Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Tolyltriazole is expected to have a similar genetic toxicity compared to the analogue Benzotriazole when investigating the gene mutation potential in mammalian cells. The genetic toxicity in respect of the gene mutation potential in mammalian cells of Benzotriazole was determined in a well performed study. The source chemical Benzotriazole is sufficiently similar to read-across towards Tolyltriazole.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: granules
Details on test material:
- Name of test material (as cited in study report): Benzotriazol Granulat
- Molecular formula (if other than submission substance): C6H5N3
- Molecular weight (if other than submission substance): 119.14 g/mol
- Smiles notation (if other than submission substance): c12c(cccc1)N=NN2
- InChl (if other than submission substance): 1S/C6H5N3/c1-2-4-6-5(3-1)7-9-8-6/h1-4H,(H,7,8,9)
- Structural formula attached as image file (if other than submission substance):


- Substance type: yellow granulat
- Physical state: solid
- Analytical purity: 99.83 %
- Lot/batch No.: 909

Method

Target gene:
HGPRT (Hypoxanthine guanine phophoribosyl transferase
Species / strain
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
Controls
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

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

1.      Source Chemical(s)

 

Benzotriazoles have two fused rings, one 1,2,3-triazole and one benzene ring. In Tolyltriazole, the benzene ring is substituted with one methyl group, while in Benzotriazole all substituents are hydrogen.

The Benzotriazoles can be deprotonated at the Nitrogen, leading to the conjugated base as sodium salt.

The differences in the chemical structure (from Benzotriazole to Tolyltriazole) are not expected to change the toxicological properties significantly.

The sodium salts are more basic than the neutral substances (Benzotriazole and Tolyltriazole) and present therefore irritant and corrosive properties, as seen in in vivo studies. Nevertheless, systemic effects are expected to be comparable between the salts and the neutral substances due to the fact that in physiological environment (pH 6-8) protonation of the slat occurs and the neutral species is yielded.

2.  purity/impurities

The impurities in the target substance do not indicate toxicological relevence to this endpoint. The impurities are all below 1 %.

 

The excess sodium hydroxide of sodium benzotriazolate and sodium tolyltriazolate increases the toxicological irritation/corrosion properties as seen in valid in vivo tests. Further on, it is assumed that no other influence then the basic reservoir is changed by this impurity

 

3.      Analogue approach justification

 

According to Annex XI, 1.5 a read-across approach can be used to fill the data gap when certain criteria are fulfilled. The fulfillment of these criteria is discussed below. The information from the REACH technical guidance document R.6 are used for this assessment as well as ECHA's Practical guide 6 on category and read-across approaches (ECHA REACH TGD; ECHA, 2009).

 

Quality of the experimental data of the analogues

 

The source chemical has been tested in a well-conducted study (according to OECD TG 476). The study results receive reliability 1.

 

Toxicokinetics

 

The source and target chemicals indicate similarity in toxicokinetic behavior based on the molecular weight (< 200), physical form (all are solids), vapor pressure (< 10 Pa) and Log POW(0-2).

 

The difference between the neutral species and the salts in respect to log POWis in the range of 1 and expected to be of minimal relevance. The charge of the Benzotriazolate anion in the salts will decrease the bioavailability but in contact with water the neutral species will be formed in dependence of the pH.

 

Reactivity towards proteins and DNA

 

(Q)SAR modeling

The (Q)SAR modeling as such is not used for predictivity but it is used for showing that the Benzotriazoles have the same toxicological profile according to these models.

The OECD (Q)SAR toolbox program is used to obtain the toxicological profile for the source and target substances.

The benzotriazole structures result in two alerts with regard to toxicity:

-         Toxic Hazard Classification by Cramer: High (Class III)

-         In vivo mutagenicity (Micronucleus) alerts by ISS: H-Acceptor-path 3-H-acceptor for DNA-binding for in vivo mutagenicity.

No alerts were found for DNA or protein binding.

 

The Toxic Hazard Classification by Cramer: High (Class III) is verified by the observed oral toxicity of the different benzotriazoles (data matrix).

 

The “in vivo mutagenicity (Micronucleus) alert by ISS” is a false positive alert as the available in vivo genetic toxicity data is negative for this endpoint. 

 

Similarities in results for toxicological endpoints between the target and the source chemical(s) to support read-across for acute dermal toxicity

 

As it is presented in the data matrix the acute oral toxicity is in the same range for the target and source chemical(s).

 

The neutral substances (Benzotriazole and Tolyltriazole) show no skin irritation, skin sensitization properties and only mild eye irritation, the salts (Sodium benzotriazolate and sodium tolyltriazolate) show severe skin irritation / corrosion which is caused by the high basicity and the pH of a solution of these substances.

 

The negative genotoxicity profile is also similar between the source and the target chemical.

For Benzotriazole the Ames test, the mammalian mutation test and the mouse micronucleus test are negative.

For Tolyltriazole the Ames test and the mouse micronucleus test are negative.

 

Systemic toxicity is seen for Benzotriazole in a two year study and for Tolyltriazole in a 28 days-repeated dose test. No target organ was identified.

A LOAELchronicof 325 mg/kg bw for Benzotriazole and a NOAELsub-acuteof 150 mg/kg bw for Tolyltriazole was established.

Toxicity to reproduction and fertility was tested in a Screening test according to OECD Guideline 421 for Benzotriazole. In this study, a NOAELReprotox-screeningof 200 mg/kg bw/day was found. Higher doses were not tested due to the systemic toxicity of the substance.

  

4.      Data matrix

 

See separate document

Applicant's summary and conclusion

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
Executive summary:

For Benzotriazole a well-conducted in vitro study is available showing no genetic toxicity for gene mutation in mammalian cells. This means that a similar result for Tolyltriazole can be anticipated.

 

Tolyltriazole is not genetic toxic in respect to gene mutation in mammalian cells.

 

A DNEL for oral, dermal and/or inhalation route can be based on this information.

Classification and labelling are / are not needed for this endpoint.

A risk characterisation will be performed because the substance is classified for orale toxicity.