Toxicological Evaluation and Food Safety Assessment of

Xylooligosaccharides Derived from Rice Straw Xylan Hydrolysis

using Recombinant Endo-β-1,4-D-Xylanase:

An Acute Oral Toxicity Study

Article

Putra Vergian

1,a

, Anak Agung Istri Ratnadewi

1,b

*

, Cahyaningtyas Tetta Riandy

1,c

, Riesma

Rukmana

1,d

, Evi Umayah Ulfa

2,e

, Tri Agus Siswoyo

3,f

, Mei Syafriadi

4,g

1

Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Jember, Jember 68121,

Indonesia

2

Faculty of Pharmacy, University of Jember, Jember 68121, Indonesia

3

Graduate School of Biotechnology and PUI-BioTIn, University of Jember, Jember 68121, Indonesia

4

Faculty of Dentistry, University of Jember, Jember 68121, Indonesia

A

bstract

Xylooligosaccharides

(XOS)

are

recognized

as

prebiotics

that

stimulate

the

proliferation

of

probiotic

bacteria and enhance short-chain fatty acid (SCFA) production. Due to their physicochemical stability,

XOS have potential applications in functional foods; however, safety evaluation is essential, particularly

when produced from lignocellulosic biomass without purification. This study evaluated the acute oral

toxicity

of

a

crude

hydrolysate

containing

XOS

obtained

from

rice

straw

xylan

hydrolyzed

using

recombinant

endo-β-1,4-D-xylanase

expressed

in

Saccharomyces

cerevisiae.

The

product

was

administered without further purification. Male Wistar rats received a single oral dose of 2000 mg/kg

body

weight

and

were

observed

for

14

days

following

OECD

Guideline

420

(Fixed

Dose

Method).

Parameters

toxicity

evaluated

included

body

weight,

clinical

signs,

hematological

and

biochemical

indices,

as

well

as

macroscopic

and

histopathological

alterations.

Signs

of

toxicity

were

observed

as

changes in stool consistency within the first 4 to 24 hours. The estimated oral LD

₅₀

was 2000 mg/kg body

weight, with treatment-related mortality observed in 3 of 5 mice at this dose level. Based on these results,

the test substance was classified into Category 4 according to the OECD Globally Harmonized System

(GHS). Histopathological examination revealed mild to moderate degenerative changes in the liver and

kidney tissues, while serum biochemical parameters remained within normal physiological ranges. These

findings indicate moderate acute toxicity of the unpurified XOS-containing hydrolysate at high doses.

Therefore,

the

observed

effects

cannot

be

attributed

solely

to

XOS

but

may

also

arise

from

other

hydrolysis by-products. Further purification and sub-chronic toxicity studies are required to confirm the

intrinsic safety of XOS for food applications.

Keywords:

Hematological, histological, recombinant xylanase, toxicity, wistar rats

*

Corresponding author

Email addresses:

istri_dewi.fmipa@unej.ac.id

(Anak Agung Istri Ratnadewi)

DOI:

https://doi.org/10.22437/chp.v10i1.49759

Received

November 13

rd

2026;

Accepted

April 07

th

2026;

Available online

May 19

st

2026

(

https://creativecommons.org/licenses/by/4.0

)

78

Graphical Abstract

Introduction

I

n

agricultural

biotechnology,

genetically

engineered products have raised significant

social

impacts

and

ethical

concerns.

Public

awareness regarding the potential effects of

biotechnological products on human health

has

increased,

highlighting

the

importance

of

consumer

protection.

Food

safety

analyses

must

be

grounded

in

scientific

evidence, and products should be rigorously

evaluated

for

their

suitability

for

human

consumption. According to the guidelines of

the

World

Health

Organization

(WHO)

and

the Food and Agriculture Organization (FAO),

all

biotechnology-derived

foods

must

undergo

assessments

for

allergenicity

and

toxicity. In Indonesia, Regulation Number 27

of 2018 issued by the Badan Pengawas Obat

dan Makanan (BPOM) outlines the standards

for

the

safety

evaluation

of

food

raw

materials.

The

introduction

of

new

technologies,

including

genetically

engineered

products,

may

influence

the

microbiological, toxicological, allergenic, and

nutritional

properties

of

raw

materials.

Consequently,

comprehensive

food

safety

assessments

are

essential

to

ensure

that

such

products

meet

regulatory

standards

and are safe for public consumption

[1,2]

Xylooligosaccharides

(XOS)

are

sugar

oligomers

composed

of

xylose

units

linked

by

β-(1

→

4)

xylosidic

bonds,

with

the

molecular

formula

C

₅ₙ

H

₈ₙ₊₂

O

₄ₙ₊₁

and

a

degree of polymerization (DP) ≤ 20

[3,4,5]

. As

prebiotics,

XOS

are

indigestible

but

exert

beneficial

effects

on

host

health

by

stimulating

the

growth

and

activity

of

intestinal microbiota

[6]

. XOS naturally occur

in

fruits

and

vegetables;

however,

their

amounts are generally insufficient to provide

measurable

health

benefits.

Therefore,

to

meet

daily

requirements,

XOS

must

be

consumed

as

a

dietary

supplement.

In

healthy adults, the minimum intake of XOS

required

to

confer

health

and

prebiotic

benefits

ranges

from

1.4

to

5.0

g

per

day

[7,8]

.

XOS,

as

prebiotics

derived

from

enzymatic

hydrolysis, have been successfully obtained

from biomass residues or agro-industrial by-

products

[9,10]

.

The

prebiotic

potential

of

cassava

pulp–derived

XOS

has

been

demonstrated

both

in

vitro

and

in

vivo,

particularly

through

its

ability

to

stimulate

79

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

the

growth

of

probiotic

bacteria

and

enhance

short-chain

fatty

acid

(SCFA)

production

[11]

.

Compared

with

other

prebiotics,

XOS

exhibit

distinct

advantages,

including

greater

stability

at

elevated

temperatures

and

tolerance

across

a

relatively broad pH range. The XOS obtained

from

biomass

residues

in

this

study

are

classified

as

genetically

engineered

products,

as

they

are

produced

via

hydrolysis using recombinant endo-β-1,4-D-

xylanase

[4]

.

The

enzyme

was

originally

identified

from

microorganisms

inhabiting

the

termite

gut,

known

for

their

efficient

lignocellulose-

degrading capability. The xylanase-encoding

gene

was

subsequently

isolated

and

introduced

into

a

heterologous

expression

system to produce a recombinant enzyme in

a

new

host,

Saccharomyces

cerevisiae

[12]

.

The

selection

of

S.

cerevisiae

as

the

expression

host

was

based

on

its

long

history of safe use in food biotechnology and

its classification as Generally Recognized As

Safe

(GRAS)

by

the

U.S.

Food

and

Drug

Administration

(FDA).

This

strategy

enables

the production of xylanase in a food-grade

host system while maintaining the catalytic

properties

of

the

original

termite-derived

enzyme.

Nevertheless,

despite

the

GRAS

status of the host organism, the safety of the

final

XOS

product

generated

through

recombinant enzymatic processing must be

independently

demonstrated

through

toxicological evaluation.

Several previous studies have reported the

prebiotic

properties

and

general

safety

of

XOS derived from various biomass sources

[13]

.

However,

toxicological

data

remain

limited

for

XOS

specifically

produced

from

rice straw xylan using recombinant xylanase

systems. Differences in substrate origin and

production methodology may influence the

physicochemical

characteristics

and

biological

responses

of

the

resulting

oligosaccharides.

To

the

best

of

our

knowledge,

no

study

has

systematically

evaluated the acute oral toxicity of rice straw

derived

XOS

produced

via

recombinant

endo-β-1,4-D-xylanase

in

a

mammalian

model.

This

represents

an

important

scientific

and

regulatory

gap

in

the

development of functional food ingredients

derived from agricultural residues.

Therefore,

the

present

study

aimed

to

evaluate the acute oral toxicity of rice straw

derived XOS by determining the LD

₅₀

value

and

assessing

hematological

parameters,

clinical

biochemical

profiles,

macroscopic

pathology, and histopathological alterations.

The

findings

of

this

study

are

expected

to

provide

novel

toxicological

evidence

supporting

the

safe

application

of

recombinant

enzyme

produced

XOS

as

a

functional food ingredient

[14,15,16]

.

Materials and Methods

Materials

The

materials

used

in

this

study

included

phosphate–citrate buffer (pH 7), chloroform

for

animal

anesthesia,

and

10%

neutral

buffered

formalin

(NBF).

Xylooligosaccharides

(XOS)

were

prepared

through

enzymatic

hydrolysis

of

xylan

derived

from

rice

straw using

recombinant

endo-β-1,4-D-xylanase

expressed

in

a

Saccharomyces cerevisiae

host. Albino Wistar

rats

of

either

sex,

aged

6–8

weeks

and

weighing

200–250

g,

were

obtained

from

Wistar

Farm,

Malang,

East

Java,

Indonesia.

The animals were housed in standard cages

and maintained under controlled laboratory

conditions.

Experimental

protocols

were

conducted in accordance with BPOM (2022)

guidelines. The experimental use of animals

was

approved

by

the

Ethics

Committee

of

the Faculty of Dentistry, Universitas Jember

(Approval No. 2882/UN25.8/KEPK/DL/2024).

Prior to experimentation, the animals were

80

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

acclimatized for one week and provided with

a standard pellet diet and water ad libitum.

Crude XOS productions

Xylan

extracted

from

rice

straw

was

dissolved in phosphate–citrate buffer (pH 7)

at

a

concentration

of

0.8%

(w/v).

The

substrate was hydrolyzed with endo-β-1,4-D-

xylanase at a ratio of 1:1 in a water bath at

40

°C

for

24

h

[11].

The

hydrolysate

containing xylooligosaccharides (crude XOS)

was centrifuged at 8,000 rpm for 15 min, and

the supernatant was concentrated by freeze-

drying.

Acute toxicity

Healthy

male

Wistar

rats

were

used

in

accordance

with

the

Regulation

of

the

Indonesian

National

Agency

of

Drug

and

Food

Control

(BPOM)

No.

10

of

2022

concerning

acute

oral

toxicity

testing.

All

animals

were

fasted

overnight

with

free

access

to

water

and

weighed

prior

to

XOS

administration. The acute oral toxicity study

was

conducted

using

the

fixed-dose

procedure.

A

total

of

11

experimental

animals were used in this acute oral toxicity

study.

Two

animals

were

assigned

to

the

sighting

study

(one

at

300

mg/kg

body

weight and one at 2000 mg/kg body weight),

while

the

remaining

nine

animals

were

allocated

to

the

main

study.

In

the

main

study,

the

animals

were

randomly

divided

into

two

groups:

a

control

group

(n

=

5),

which

received

distilled

water,

and

a

treatment group (n = 4), which received the

limit dose of XOS. Animals were observed for

mortality,

acute

toxicity

signs,

and

behavioral

changes

(e.g.,

aggression,

abnormal

vocalization,

agitation,

sedation,

somnolence,

convulsions,

tremors,

ataxia,

catatonia,

paralysis,

fasciculation,

prostration,

unusual

locomotion,

and

asphyxia) during the first 30 min and the first

hour, followed by hourly observations for 5

h,

and

then

periodically

up

to

48

h.

Daily

observations were conducted for 14 days to

monitor

general

behavior,

body

weight

changes, adverse symptoms, and mortality.

According

to

the

guideline,

the

LD

50

is

greater

than

5

g/kg

if

three

or

more

rats

survive.

At

the

end

of

the

experimental

period, all animals were weighed, sacrificed

by

cervical

dislocation,

and

subjected

to

necropsy for organ collection.

Hematological and biochemial analyzes.

At the

end

of

the

experimental

period,

blood

samples

were

collected

from

the

surviving

animals

via

the

abdominal

aorta

under

chloroform

anesthesia.

Hematology

samples

were

placed

into

EDTA-treated

tubes and analyzed using a CA-500 counter

(Drew

Scientific

Hemavet

950,

USA).

Parameters

measured

included

red

blood

cell count (RBC), hemoglobin concentration

(HGB), hematocrit (HCT), mean

corpuscular

volume

(MCV),

mean

corpuscular

hemoglobin

(MCH),

mean

corpuscular

hemoglobin

concentration

(MCHC),

platelet

count

(PLT),

and

white

blood

cell

count

(WBC). For clinical chemistry, blood samples

were collected into anticoagulant-free tubes,

allowed

to

clot

at

room

temperature,

and

centrifuged

at

3000

rpm

for

15

min

to

separate

the

serum.

Serum

biochemical

analyses

included

total

cholesterol

(T.Cho),

serum

glutamate

oxaloacetate

transaminase (SGOT), and serum glutamate

pyruvate transaminase (SGPT).

Macroscopic and Histological examination

Five

major

organs—kidneys,

spleen,

liver,

lungs,

and

heart—were

carefully

excised

using

sterile

surgical

instruments.

Each

organ

was

subjected

to

macroscopic

examination,

including

assessment

of

size,

color,

texture,

and

the

presence

of

visible

lesions

or

abnormalities.

Following

macroscopic

evaluation,

the

organs

were

placed into containers filled with 10% neutral

81

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

buffered

formalin

(NBF)

for

fixation.

Histological

preparation

and

microscopic

examination of the organs were conducted

at

the

Faculty

of

Pharmacy,

University

of

Jember.

Statistical analyses

All values are expressed as mean ± SD (n =

5). Statistical analyses were performed using

Jamovi

statistical

software

(version

2.3.28).

One-way

analysis

of

variance

(ANOVA)

was

applied, followed by Tukey’s post hoc test for

multiple

comparisons.

A

value

of

p

<

0.05

was considered statistically significant[17].

3. Result and Discussion

3.1. LD

50

Value and Weight Change

The results of the sighting study showed that

Wistar rats administered crude XOS at doses

of

300

and

2000

mg/kg

body

weight

(BW)

exhibited

no

mortality

or

moribund

conditions;

however,

mild

toxic

signs

were

observed. Within the first 4 hours following

oral

administration

of

crude

XOS,

rats

in

both

dose

groups

displayed

signs

of

weakness and changes in stool consistency.

In

the

300

mg/kg

BW

group,

stool

consistency

returned

to

normal

within

24

hours. Based on the findings of the sighting

study, a dose of 2000 mg/kg BW was selected

for the main study. The results of the main

study

demonstrated

changes

in

stool

consistency in all rats within the first 4 hours

after administration, followed by recovery to

normal

conditions.

During

the

14-day

observation period, three Wistar rats in the

main

study

were

found

dead.

Accordingly,

the

oral

LD

₅₀

value

of

crude

XOS

was

estimated to be <2000 mg/kg BW. According

to Regulation No. 10 of 2022 issued by the

Badan

Pengawas

Obat

dan

Makanan

(BPOM),

substances

with

an

LD

₅₀

in

the

range of >300–2000 mg/kg BW are classified

as moderately toxic. It should be noted that

administration of XOS at an extremely high

dose may induce marked osmotic effects in

the gastrointestinal tract. As a non-digestible

oligosaccharide,

unabsorbed

XOS

can

substantially

increase

luminal

osmotic

pressure,

leading

to

excessive

water

retention, accelerated intestinal transit, and

severe osmotic diarrhea. At such high dose

levels,

these

effects

may

cause

significant

gastrointestinal imbalance and dehydration,

indicating

that

XOS

cannot

be

considered

safe when administered at excessively high

doses.

Rapid

fermentation

of

large

quantities

of

XOS

by

intestinal

microbiota

may further generate short-chain fatty acids

and

gases,

potentially

resulting

in

gastrointestinal

stress

and

systemic

physiological

burden.

This

result

contrasts

with the findings reported by Boonchuay et

al.,

(2021)

who

demonstrated

that

purified

corncob-derived XOS exhibited an LD

₅₀

value

greater

than

5,000

mg/kg

body

weight,

suggesting

a

practically

non-toxic

classification

[3]

.

The

primary

distinction

between the two studies lies in the degree of

product

refinement.

While

the

evaluated

XOS

that

had

undergone

purification

processes

to

remove

residual

impurities, the present study utilized crude

XOS without purification.

Wistar rats that survived the main test after

14

days

showed

no

other

toxic

symptoms.

Changes in fecal consistency ceased on the

second day of observation. Body weight gain

occurred in line with the age increase of the

test animals. Data on changes in body weight

indicated that crude XOS at a dose of 2000

mg/kg BW had a considerable effect on the

growth

of

Wistar

rats.

The

graph

of

body

weight

changes

in

Wistar

rats

in

the

main

test can be seen in Figure 1.

82

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

Figure 1.

Body weight chart of male Wistar rats during the main test (administration of crude

XOS at a dose of 2000 mg/kg BW resulted in an increase in the body weight gain of test rats)

Clinical biochemistry tests

Clinical

biochemistry

tests

are

a

series

of

laboratory

tests

used

to

evaluate

the

chemical components in biological samples

such as blood, urine, or serum. These tests

provide

information

about

organ

function,

enzyme

levels,

nutrient

concentrations,

electrolyte

balance,

and

metabolic

conditions in the body [18]. Blood sampling

was

carried

out

on

the

last

day

of

the

observation period. Blood samples obtained

from the heart, or through cardiac puncture,

are

intended

to

collect

a

large

quantity

of

high-quality

blood

at

the

final

stage

of

the

study

[19]

.

Blood

serum

can

provide

information

regarding

organ

function.

The

Clinical

Biochemistry

examination

in

this

study

aimed

to

evaluate

the

risk

of

cardiovascular disease in the liver using liver

enzyme tests such as SGOT and SGPT, as well

as

total

cholesterol

measurement.

The

results

of

the

clinical

biochemistry

examinations

of

Wistar

rats

administered

the crude XOS are presented in Table 1.

The biochemical analysis showed that total

cholesterol levels in both the control group

(26.07

mg/dL)

and

the

crude

XOS-treated

group (32.71 mg/dL) were below the normal

reference range (37.0–85.0 mg/dL). Although

a slight increase was observed in the treated

group

compared

to

the

control,

the

values

remained below physiological norms and did

not

indicate

treatment-related

lipid

disturbances.

Alterations

in

serum

lipid

parameters

are

commonly

used

to

assess

systemic metabolic effects in toxicity studies.

Serum

SGOT

levels

in

both

groups

were

within

the

normal

reference

range,

with

comparable

values

between

the

control

(137.29

U/L)

and

treated

animals

(138.79

U/L),

suggesting

no

evidence

of

hepatocellular

injury.

In

toxicological

evaluations, increases in SGOT and SGPT are

considered

sensitive

indicators

of

liver

cell

damage.

Although

SGPT

levels

in

both

groups were above the stated normal range,

the

treated

group

exhibited

lower

SGPT

levels

(67.43

U/L)

compared

to

the

control

group

(92.47

U/L).

Since

hepatotoxicity

is

typically

characterized

by

a

treatment-

in

transaminases

relative

to

controls,

the

findings

do

not

indicate

hepatic

injury

attributable

to

crude

XOS.

Overall, these biochemical findings suggest

that crude XOS administered at 2000 mg/kg

body

weight

did

not

produce

significant

alterations

in

liver

function

markers.

83

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

According to internationally accepted acute

toxicity

testing

guidelines,

biochemical

parameters

should

be

interpreted

in

conjunction with clinical signs and mortality

data to determine treatment-related effects

[20].

Table 1.

Clinical biochemistry results of male Wistar rats

Parameters	Control (n=5)	Crude XOS 2000 mg/kgBW (n=2*)	Normal Range
Total cholesterol (mg/dL)	26.07 ± 0.05	32.71 ± 11.47	37.0 – 85.0
SGOT (U/L)	137.29 ± 0.21	138.79 ± 6.05	85.71 – 213.33
SGPT (U/L)	92.47 ± 0.33	67.43 ± 6.26	22.68 – 45.64

*

Values represent surviving animals in the 2000 mg/kg BW group

Hematological testing

Hematological

testing

in

toxicity

studies

is

crucial

because

it

provides

detailed

information

about

the

toxic

effects

of

a

substance

on

the

blood

system

and

body

metabolism.

Hematological

examination

involves

analyzing

leukocytes,

erythrocytes,

hemoglobin,

platelets,

and

hematocrit,

which

can

reveal

abnormalities

or

changes

that

occur

due

to

exposure

to

toxic

substances. The results of the hematological

analysis

are

presented

in

Table

2.

In

the

hematological parameters of male rats, the

leukocyte

and

MCV

values

in

test

rat

2

showed

a

slight

increase,

while

the

MCHC

value showed a slight decrease, all within the

BPOM reference range. Meanwhile, all other

hematological

parameters

showed

no

significant

differences

between

the

control

and

test

rats,

and

all

remained

within

the

BPOM reference range. The slight increases

and decreases in hematological parameters

of the male rats were still within the normal

range,

indicating

that

there

were

no

significant

disturbances

in

the

blood

cell

formation system [21].

Table 2.

Hematological parameters of male Wistar rats.

Parameters

Control (n=5)

Crude XOS 2000

mg/kgBW (n=2*)

Normal

Range

WBC (10

3

/mm

3

)

13.58 ± 0.39

13.46 ± 5.41

3.0 – 17.0

HGB (g/dL)

14.50 ± 0.10

13.90 ± 0.14

11.0 – 19.0

RBC (10

6

/mm

3

)

7.23 ± 0.16

7.30 ± 0.35

5.0 – 10

PLT (10

3

/mm

3

)

781 ± 0.50

929.5 ± 19.09

200 - 1500

HCT (%)

42.53 ± 0.35

42.20 ± 0.00

35.0 – 57.0

MCH (pg)

19.10 ± 0.05

19.00 ± 1.13

18.0 – 23.0

MCHC (g/dL)

32.53 ± 0.35

32.85 ± 0.21

31.0 – 40.0

MCV (fl)

57.9 ± 0.25

57.90 ± 2.82

46.0 – 65.0

Note: WBC, white blood cell; HGB, hemoglobin; RBC, red blood cell; PLT, platelet count; HCT, red blood cell specific

volume; MCH, mean corpuscular hemoglobin;

MCHC, mean corpuscular hemoglobin concentration; MCV, mean

corpuscular volume, *

Values represent surviving animals in the 2000 mg/kg BW group

84

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

Macropathological testing

Macropathological

observation

aims

to

identify physical and morphological changes

in

organs

due

to

exposure

to

the

test

compound.

This

observation

provides

an

initial overview of the toxic effects that may

occur

[22].

Macropathological

examination

of

the

organs

in

rats

was

conducted

by

performing

macroscopic

observations

and

calculating

the

relative

organ

weights.

The

organs observed included the liver, kidneys,

heart,

spleen,

and

lungs.

Relative

organ

weight is the ratio between the organ weight

and the total body weight of the test animal,

usually

expressed

as

a

percentage.

This

parameter is commonly used in toxicological

studies

to

assess

the

effects

of

the

test

substance on organ function and condition,

as well as physiological disorders induced by

exposure

to

chemicals

or

drugs

[23].

The

results of relative organ weight observations

in rats are presented in Table 3. The changes

in relative organ weight were not significant,

indicating

that

administration

of

the

crude

XOS at a dose of 2000 mg/kg did not cause

any

meaningful

changes

in

the

liver.

The

macroscopic observations of each organ in

the control group and test rat are shown in

Figure 2.

Table 3.

Relative organ weight of male Wistar rats.

Test Animals

Liver (%)

Kidney (%)

Heart (%)

Spleen (%)

Lung (%)

Control (n=5)

3.53 ± 0.81

0.53 ± 0.06

0.56 ± 0.10

Crude

XOS

2000

mg/kgBW (n=2*)

4.10 ± 0.14

0.57 ± 0.05

0.57 ± 0.13

0.63 ± 0.04

*Values represent surviving animals in the 2000 mg/kg BW group

Assessment

of

relative

organ

weights

revealed

no

statistically

significant

differences

between

the

control

and crude

XOS-treated

groups

(2000

mg/kg

BW)

(p

>

0.05). The relative liver weight in the treated

group (4.10 ± 0.14%) was slightly higher than

that

of

the

control

group

(3.53

±

0.81%);

however, the difference was not statistically

significant.

Since

alterations

in

liver

weight

may

reflect

hepatocellular

hypertrophy,

congestion,

or

inflammatory

changes,

the

absence of significant variation suggests that

crude XOS did not induce hepatotoxic effects

at the tested dose

[24]

.

The

relative

kidney

weights

were

0.53

±

0.06% in the control group and 0.57 ± 0.05%

in

the

treated

group,

with

no

significant

difference

observed

(p

>

0.05).

Changes

in

kidney weight are commonly associated with

nephrotoxicity,

edema,

or

inflammatory

processes. The lack of significant alteration

indicates that crude XOS administration did

not

adversely

affect

renal

integrity

under

acute exposure conditions

[24]

.

Similarly,

the

relative

heart

weights

in

the

control

and

treated

groups

were

0.53

±

0.06%

and

0.57

±

0.05%,

respectively,

showing no statistically significant difference

(p > 0.05). Cardiac enlargement may indicate

hypertrophy

or

toxic

stress;

therefore,

the

absence of significant changes suggests that

crude

XOS

did

not

produce

cardiotoxic

effects

[25]

.

85

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

Figure 2.

Macroscopic examination of organs in control and test rats: (a) liver in control rat, (b)

liver in test rat 1, (c) liver in test rat 2, (d) kidney in control rat, (e) kidney in test rat 1, (f) kidney

in test rat 2, (g) heart in control rat, (h) heart in test rat 1, (i) heart in test rat 2, (j) spleen in control

rat, (k) spleen in test rat 1, (l) spleen in test rat 2, (m) lung in control rat, (n) lung in test rat 1, (o)

lung in test rat 2.

For the spleen and lungs, the relative organ

weights were 0.53 ± 0.06% and 0.56 ± 0.10%

in the control group, and 0.57 ± 0.13% and

0.63

±

0.04%

in

the

treated

group,

respectively.

Statistical

analysis

demonstrated

no

significant

differences

between

groups

(p

>

0.05),

indicating

that

crude XOS did not induce treatment-related

86

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

effects

on

immune

or

respiratory

organs.

Macroscopic

examination

corroborated

these

findings.

All

examined

organs

exhibited normal morphology, characterized

by smooth surfaces, normal coloration, and

absence

of

visible

lesions,

hemorrhage,

congestion, or enlargement. No macroscopic

damage was observed in the treated animals

compared with the control group

[26]

.

Histopathology

is

the

analysis

of

organ

or

tissue

conditions

through

microscopic

observation of morphological and structural

changes,

as

well

as

indications

of

damage,

infection,

or

other

mutagenic

processes

caused by disease or toxic agents [27]. Based

on

the

histopathological

evaluation

of

the

kidneys

(Figure

3),

differences

were

observed in test rat 1 (male) and test rat 2

(male) administered XOS at a dose of 2000

mg/kg BW compared with the control group.

The kidneys of test rat 1 (male) and test rat 2

(male) were structurally preserved; however,

mild abnormalities were observed in several

glomeruli,

characterized

by

glomerular

swelling

(black

arrows).

Normal

glomeruli

(green

arrows)

showed

uniform

size

and

a

clearly

defined

Bowman’s

space.

The

swelling

was

limited

to

a

small

number

of

glomeruli,

indicating

a

localized

alteration

that

is

unlikely

to

cause

significant

impairment of overall renal function.

(a)

(b)

(c)

Figure 3.

Histopathological observation of the kidney: (a) kidney in normal control;

(b)

kidney

in

test

rat

1;

(c)

kidney

in

test

rat

2.

(Green

arrows

indicate

normal

glomeruli, black arrows indicate swollen glomeruli).

87

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

(a)

(b)

(c)

Figure 4.

Histopathological observation of the spleen: (a) spleen in normal control; (b) spleen

in test rat 1; (c) spleen in test rat 2. (Red circles indicate red pulp, black circles indicate white

pulp).

(a)

(b)

(c)

Figure 5.

Histopathological observation of the liver: (a) liver in normal control; (b) liver in test

rat 1; (c) liver in test rat 2. (Black circles indicate cytoplasmic condensation).

88

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

(a)

(b)

(c)

Figure 6.

Histopathological observation of the lungs: (a) lung in normal control; (b) lung in test

rat 1; (c) lung in test rat 2. (Green arrows indicate normal alveoli, black arrows indicate dilated

alveoli, black circles indicate hemorrhage).

Histopathological examination of the spleen

(Figure 4) revealed no significant differences

between

the

control

group

and

test

rats

1

(male) and test rat 2 (male), which received

XOS at a dose of 2000 mg/kg BW. The spleen

tissue

of

both

control

and

treated

rats

demonstrated

typical

architecture

composed of red pulp and white pulp. The

white pulp consisted of lymphocytes with a

purplish

hue,

indicated

by

black

circles,

whereas

the

red

pulp

was

composed

of

erythroblasts, indicated by red circles. In test

rat 1 (male), the lymphoid tissue

remained

well

preserved;

however,

some

sections

appeared

less

distinct

due

to

artifacts

caused

by

tissue

slicing.

Overall,

no

structural

damage

was

detected

in

the

spleens

of

treated

rats,

and

their

cellular

morphology was comparable to that of the

control group.

In contrast, histopathological examination of

the

liver

(Figure

5)

demonstrated

mild

alterations.

Control

rats

demonstrated

normal

hepatic

architecture.

In

contrast,

liver sections from test rat 1 (male) and test

rat 2 (male) revealed focal areas of increased

staining

intensity

(black

circles),

corresponding to cytoplasmic condensation

in

hepatocytes.

These

alterations

were

localized predominantly in zone 1 (periportal

region)

of

the

hepatic

lobule.

Cytoplasmic

condensation in the absence of necrosis or

extensive

cellular

damage

is

generally

considered

indicative

of

mild,

early-stage

hepatocellular

degeneration,

representing

the

earliest

detectable

pathological

change

in hepatocytes [28].

Histopathological

examination

of

the

lungs

(Figure 6) in test rat 1 (male) and test rat 2

(male) revealed focal inflammatory changes,

89

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

indicating

structural

alterations

in

the

pulmonary

tissue.

The

affected

sections

showed

distended

alveoli

with

widened

interalveolar spaces (black arrows), whereas

normal

alveoli

(green

arrows)

exhibited

uniform

architecture

without

apparent

enlargement.

The

widening

of

alveolar

spaces

was

associated

with

inflammatory

cell infiltration. In addition, hemorrhagic foci

(black circles) were observed, characterized

by

the

presence

of

erythrocytes

within

the

interalveolar spaces. The hemorrhage likely

resulted

from

vascular

dilation

and

increased

vascular

permeability,

allowing

blood

cells

to

extravasate

into

the

surrounding alveolar tissue [29].

(a)

(b)

(c)

Figure 7.

Histopathological observation of the heart: (a) heart in normal control; (b) heart in

test rat 1; (c) heart in test rat 2. (Green arrows indicate normal heart wall).

Considering that the route of administration

was

oral,

the

pulmonary

lesions

observed

are unlikely to be attributable to inhalation

of

XOS.

The

findings

may

instead

reflect

secondary

systemic

effects,

aspiration-

during

oral

gavage,

or

nonspecific inflammatory responses.

Based

on

histopathological

observation

of

the

heart,

no

differences

were

observed

between the control rats and test rat 1 (male)

and test rat 2 (male) administered XOS at a

dose

of

2000

mg/kg

BW.

Histopathological

evaluation of the heart (Figure 7) in control

rats and test rats 1 and 2 (male) showed well-

preserved

heart

walls,

with

no

evidence

of

damage or abnormalities.

Conclusion

The acute oral toxicity study demonstrated

that

crude

XOS

administered

at

a

dose

of

2000

mg/kg

body

weight

resulted

in

mortality in 3 out of 5 rats during the 14-day

observation period. Based on these findings,

the oral LD

₅₀

of crude XOS was estimated to

be <2000 mg/kg body weight. According to

the

classification

criteria

of

the

United

90

P Vergian et al.,

Chempublish Journal, 10(1) 2026, 78-93

Nations Globally Harmonized System (GHS),

the

substance

falls

under

Category

4

for

acute

oral

toxicity.

Biochemical

evaluation

revealed

no

marked

elevation

of

hepatic

transaminases (SGOT and SGPT) relative to

controls,

suggesting

the

absence

of

significant

hepatocellular

injury.

Histopathological examination showed mild

and

localized

degenerative

changes

in

the

liver,

focal

glomerular

swelling

in

the

kidneys, and inflammatory and hemorrhagic

foci

in

the

lungs,

without

evidence

of

extensive

necrosis

or

severe

structural

damage.

These

findings

suggest

that

although

mortality

occurred

at

the

tested

dose,

overt

target-organ

toxicity

was

not

strongly

demonstrated.

Given

that

the

test

material

was

crude

XOS,

the

observed

toxicity may be associated not only with the

oligosaccharides

themselves

but

also

with

residual

impurities,

by-products,

or

endotoxin

contamination

remaining

from

the

production

process.

Therefore,

further

purification

and

compositional

characterization

of

XOS

are

recommended

to

reduce

potential

impurities

and

better

evaluate its intrinsic safety profile. Additional

studies using purified XOS preparations are

necessary to clarify the relationship between

purity level and acute toxicity outcomes.

Acknowledgement

This

research

supported

by

Hibah

Inovasi

2024

Universitas

Jember

through

Anak

Agung Istri Ratnadewi.

Author Contributions

Conceptualization,

AAI

Ratnadewi

and

Evi

Umayah; Methodology, AAI

Ratnadewi, Mei

Syafriadi;

Validation,

Cahya,

Reisma

and

Putra; Formal Analysis, Tri Agus Siswoyo and

Putra;

Investigation,

AAI

Ratnadewi;

Data

Curation, Cahya, Reisma and Putra; Writing –

Original

Draft

Preparation,

Cahya,

Reisma

and

AAI

Ratnadewi;

Writing

–

Review

&

Editing, Evi Umayah.

Conflict of Interest

The authors declare no conflict of interest

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Chempublish Journal, 10(1) 2026, 78-93

Toxicological Evaluation and Food Safety Assessment of Xylooligosaccharides Derived from Rice Straw Xylan Hydrolysis using Recombinant Endo-β-1,4-D-Xylanase: An Acute Oral Toxicity Study

Putra Vergian 1,a , Anak Agung Istri Ratnadewi 1,b * , Cahyaningtyas Tetta Riandy 1,c , Riesma Rukmana 1,d , Evi Umayah Ulfa 2,e , Tri Agus Siswoyo 3,f , Mei Syafriadi 4,g

A bstract

Graphical Abstract

Introduction

Toxicological Evaluation and Food Safety Assessment of

Xylooligosaccharides Derived from Rice Straw Xylan Hydrolysis

using Recombinant Endo-β-1,4-D-Xylanase:

An Acute Oral Toxicity Study

Putra Vergian

1,a

, Anak Agung Istri Ratnadewi

1,b

*

, Cahyaningtyas Tetta Riandy

1,c

, Riesma

Rukmana

1,d

, Evi Umayah Ulfa

2,e

, Tri Agus Siswoyo

3,f

, Mei Syafriadi

4,g

A

bstract