Ceratonia siliqua, ext.

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2017 - 15 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 2009

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Physical appearance: Dark brown to black liquid
- Storage of test material: At room temperature

Specific details on test material used for the study:

pH: 5.0

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: males: 293 to 341 g; females: 190 to 213 g
- Fasting period before study: no
- Housing:
* on arrival and following assignment to the study: group housing (up to 5 animals of the same sex and exposure group/cage) in polycarbonate cages containing sterilized sawdust as bedding material
* during the study: individually housed and moved to an inhalation area in order to perform the exposure. Upon return to the cages, the bedding material was covered with a sheet to prevent suffocation.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: municipal tap-water was freely available to each animal via water bottle, except during exposure when there was no access to water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24 (actual: 19-20)
- Humidity (%): 40-70 (actual: 49-55)
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01 December 2017 - 15 December 2017

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Water (elix)

Mass median aerodynamic diameter (MMAD):

> 1 - < 4 µm

Geometric standard deviation (GSD):

> 1.5 - < 3

Remark on MMAD/GSD:

The indicated ranges are target values for MMAD and GSD.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the design of the exposure chamber is based on the directed flow nose only inhalation chamber
- Exposure chamber volume: not indicated
- Method of holding animals in test chamber: polycarbonate restraining tubes connected to the exposure chamber
- Source and rate of air: at least 1 L/min
- Method of conditioning air: not indicated
- System of generating particulates/aerosols: an aerosol was generated by nebulization of the test item (diluted 1:1 with water) with pressurized air. For this, the test item was transferred to a Collison nebulizer by means of a rotating pump (type VL500 digit, VERDER Lab Tec GmbH & Co. KG, Haan, Germany). The primary aerosol was diluted with pressurized air before it entered the exposure chamber. The mean total airflow was 10 L/min.
- Method of particle size determination: The particle size distribution was characterized twice during each exposure period. The samples were drawn with a flow of 2 L/min. from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor. Amounts of test item collected were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined based on OECD guidance document No 39.
Graphs of the cumulative mass of test item collected (percentage of total collected) against the cut points of the impactor stages were drawn on log-normal paper. When drawing the graphs more weight was given to the cut points where the cumulative mass sampled was within the range of 5 to 95%. The Mass Median Aerodynamic Diameter (MMAD), i.e. the particle size where 50% of the particle mass was borne by particles smaller than the MMAD and the σ84%, (the particle size where 84% of the particle mass was borne by particles smaller than the σ84% was read from the graph. The geometric standard deviation (gsd) was calculated as σ84% / MMAD.
- Treatment of exhaust air: not indicated
- Temperature and humidity in air chamber: measured at 30 minute intervals after initiation of exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The nominal concentration was calculated by dividing the amount of test item used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volume of air was calculated from the average air flow (which was measured by means of thermal mass flow meters and recorded regularly, preferably in 30 minute intervals) and the exposure time.
- Samples taken from breathing zone: yes

VEHICLE: the test item was diluted in Elix water (1:1) with a specific gravity of 1.0.

CLASS METHOD
- Rationale for the selection of the starting concentration: the target concentration (5 mg/L) were based on the cut off concentration values specified in the UN and EC classification guidelines.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

In a stepwise manner: three animals of each sex were exposed to a test item target concentration of 5 mg/L. Based on the results, no more animals were exposed.

No. of animals per sex per dose:

Three male and three female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
* body weights: on day 1 (pre exposure), 2, 4, 8 and 15.
* mortality: twice daily
* clinical observations during exposure: at least three times
* clinical observations post exposure: at periodic intervals on the day of exposure (at least two times) and once daily thereafter.
- Other examinations performed: macroscopic observations after necropsy

Statistics:

No statistical analysis performed.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 other: mg/L (nominal)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred.

Clinical signs:

other: During exposure, quick breathing was seen in all animals and shallow respiration was noted for one animal at the first of the three observations (not presented in the table). After exposure, no clinical signs were noted.

Body weight:

Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Other findings:

TEST ATMOSPHERE: see table 1
MMAD were

Any other information on results incl. tables

Table 1 Details on exposure concentrations

Dose (mg/L)	TWA actual exposure concentration (mg/L)	Nominal concentration (mg/L)	Generation efficiency (%)	MMAD (μm)
				1st measurement (gsd)	2nd measurement + gsd
5	5.2 ± 0.09	22.3	47*	3.3 (2.1)	3.5 (2.0)

* the ratio of actual and nominal concentration was 23.5% (generation efficiency) but as the test item was diluted in a 1:1 ratio, the generation efficiency of the test item was estimated at 47%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008.

Conclusions:

The inhalation 4h-LC50 of Carob Bean Extract in Wistar rats was established to exceed 5 mg/L. Based on this result, the substance is not classified for acute inhalation toxicity according to GHS and Regulation (EC) No. 1272/2008.