Benzotriazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence for genetic toxicity was found in the in vitro bacterial reverse mutation assay and mammalian cell reverese mutation assay reported in the dossier.

 

Mutagenicity of Benzotriazole is discussed in scientifc literature. A survey by Zeiger et al, (1987) is used often as reference, as this survey was used by Kirkland in various publications on test performance asssessment. However the available documentation is limited and not sufficent for a thorough evaluation.

A Publication by Zwart et al. (2020) reports a downscaled luminescent Ames assay with the Strains TA98lux and TA100lux. The authors are testing fractions of environmental samples, one fraction is claimed to contain Benzotriazole (analytically determined) and showing mutagenic properties. However, it is not clear whether other components, not detected and contained in the sample contribute to the observations. Furthermore, the available Ames test reported in the dossier (Andres, 2012) investigates the strains TA98 and TA100 and no mutagenic effect with the tested substance is observed.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guidance study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA1535

Metabolic activation:

with and without

Test concentrations with justification for top dose:

first experiment
50/150/500/1501/5003 µg/plate
second experiment
313/625/1250/2500/5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

DMSO is chosen because 1H-benzotriazole is completly soluble and the vehicle does not have any effects on the viability of the bacetria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylenediamine; 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) with and without preincubation

DURATION
- Preincubation period: 20 min

NUMBER OF REPLICATIONS: 4

Evaluation criteria:

The colonies were counted visually.
The increase factor f(I) of revertant induction and the absolute number ov refertants were calculated.
If a significant, reproducible increase of revertant colonies (f(I) > 2) can be observed, 1H-benzotriazole is considered mutagenic.

Statistics:

mean values and standard deviations of each treatment, solvent control and positive control are calculated. Calculations were performed with unrounded values

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative, vehicle and positive controls were compared to historical data and
found to be in the range of this data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table: Mean revertants first experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	118	121	15	19	90	101	145	144	16	17
	sd	8	6	3	2	9	9	9	20	3	2
DMSO	Mean	126	121	19	20	97	94	127	140	17	15
	sd	9	7	3	3	12	14	9	8	2	3
Positive controls	Mean	563	562	236	220	452	470	583	631	193	186
	sd	69	37	34	12	41	26	102	66	13	14
	f(I)	4.47	4.64	12.42	11.00	5.02	5.00	4.59	4.51	12.06	12.40
5003 µg/pl.	Mean	107	125	18	15	99	102	149	141	16	14
	sd	5	4	2	4	18	6	10	9	5	1
	f(I)	0.85	1.03	0.95	0.75	1.02	1.09	1.17	1.01	0.94	0.93
1501 µg/pl.	Mean	123	124	17	16	103	103	143	146	17	17
	sd	10	5	2	4	11	7	7	7	3	2
	f(I)	0.98	1.02	0.89	0.80	1.06	1.10	1.13	1.04	1.00	1.13
500 µg/pl.	Mean	120	118	15	16	89	110	139	130	15	14
	sd	5	10	5	1	8	3	16	11	4	1
	f(I)	0.95	0.98	0.79	0.80	0.92	1.17	1.09	0.93	0.88	0.93
150 µg/pl.	Mean	116	121	14	18	88	101	136	141	15	15
	sd	3	7	1	3	21	10	5	11	2	1
	f(I)	0.92	1.00	0.74	0.90	0.91	1.07	1.07	1.01	0.88	1.00
50 µg/pl.	Mean	119	108	16	20	83	97	137	128	15	15
	sd	5	7	3	3	19	6	4	15	2	2
	f(I)	0.94	0.89	0.84	1.00	0.86	1.03	1.08	0.91	0.88	1.00

Table: Mean revertants second experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	118	117	15	15	100	87	166	153	16	15
	sd	4	3	3	1	8	10	14	11	2	2
DMSO	Mean	117	115	18	16	98	96	149	154	18	16
	sd	3	6	3	1	3	3	23	7	3	2
Positive controls	Mean	517	592	217	222	416	473	621	611	210	241
	sd	71	13	12	15	38	26	25	43	10	13
	f(I)	4.42	5.15	12.06	13.88	4.16	4.93	4.17	3.97	13.13	15.06
5002 µg/pl.	Mean	104	112	15	14	95	93	139	146	15	14
	sd	7	7	2	3	7	7	12	5	2	3
	f(I)	0.89	0.97	0.83	0.88	0.97	0.97	0.93	0.95	0.83	0.88
2501 µg/pl.	Mean	112	113	14	15	84	79	145	157	15	13
	sd	3	3	3	3	8	9	5	16	2	1
	f(I)	0.96	0.98	0.78	0.94	0.86	0.82	0.97	1.02	0.83	0.81
1251 µg/pl.	Mean	117	109	14	16	82	79	143	150	17	16
	sd	4	6	2	2	8	6	11	16	4	4
	f(I)	1.00	0.95	0.78	1.00	0.84	0.82	0.96	0.97	0.94	1.00
626 µg/pl.	Mean	116	116	15	17	92	87	156	157	15	15
	sd	4	4	1	3	16	9	14	12	2	3
	f(I)	0.99	1.01	0.83	1.06	0.94	0.91	1.05	1.02	0.83	0.94
313 µg/pl.	Mean	115	114	14	17	85	85	146	151	16	14
	sd	2	2	2	3	11	7	5	8	2	2
	f(I)	0.98	0.99	0.78	1.06	0.87	0.89	0.98	0.98	0.89	0.88

Conclusions:

1H-benzotriazole did not show mutagenic effects in the two conducted experiments.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guidance study with minor deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

test article treatment is 4 hours instead of 5; whole fetal bovine serum is used instead of dialyzed calf serum

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HGPRT (Hypoxanthine guanine phophoribosyl transferase

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Clone: CHO-K1BH4
- Type and identity of media: Ham's Nutrient Mixture F10 supplemented with L-glutamine, penicillin G, strptomycin sulfate, fungizone fetal bovine serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate

Test concentrations with justification for top dose:

50 to 1000 µg/ml see tables

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

other: 5-Bromo-2'-deoxyuridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6 to 7 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

Evaluation criteria:

Relative survival
Relative Population Growth
Absolute cloning efficiency
Mutant Frequency

Statistics:

mean values and standard deviation; significance calculated

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
concentrations ranging from 2 to 1000µg/ml were tested for cytotoxic effects.
Without activation, only in the highest concentration a decrease in the relative survival to 80 % was obtained.
With activation, a concentration of 125 µg/ml lead to a survival of 70 % and at the highest concentration the rate was 0%.

MUTAGENICITY STUDY:
Non activation
Three trials were performed under nonactivation conditions, but the first trial was not used for evaluation, because the cloning efficiency of the vehicle controls was too low. In the second trial the assayed cultrues showed a low toxicity up to 1000 µg/ml. The lowest two concentrations were not assyed, because enough concentrations were left for cloning. None of the ten treated cultrues showed increases in mutant frequency that were statistically significantly elevated over the concurrent vehicle controls. Therefore, this nonactivation assay was evaluated as negative. In the independent repeat test concentrations from 400 to 1000 µg/ml were cloned, inducing low toxicities. The mutatnt frequencies varied for concentrations up to the maximum applied concentration of 1000µg/ml. The test material was therefore evaluated as nonmutagenic in this trial.

Activation

Conclusions:

Interpretation of results (migrated information):
negative

In this study the toxicity and mutagenicity of Benzotriazole was assayed.
Under non-activating conditions only low toxicity could be induced up to the maximum applied concentration (1000 µg/ml).
With S9 metabolic activation the substance is more toxic to the used cells as seen by decreases in relative survival and relative population growth.
In all assays the mutant frequencies were within or near the range that is typical of vehiclecontrol variation between trials (1-15*10^-6), while no dose-respons relationships were evident.
Based on these conclusions, the substance 1H-benzotriazole is considered to be nonmutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

One study for the endpoint in vivo genetic toxicity is reported. The Micronucleus test does not report any genotoxic properties of the substance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD-Study with insufficient discussions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 28 - 42 g
- Assigned to test groups randomly: yes, using a randomisation plan produced by teh Institute of Biometrics, BAYER AG, Wuppertal
- Fasting period before study: no
- Housing: Makrolon cages type I and II, bedding of soft wood granules
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24 °C
- Humidity (%): 50 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

other: oral stomach tube

Vehicle:

polyethylene glycol 400

Details on exposure:

a pilot study indicated 800 mg / kg bw as concentration for the test.
800 mg /kg bw were dissolved in polyethylene glycol 400.
Via stomach tube 5 ml / kg bw were administered.

Frequency of treatment:

once

Post exposure period:

24 to 72 hours

Remarks:

Doses / Concentrations:
800 mg / kgbw
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

10 animals, 5 male, 5 female

Control animals:

yes

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide is a known clastogen
- Route of administration: oral via stomach tube
- Doses / concentrations: 20 mg / kg bw, dissolved in demineralised water, 10 ml / kg were administered

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:

1. At least one intact femur was prepared from each
sacrificed animal (not pretreated with a spindIe
inhibitor). A suitale instrument was used to
sever the pelvic bones and calf area.
2. The femur was separated from muscular tissue.
3. The lowerleg stump with knee and all attached
soft parts in the distal epiphyseal cartilage
were remtoved by a gentIe pull at the distal end.
4. The proximal end of the femur was opened at its
extreme end with a suitable instrument, e.g.
fine scissors, so that a small opening of the
bone marrow channel became visible.
5. A suitable tube Has filled with sufficient fetal
calf serum.
6. Some serum was drawn from the tube into a suitable
syringe, with thin cannula.
7. The cannula was pushed into the open end of the
marrow cavity.
8. The femur was then completely immersed in the calf
serum and pressed against the wall of the tube,
so that it could not slip off.
9. The contents were then flushed several times, and
the bone marrow passed into the serum as a fine
suspension.
10. FinaIly, the flushing could be repeated from the
other end after it was opened.
11. The tube containing the serum and bone marrow
was centrifuged in a suitable centrifuge at
approximately 1000 rpm for five minutes.
12. The supernatant was removed with a suitable
pipette (e.g. Pasteur pipette) except for a
small resldue.
13. The sediment was mixed until the suspension
was homogeneous.
14. one drop of the viscous suspension was placed
on the thoroughly cleaned slide and spread
with a suitable object, e.g. a slide, so that
the smear could be properly evaluated.
15. The labeled slides were dried overnight.
If fresh smears are to be stained, they must
be dried for a short period with heat.

Staining of smears
The mears were stained automatically with an Ames Hema-Tek
Slide Stainer of Miles company. The slides were then
"destained" with methanol and rinsed with deionized water.
They were then le ft to dry.

METHOD OF ANALYSIS:
the slides were analysed with a light microscope at a magnification of 1000

Evaluation criteria:

ratio of polychromatic to normochromatic erythrocytes

Statistics:

The Preventol CI8-100 group with the highest mean, if this
superceded the negative control mean, and the positive
control were checked by Wilcoxon's non-parametric rank sum
test in respect to the count of polychromatic erythrocytes
with micronuelei and the rate of normochromatic
erythrocytes. A variation is considered statistically
significant if its error probability is below 5% and the
treatment group figure is higher than the negative
control's .
The rate of normochromatic erythrocytes with micronuclei is
examined if the micronucleus rate for polychromatic
erythrocytes was allready relevantly increased. In this case,
the group with the highest mean 1s compared with the
negative control using the one-sided chi 2-test. A variation
wss considered statistically significant if the error
probability is below 5% and the treatment group figure is
higher than the negative control's.

In addition, standard deviations (1s ranges) were calculated
for all the means.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500/750/850/1000 mg/kg bw
- Clinical signs of toxicity in test animals: apathy, reduced motility, unkempt coat, lateral position, abdominal position, cramp, convulsion, rapid breathing

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No, 0.9 - 1.3 micronucleated cell per 1000 NCE/PCE
- Ratio of PCE/NCE (for Micronucleus assay): 1.028 - 1.084

Positive control
- Induction of micronuclei (for Micronucleus assay): No, 0.5 / 13.4 micronucleated cell per 1000 NCE/PCE
- Ratio of PCE/NCE (for Micronucleus assay): 1.133

Conclusions:

Interpretation of results (migrated information): negative
There is no sign of genotoxicity arising from this study.
In comparison with the negative control, no alteration in the ratio of polychromatic to normochromatic erythocytes was observed.
Further, there is no variation in regard to incidence of micronucleated cells between the negative control and the test groups.
In the positive control group there is a significant change in the incidence of micronuclei compared to the negative control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The three performed studies were GLP compliant and of high quality (Klimisch score = 1). Therefore, there is no reason to believe that these results would not be applicable to humans.

Justification for selection of genetic toxicity endpoint
well performed study according to OECD Guidelines

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.