Fructofuranosidase, β-

Endpoint summary

Description of key information

Acute oral toxicity study (OECD 401), male and female rat: LD50 > 2000 mg/kg bw

Read-across from the source substance: endoxylanase (CAS 9025 -57 -4)

Acute inhalation toxicity study (OECD 403), male and female rat: LC50 > 5.13 mg/L air

Read-across from the source substance: endoxylanase (CAS 9025 -57 -4)

Acute toxicity via dermal route is waived based on exposure considerations and the known properties of the substance.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths observed during the study.

Clinical signs:

There were no clinical signs observed.

Body weight:

The animals showed expected gains in body weight over the observation period.

Gross pathology:

No abnormalities were noted at the macroscopic examination at study termination.

Interpretation of results:

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

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their oral toxicity potential. Based on the results of the available acute oral toxicity study conducted with the source substance endoxylanase (CAS 9025-57-4) the oral LD50 was >2000 mg/kg bw in male and female rats. Thus the target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to be hazardous following acute exposure via the oral route.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March - 6 April 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 24 February 1987

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

adopted 17 December 2001

Deviations:

yes

Remarks:

animal number and schedule of administration: 5 animals per sex were used (Limit test) and a preliminary test was conducted using 2 females administered doses of 400 or 800 mg/kg bw/day

GLP compliance:

yes

Limit test:

yes

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: 5 weeks
- Weight at study initiation: 109 - 136 g (males), 99 - 117 g (females)
- Fasting period before study: overnight
- Housing: 5 animals per sex in stainless steel grid cages (RS Biotech, Northants, England)
- Diet: complete pelleted rodent diet (RM 1 (E) SQC,Special Diets Services Limited, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purifed (obtained by the reverse osmosis of water)

Details on oral exposure:

VEHICLE
- purified water (obtained by the reverse osmosis of water)
- Concentration in vehicle: the test material was prepared at the appropriate concentration in purified water

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bw

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

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 two times within the first hours after test substance application and two forther ones at Day 1. From Day 2 onwards animals were inspected twice daily and bodyweights were determined on the the day before dosing and on Day 1, 8, and 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, other: larger organs were sectioned and gastrointestinal tract was opened at intervals for examination of the mucosal surfaces

Preliminary study:

A preliminary study was carried out using two female rats given an oral administration of [test substance] at a dosages of 400 or 800 mg/kg bw, at a constant volume-dosage of 20 mL/kg bw in purified water. In this study no deaths occurred.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths observed during the study.

Clinical signs:

There were no clinical signs observed.

Body weight:

The animals showed expected gains in body weight over the observation period.

Gross pathology:

No abnormalities were noted at the macroscopic examination at study termination.

Interpretation of results:

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

Conclusions:

In this acute oral toxicity study in rats a LD50 value of > 2000 mg/kg bw was determined. CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Mass median aerodynamic diameter (MMAD):

µm

Duration of exposure:

h

Preliminary study:

A range-finding test was performed using a group of one male and one female rat exposed to an atmosphere generated from the test material at the oncentration of 3.92 mg/mL. The results of this exposure, which are not reported in detail, indicated that exposure of the main study group should target the test guideline limit chamber concentration (5 mg/L) of GBR 50015.

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 minutes onwards. Exaggerated respiratory movements were observed from 150 minutes onwards in 3 males and 3 females, respectively.and was seen for three males and t

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.

Interpretation of results:

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

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their inhalation toxicity potential. Based on the results of the available acute inhalation toxicity study conducted with the source substance endoxylanase (CAS 9025-57-4) the inhalation LC50 was > 5.13 mg/L air was determined (4h) in male and female rats. The target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to be hazardous following acute exposure via the inhalation route.

Reference 2

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted: May 1981

Deviations:

no

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

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

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.

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.

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.

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across:

There are no experimental data available regarding the acute oral and inhalation toxicity of β-fructofuranosidase (CAS 9001-57-4). It must be noted however that β-fructofuranosidase is produced by a classical strain of Saccharomyces cerevisiae, which has a long history of safe use in food applications globally. Read-across from an appropriate analogue substance endoxylanase (CAS 9025-57-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.1.Common functional groups and structural similarities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Acute oral toxicity

An acute oral toxicity study with the analogue substance endoxylanase (CAS 9025-57-4) was performed according to OECD guideline 401 and in compliance with GLP (Rees, 1995).

The acute oral toxicity of the test substance was assessed in 5 male and 5 female CD rats. Rats were administered the test substance dissolved in water orally by gavage at a dose of 2000 mg/kg bw. Selection of this dose level was based on a preliminary study in which two female rats were dosed at 400 and 800 mg/kg bw, respectively and did not show any signs of toxicity or deaths.

Also, in the main study, no mortality or clinical signs of systemic toxicity occurred. The animals showed expected gains in body weight and no abnormalities were noted at the macroscopic examination at study termination. Therefore a LD50 >2000 mg/kg bw was determined for male and female rats.

 

The target and source substances are considered unlikely to differ in their acute oral toxicity potential. Therefore, an acute oral LD50 value of > 2000 mg/kg bw was considered for the hazard assessment and classification and labelling purposes for the target substance β-fructofuranosidase (CAS 9001-57-4).

 

Acute inhalation toxicity

An acute inhalation toxicity study with the analogue substance endoxylanase (CAS 9025-57-4) was performed according to OECD guideline 403 and in compliance with GLP (Jackson, 1995).

The acute inhalation toxicity of the test substance, was assessed in 5 male and 5 female CD rats by a 4 h snout-only exposure to an atmosphere targeted to contain a chamber concentration of 5 mg/L air. Selection of this dose level was based on a preliminary dose range-finding study in which one male and one female rat were exposed to an atmosphere generated from the test material at a concentration of 3.92 mg/mL. In this study, no deaths occurred. A control group was exposed to compressed air only. All animals were observed for the occurrence of mortality, clinical signs of toxicity and effects on body weights within a period of 14 days. At necropsy, organ weights were determined and macroscopic analyses were performed.

The chamber atmosphere was characterized as followed: 5.13 ± 0.07 mg/L was the mean achieved exposure level, 35.3 ± 2.0 % of the atmosphere had an equivalent aerodynamic diameter (E.A.D.) < 6 µm and the mass median E.A.D ± geometric standard deviation was 8.00 ± 2.56 µm.

During the observation period, no deaths were observed. Clinical signs were only observed during the exposure period, but not thereafter. During exposure, the respiratory rate was reduced in all animals and respiratory movements were exaggerated in 3/5 males and females, respectively. Exaggerated movements were considered a non-specific response to the inhalation of a high concentration of particulate material. In addition, body weight loss or reduced body weight gain was observed in male animals on the day after exposure. In females, body weight gains were not affected. Macroscopic analyses did not reveal any effects and organ weights (lung, liver and kidneys) were not altered. Based on these data, a LC50 of > 5.13 mg/L for the test substance in air was determined.

 

The target and source substances are considered unlikely to differ in their acute inhalation toxicity potential. Therefore, an LC50 value of > 5.13 mg/L air was considered for the hazard assessment and classification and labelling purposes for the target substance β-fructofuranosidase (CAS 9001-57-4).

Justification for classification or non-classification

No mortality or clinical signs of toxicity were observed in the acute oral toxicity study with the read across substance endoxylanase (CAS 9025-57-4) up to the limit dose. Also, in the inhalation toxicity study, no deaths were observed, clinical signs were limited to the exposure period and partially not considered test substance-related. Effects on body weights were limited to males and the first day after exposure. Thus, the available read across data on acute oral and inhalation toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification of the target substance.