Fructofuranosidase, β-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March - 13 April 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

For deteails, please refer to guideline deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted: May 1981

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted: September 2009

Deviations:

yes

Remarks:

mass median aerodynamic diameters (MMAD) is not ranging from 1 to 4 μm, due to test substance properties

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Test material

Test material information

1

Reference substance name:

Xylanase, endo-1,4-

EC Number:

232-800-2

EC Name:

Xylanase, endo-1,4-

Cas Number:

9025-57-4

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

endo-1,4-beta-xylanase

Test animals

Species:

rat

Strain:

other: CD strain (remote Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK), Margate, England
- Age at study initiation: Arrival: 5 - 6 weeks (males), 8 -9 weeks (females), Main study: approximately 7 weeks (males), approximately 10 weeks (females)
- Weight at study initiation: Arrival: 144 - 161 g (males), 175 - 184 g (females), Main study: 213 - 248 g (males), 210 - 236 g (females)
- Housing: 5 animals per sex in Type TRI 8 (Modular Systems and Developments Co. Ltd. Hereford, England), which were made of a stainless steel body measuring 51 x 36 x 21 cm with a stainless steel mesh lid and floor.
- Diet: complete pelleted rodent diet (RM 1 (E) SQC,Special Diets Services Limited, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks:

oil-free

Mass median aerodynamic diameter (MMAD):

8 µm

Geometric standard deviation (GSD):

2.56

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chamber consisted of 30 cm diameter aluminium alloy cylinder. The cylinder incorporated three animal exposure sections each having 20 exposure ports (ADG Instruments Ltd., Codicote, Hitchin, Hertfordshire,
England). The exposure chamber and generation apparatus were positioned in a large cabinet equipped with an extract fan exhausting to atmosphere through a collection filter.
- Exposure chamber volume: 60 L
- Method of holding animals in test chamber: rodent polycarbonate restraining tube
- Source and rate of air: 16,5 L/min (dry oil free compressed air with test substance), 2.0 L/min filtered air (additional air from the environment)
- Method of conditioning air: wright dust feed mechanism
- System of generating particulates/aerosols: the test substance as supplied by the Sponsor was not suitable for atmosphere generation. Therefore the sample used in this study was milled before use. Milled test substancec was analyzed by the sponsor to demonstrate the integrity.
- Method of particle size determination: Sierra-Marple Cascade Impactor
- Treatment of exhaust air: vacuum pump used for exhaustion of air 18.5 L/min
- Temperature, humidity, pressure in air chamber: control group: 21.2 ± 0.4 °C, 51.2 ± 1.7%; test group: 21.3 ± 0.4°C, 52.8 ± 0.4%

TEST ATMOSPHERE
- Brief description of analytical method used: chamber concentration was determined by the gravimetric analysis of five samples of chamber air taken during the exposure. The nominal chamber concentration was calculated as the mass of test material that entered the inhalation exposure chamber divided by the total volume of air delivered to the chamber.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: particle size distribution of the chamber atmosphere was measured on 4 occasions during the exposure period. The samples were analysed gravimetrically by drawing a continuous atmosphere sample at 2 L/min through a cascade impactor (Sierra-Marple Model 296., Sierra Instruments, Incorporated, Carmel Valley, California, USA) which was located in a spare animal exposure port (Please refer to table 2 in the 'Any other infromation on results incl. tables section)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 8.00 ± 2.56 µm (total aerosol concentration was determined gravimetrically on 5 occasions during the exposure) (Please refer to table 1 in the 'Any other infromation on results incl. tables section)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5.13 mg/L

No. of animals per sex per dose:

5 control
5 treatment

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: clinical observations and inspections for morbidity / mortality were performed 15 min and 30 min after the start of the exposure and at 30 min intervalls therafter until end of the exposure period. During observation period animals were examined in 30 min intervals during the first 2 h and subsequently twice daily until completion of the observation period. Bodyweights were determined daily from the day of delivery until the end of the observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights (lung with bronchi and trachea, liver, kidneys), Other: tissues preserved in fixative: larynx, lungs with bronchi and trachea, liver, kidneys

Statistics:

Group mean values were calculated from individual values.
Standard deviations were calculated where appropriate.

Results and discussion

Preliminary study:

A range-finding test was performed using one male and one female rat. The animals were exposed to an atmosphere generated from the test material at an concentration of 3.92 mg/mL. The results of this exposure, which were not reported in detail, indicated that exposure of the main study group should target the test guideline limit chamber concentration of 5 mg/L.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.13 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths observed.

Clinical signs:

other: Clinical signs during exposure: reduced repiratory rate was observed in all animals from 30 min onwards. Exaggerated respiratory movements were observed from 150 min onwards in 3 males and 3 females, respectively.

Body weight:

Reduced body weight or reduced rate of weight gain compared to controls, was observed in male animals on the day following exposure. Afterwards the weight gain was unaffected. The body weight gain was unaffected in females (Please refer to table 4 in the 'Any other infromation on results incl. tables section')

Gross pathology:

There were no macroscopic findings in any animal.

Other findings:

- Lung, liver and kidney weights were unaffected by test substance exposure.

- A relatively small percentage of test material that was of inhalable aerodynamic particle size, which was attributable to the physical characteristics of the milled test material.
- The total atmosphere generated did not meet the US Environmental Protection Agency, Health Effects Division (Interim Policy for Particle Size Distribution and Limit Concentration Issues; dated February 1994), requirement that the M.M.E.A.D. should be less than 4 μm. However, the exposure concentration employed to meet other regulatory guidelines was 2.57 x the limit concentration required by the EPA. Based on the slope of the graph of the particle size data (Probability; logarithm plot) 23% by weight ( equivalent to 1.18 mg/L) of the particles generated were less than 4 μm in aerodynamic diameter.

Any other information on results incl. tables

Table 1: Aerosol characterization: chamber concentration

Group	Mean achieved chamber concentration ± standard deviation (mg/L)	Nominal chamber concentration (mg/L)	Generation efficiency (%)
test substance	5.13 ± 0.07	14.87	34.5
Group	Sample number	Sample time (minutes)	Chamber concentration (mg/L)
test substance	2.1	15	5.03
	2.2	52	5.11
	2.3	115	5.10
	2.4	170	5.21
	2.5	231	5.19

 

Table 2: Aerosol characterization: particle size determination by Sierra-Marple 296

Group	Sample number	Sample time (minutes)	Percentage on stage (cumulative)
			Filter < 0.52µm E.A.D	6 < 0.93µm E.A.D	5< 1.55µm E.A.D	4 < 3.5 µm E.A.D	3 < 6.0µm E.A.D	2 < 9.8µm E.A.D	Mass median E.A.D. (µm) (geometric SD)
test substance	2.1	15	1.6	2.1	4.1	16.5	37.7	60.5
	2.2	85	1.4	2.0	4.3	17.5	36.1	59.0
	2.3	150	1.8	2.6	4.7	16.4	33.6	55.8
	2.4	205	2.1	2.8	5.0	16.5	33.6	54.8
		Mean	1.7	2.4	4.5	16.7	35.3	57.5	8
		Standard deviation	0.3	0.4	0.4	0.5	2.0	2.7	2.56

E.A.D.Equivalent Aerodynamic Diameter.

Table 3: Clinical signs

Exposure period
Group	sex	Observation	Time during exposure (min)
			0	15	30	60	90	120	150	180	210	240
control	male	wet fur	0	0	0	0	0	0	0	0	2	2
test substance	male	reduced respiratory rate	0	0	2	3	4	4	4	4	5	5
		exaggerated respiratory movements	0	0	0	0	0	0	1	2	3	3
		wet fur	0	0	0	0	0	1	2	2	3	3
control	female	wet fur	0	0	0	0	0	0	1	2	2	3
test substance	female	reduced respiratory rate	0	0	3	3	5	5	3	4	5	5
		exaggerated respiratory movements	0	0	0	0	0	1	1	2	2	3
		wet fur	0	0	0	0	0	1	1	1	2	2
Observation period
Group	sex	Observation	Day of observation
			1 (0-2 h after exposure)	1 (18 h after exposure)	2-14
test substance	male	wet fur	1	0	0
control	female	wet fur	2	0	0
test substance	male	wet fur	2	0	0

 

Table 4: Body weight - group mean values (g)

Day of observation	control (mean ± standard deviation)	test substance (mean ± standard deviation)	control (mean ± standard deviation)	test substance (mean ± standard deviation)
	male	male	female	female
-5	181 ± 4	184 ± 10	208 ± 3	209 ± 6
-4	188 ± 4	193 ± 11	212 ± 7	209 ± 7
-3	196 ± 2	202 ± 12	215 ± 10	215 ± 7
-2	204 ± 5	212 ± 11	218 ± 7	216 ± 6
-1	215 ± 5	220 ± 14	221 ± 5	222 ± 6
0	222 ± 5	231 ± 14	225 ± 7	221 ± 7
1	227 ± 5	230 ± 15	224 ± 10	223 ± 7
2	232 ± 4	240 ± 14	229 ± 7	229 ± 7
3	240 ± 5	248 ± 17	228 ± 5	229 ± 8
4	249 ± 5	258 ± 17	233 ± 6	230 ± 8
5	254 ± 5	262 ± 17	232 ± 10	234 ± 9
6	258 ± 6	272 ± 16	236 ± 9	240 ± 8
7	268 ± 8	277 ± 15	236 ± 10	238 ± 9
8	275 ± 8	288 ± 19	240 ± 10	242 ± 6
9	282 ± 9	292 ± 17	239 ± 13	244 ± 8
10	289 ± 8	300 ± 19	243 ± 11	249 ± 9
11	292 ± 8	304 ± 19	244 ± 11	248 ± 8
12	300 ± 12	308 ± 19	248 ± 12	247 ± 6
13	305 ± 9	317 ± 22	247 ± 14	251 ± 9
14	312 ± 11	321 ± 23	248 ± 10	254 ± 10

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

In this acute inhalation toxicity study in rats a LC50 value of > 5.13 mg/L air was determined (4h).