Sodium 4(or 5)-methyl-1H-benzotriazolide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Sodium Tolyltriazolate is expected to have a similar genetic toxicity compared to the analogues Benzotriazole and Tolyltriazole when investigating the clastogenic potential in vivo. The genetic toxicity of Benzotriazole and Tolyltriazole was determined in well performed micronucleus studies. The source chemicals Benzotriazole and Tolyltriazole are sufficiently similar to read-across towards Sodium Tolyltriazolate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl-1H-benzotriazole

EC Number:

249-596-6

EC Name:

Methyl-1H-benzotriazole

Cas Number:

29385-43-1

Molecular formula:

C7H7N3

IUPAC Name:

4(or5)-methyl-1H-1,2,3-benzotriazole

Constituent 2

Reference substance name:

1H-4/5-Methyl-1,2,3-Benzotriazole

IUPAC Name:

1H-4/5-Methyl-1,2,3-Benzotriazole

Test material form:

solid: pellets

Details on test material:

- Name of test material (as cited in study report): Preventol CI 7-100
- Molecular formula (if other than submission substance): C7H7N3
- Molecular weight (if other than submission substance): 133.15 g/mol
- Smiles notation (if other than submission substance): CC1=CC=C2NN=NC2=C1; CC1=C2N=NNC2=CC=C1
- InChl (if other than submission substance): InChI=1S/C7H7N3/c1-5-3-2-4-6-7(5)9-10-8-6/h2-4H,1H3,(H,8,9,10); InChI=1S/C7H7N3/c1-5-2-3-6-7(4-5)9-10-8-6/h2-4H,1H3,(H,8,9,10)
- Structural formula attached as image file (if other than submission substance):
- Physical state: solid
- Analytical purity: 99.3 %
- Composition of test material, percentage of components: 40 % 4-Methylbenzotriazole and 60 % 5-Metyhlbenzotriazole
- Storage condition of test material: at 4 °C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 31 - 44 g
- Assigned to test groups randomly: [yes, under following basis: company randomising plan]
- Housing: standard Kakrolon cages type I and II
- Diet: Altromin 1324 ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2°C
- Humidity (%): 50 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Polyethylenglycole
- Justification for choice of solvent/vehicle: suitable solvent for Tolyltriazole
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test item was solved in polyethylene glycol and
administered by gavage

Duration of treatment / exposure:

not applicable

Frequency of treatment:

once

Post exposure period:

24 / 48 / 72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
600 mg / kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): common substance, reommended by the OECD Guideline
- Route of administration: gavage
- Doses / concentrations: 20 mg / kg bw

Examinations

Tissues and cell types examined:

bone marrow from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a range finding study indicated the dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:
The bonemarrow is purged with fetal calve serum
Dyeing with an Ames Hema-Tek Slide Stainer

METHOD OF ANALYSIS:
manual counting with a microscope (magnification 1000)

Evaluation criteria:

for the test item the number of polychromatic cells with micro nuclei is compared to the controls
A difference is significant, if the error probability is below 5 %

Statistics:

distrribution free rank sum test according to wilcoxon
Chi2-Test
Mean values and 1s-range

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 1000 mg/kg bw
- Clinical signs of toxicity in test animals: apathy, horrent fur, narcotic state, prone position, spasm, breathlessness

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): normal
- Ratio of NCE/PCE (for Micronucleus assay): 1000:1715

Any other information on results incl. tables

Group	evaluable polychromatic Erythrocytes	Ratio NCE/PCE	Micronuclei per 1000 NCE	Micronuclei per 1000 PCE
negative control	10000	1320	0.5	1.2
Tolyltriazole, 24 h	10000	1540	0.9	1.7
Tolyltriazole, 48 h	10000	1715	0.6	1.4
Tolyltriazole, 72 h	10000	994	0.5	1.0
positive control	10000	1393	0.8	14.7

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 407). 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 P_OW(0-2).

 

The difference between the neutral species and the salts in respect to log P_OWis 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 LOAEL_chronicof 325 mg/kg bw for Benzotriazole and a NOAEL_sub-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 NOAEL_{Reprotox-screening}of 200 mg/kg bw/day was found. Higher doses were not tested due to the systemic toxicity of the substance.

 

4. Data matrix (IUCLID: „Results and discussion“)

 

Separate document

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Tolyltriazole does not show genetic toxicity in this study.
Toxic effects were observed in a range finding study at 750 mg /kg bw

Executive summary:

For Benzotriazole and Tolyltriazole well-conducted in vivo studies are available showing no genetic toxicity for micronucleus formation in mice. This means that a similar result for Sodium Tolyltriazolate can be anticipated.

 

Sodium Tolyltriazolate is not genetic toxic in respect to micronucleus formation.

 

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

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

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