p.a.


Furthermore, the following sign of systemic toxicity occurred in 1 of 3 female animals in test week 2 p.a.:


Ptosis and corneal oedema was noted in female no. 6 f on test day 8 or 9 and lasted until test day 11 or 13, respectively.


 


Feasibility test results summary

































Feasibility


test


 


 



Initial


concentration


 


 



MMAD


 


 


 



GSD


 


 


 



Cumulative mass of particles


(approximate values)


less than



< 1 µm



< 4 µm



1st



5.20 mg/L air



9.124 µm



5.443



10%



31%



2nd



2.06 mg/L air



9.768 µm



2.057



0.08%



11%



 


Limit study test results summary



























Nominal concentration



Gravimetric


(actual) concentration


and standard deviation



Mass median aerodynamic diameter


(MMAD)



Geometric standard deviation


(GSD)



Relation of actual to nominal concentration



[mg/L air]



[mg/L air]



[µm]



 



 



49.88#



5.02 ± 0.02



9.205



4.913



0.10


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an acute inhalation toxicity study in male/female Crl: CD(SD) rats, the LC50 of Vinyltoluene was > 5.02 mg/L air.
Executive summary:

In an acute inhalation toxicity study (OECD 403/GLP), a group of young adult Crl: CD(SD) rats (3/sex) were exposed to a test atmosphere of Vinyltoluene (99.66%; 3-Vinyltoluene CAS No. 100-80-1: 64.3 %; 4-Vinyltoluene CAS No. 622-97-9: 35.7 %) in air (aerosol) for 4 hours (nose only) at the target concentration of 5 mg/L air (limit test). Animals were then observed for 14 days.


 


LC50 male/female  > 5.02 mg/L air. 


 


The determination of the aerosol particle distribution did not reveal the range for MMAD (1 - 4 µm) and GSD (1.5 - 3.0) as recommended in the guideline. The feasibility tests revealed that the MMAD at a concentration of 2 mg/L air (9.768 µm) was even slightly worse compared to a concentration at 5 mg/L air (9.124 µm).The cumulative mass of particles less than 4 µm was approximately 31% at a concentration at 5 mg/L air and only 11% at a concentration at 2 mg/L air. Hence, the use of 5 mg/L air is from a toxicological point of view more suitable than employing 2 mg/L air, as at a concentration of 5 mg/L air the absolute concentration of smaller particles reaching the alveoli is considered to be larger than at 2 mg/L air. The gravimetric (actual) concentration in the limit test was 5.02 ± 0.02 mg/L air. The mean MMAD and GSD were 9.205 μm and 4.913, respectively.


 


The test item led to mortality/morbidity in only 1 of the 6 animals. One female rat revealed a pronounced sedation until 60 minutes p.a. and was sacrificed prematurely 2 hours after end of exposure due to animal welfare reasons. Systemic toxicity was noted in in form of reduced motility, ataxia, tremor, a reduced respiratory rate, tonic and clonic convulsions, and/or lacrimation immediately after end of exposure up to 3 hours after end of exposure in all 3 of 3 male and up to 3 of 3 female animals. Pilo-erection was noted up to 48 hours after end of exposure. The body weight did not reveal test item-related changes in any of the rats at the end of the study.The macroscopic examination at necropsy did not reveal any changes. Hence, no histopathological examination was performed. No influence on the lung weights was observed.

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