4,5-bis(hydroxymethyl)-2-phenyl-1H-imidazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: Negative (OECD 471, GLP, K, rel.1)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Aug 2021 - April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to the OECD TG 471 without any deviation affecting the reliability of the assay.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21st, 1997 corrected 26.06.2020

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. See 'additional information on methods' for details.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene | E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

WP2 (uvr A ̄) (pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: MOLTOX (S9 Moltox-11101-5-4244 validated on 21.08.2020 – expiry date: 28.04.2022).
- method of preparation of S9 mix: prepared from Sprague Dawley rat liver homogenate,
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % (v/v), 500 µL per plate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): MOLTOX quality control and production certificate 28 Apr 2020

Test concentrations with justification for top dose:

Cytotoxicity assessment: 5000 μg /plate, 1500 μg /plate, 500 μg /plate, 150 μg /plate, 50 μg /plate | No cytotoxicity was observed to the highest dose, so the same doses were used for the main experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: commonly used vehicle in which the test item was partially soluble (precipitate confirmed not to interfere with scoring)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

See Table 1 for details / strain. Solvent used to solubilize positive controls: Acetone, DMSO, NaCl 0.15 M

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-9 x10E9 bacteria/mL
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30min (preincubation assays only)
- Exposure duration/duration of treatment: incubation at 37° over a 48-72-hour period

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS)
- Any supplementary information relevant to cytotoxicity: In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less.

METHODS FOR MEASUREMENTS OF GENOTOXICITY: dose-related increase in the number of revertant colonies compared to the solvent control

Rationale for test conditions:

Dose levels were selected based on the result of the bacteriostatic activity control test.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dose-response relationship between concentration and number of revertants is obtained in one, or several of the 5 strains, without and/or with metabolic activation. A mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two-fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three-fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: n.a.
- Data on osmolality: n.a.
- Possibility of evaporation from medium: n.a.
- Water solubility: n.a.
- Precipitation and time of the determination: none reported
- Definition of acceptable cells for analysis: n.a.
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES (if applicable):
Results of the bacteriostatic activity control test showed no toxicity in presence of the test item. Therefore, the test item was tested at the following doses: 5 000, 1 500, 500, 150 and 50 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
- Signs of toxicity: none reported
- Individual plate counts: cf. Table of Results 'Attachments')
- Mean number of revertant colonies per plate and standard deviation: cf. Table of Results ('Attachments')
There is no evidence of any increase in the number of revertant colonies in the presence of the test item without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2(uvr A¯)(pKM101) strain

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf. Table of Results 'Attachments')
- Negative (solvent/vehicle) historical control data: cf. Table of Results 'Attachments')
There is no difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory

Results tables and HCD are included in attachment.

Conclusions:

Under the assay conditions, the test item did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2(uvr A ̄)(pKM101) strain without, or with metabolic activation, according to the OECD Guideline n° 471.

Executive summary:

Suspensions obtained from CUREZOL 2PHZ-PW (the test item) have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A ̄)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

 

For assay n° 1, various concentrations of test item were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

 

For assay n° 2, various concentrations of test item were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

 

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

 

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (5 000, 1 500, 500, 150 and 50 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA ̄) (pKM 101).

 

These results validate the two tests.

 

Conclusion:

Under the assay conditions the test item did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2(uvr A ̄)(pKM101) strain without, or with metabolic activation, according to the OECD Guideline n° 471.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6.1/1 : Summary of genotoxicity tests:

 

Test n°	Test Guideline / Reliability	Focus	Strains  / cells tested	Metabolic activation	Test concentration	Statement
1 (2022)	Ames Test (OECD 471, GLP) K, rel.1	Gene mutation	S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2(uvrA-)	-S9 +S9	Tested up to recommended limit concentrations	-S9: not mutagenic + S9: not mutagenic

Gene mutation Assay (Test n° 1)

A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation. The test indicate that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test. 

Justification for classification or non-classification

Harmonised classification: 

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification: 

Based on the available information, no additional classification is proposed according to the CLP and the GHS.