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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Bacterial Reverse Mutation Test, which is analogous to the OECD Test Guideline No. 471, the test item methylhydroquinone was non mutagenic for all the used indicator strains with and without metabolic activation.

In a micronucleus test according to OECD Test No. 487 the test item methylhydroquinone had no genotoxic effects in the human peripheral blood lymphocytes with as well as without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06.03.2018 - 27.04.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
yes
Remarks:
Two cytotoxic concentrations were observed in the second experiments in Salmonella typhimurium TA 98. As 6 concentrations were used and 4 of them were acceptable this deviation had no impact on the outcome of study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the first cytotoxicity experiment (in TA 98), the first mutagenicity experiments were performed as plate incorporation test with and without metabolic activation in all the strains. The concentration of 10 µg per plate was then used as maximum in the, whereas the cytotoxicity was expected in the highest concentration only. Further doses were diluted with factor approximately 2-√10 with resulting concentration range 10.0, 3.0, 1.0, 0.3 and 0.1 µg per plate.
The second mutagenicity experiments were performed as plate incorporation test. Firstly the maximum concentration was increased by one concentration which was higher by √3 approximately i.e. 30 μg per plate. Experiments were performed in Salmonella typhimurium TA 100 and TA 98. Neither this concentration was cytotoxic for Salmonella typhimurium TA 100.
Experiments in the other indicator strains including repeated experiments in Salmonella typhimurium TA 100 (= third mutagenicity experiments) were performed with concentrations 10, 30, 75, 150 and 300 μg per plate (concentrations based on the second cytotoxicity experiment).
Some higher concentrations were cytotoxic for indicator strains Salmonella typhimurium TA 1535 and TA 1537 without metabolic activation. These experiments were repeated (third mutagenicity experiments) using the series of lower concentrations 2.5, 5.0, 10.0, 25.0 and 50.0 μg per plate.
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate
Details on test system and experimental conditions:
Bacterial strains: Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 0102201220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732).
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains: Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9: The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20, 30, 50 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7, 9.1 or 16.7%). In experiments without metabolic activation only buffer was added to the top agar.
Volume of 20 µL (3.8 % of S9 in S9 mix) of S9 is used for positive controls in all strains with except of E. coli (reason – to keep constant conditions for database of historical values).
Volume of 30 µL (5.7% of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the first experiments in all strains with except of E. coli.
Volume of 50 µL (9.1% of S9 in S9 mix) of S9 was used for test item, solvent and negative control plates in the second experiments in all strains with except of E. coli
Volume of 100 µL (16.7% of S9 in S9 mix) of S9 is used for all plates in both experiments in E. coli. The reason is small number of induced revertants in positive control at lower volumes of S9.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Evaluation of Results
The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods. Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statistical evaluation of the results is not necessary.

Acceptability of Results
Minimum two experiments will be performed in each indicator strain. Only experiments complying with validity requirements will be involved in evaluation. When validity criteria are not met than concerned experiment was repeated.
The following conditions shoud be fulfilled:
1. Every strain should have appropriate properties (uvr mutation, rfa mutation, plasmids),
2. Average value of negative control (spontaneous reversion) should fall to historical limits of historical average negative control (spontaneous reversion),
3. Every test should have at least 4 acceptable doses except of negative control (acceptable dose = 2 countable dishes at least, non toxic),
4. Average value of positive control should fall to historical limits of historical average positive control,
5. The test should have most 1 unacceptable dose.
Conclusions:
Under the above-described experimental design, the test item, Methylhydroquinone, was non mutagenic for all the used indicator strains with and without metabolic activation.
Executive summary:

The test item, Methylhydroquinone, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test item was dissolved in water for injection and assayed in concentratins given further, which were applied to plates in volume of 0.1 mL.

The first mutagenicity experiments were performed as plate incorporation test without and with metabolic activation using a supernatant of rat liver (S9; 30 μL or 100 μL per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 0.1, 0.3, 1.0, 3.0 and 10 mg per plate.

The result of first series of mutagenicity experiments were negative, but the test item was cytotoxic in the highest concentration in Salmonella typhimurium TA 98 only, so concentration in the second mutagenicity must have been increased to subtoxic concentrations in the other indicator strains.

As the test item was differentially cytotoxic for differrent indicator strains, concentrations in the other mutagenicity experiments were differrent for Salmonella typhimurium TA 98 (0.1-30 mg per plate ) Salmonella typhimurium TA 100 and E. coli (concentrations 10-300 mg per plate) and Salmonella typhimurium TA 1535 and TA 1537 (concentrations 10-300 mg per and 2.5-50 mg per plate).

Volume of S9 in the subsequent experiments was increased from 30 to 50 μL per plate.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

In the arrangement given above, the test item, Methylhydroquinone, was non-mutagenic for all the used bacterial strains.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.04.2018 - 10.05.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Adopted 29th July, 2016
Deviations:
yes
Remarks:
3 Deviations with no impact on the outcome of the study
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: peripheral blood lymphocytes
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
At first, the preliminary preparations of the test item were done. The test item was low soluble in RPMI medium as well as in DMSO. It was necessary to prepare the test item stock solution in concentration, in whitch the test item is soluble. RPMI medium is better for preparation of the stock solution, because a larger volume of test item solution can be added to the tube with cultivated blood. So for the testing the test item was solubled in RPMI.
In the 1st experiment 100 mg of the test item was dissolved in 2 mL of RPMI medium in volumetric flask. The starting stock solution (50 mg/mL) was diluted and concentration line (1.563, 3.125, 6.25, 12.5, 25 mg/mL) was prepared.
Then, 100 µL of each stock concentration was added to the tube with cultivated blood, so the final concentrations of the test item were 62.5, 125, 250, 500, 1000 and 2000 µg/mL - according OECD TG 487 paragraph 31. Fresh solutions of the test item were prepared before each experiment.
In the highest prepared concentration (final concentration 2000 g/mL) the pH was measured.
In the 2nd and 3rd experiment 31.25 mg of the test item was dissolved in 2 mL of RPMI medium in volumetric flask. The starting stock solution (15.6 mg/mL) was diluted in proportion 1:10 in RPMI medium to concentration 1.56 mg/mL and than concentration line (0.024, 0.049, 0.098, 0.195, 0.391, 0.781 mg/mL) was prepared in the 2nd experiment. In the 3rd experiment concentration 0.012 mg/mL was added.
Then, 50 µL of each stock concentration was added to the tube with cultivated blood, so the final concentrations of the test item were 0.488, 0.977, 1.953, 3.906, 7.813, 15.625 and 31.25 µg/mL - according OECD TG 487 paragraph 31. Fresh solutions of the test item were prepared before each experiment.
Untreated negative controls:
yes
Remarks:
untreated culture; in case of experiment with metabolic activation system – untreated culture with S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: colchicine
Details on test system and experimental conditions:
The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking females (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medical laboratory in the morning and transported into the test facility as soon as possible.
Evaluation criteria:
Criterion 1
Cell cycle length of lymphocytes from donors was evaluated every 6 months. Results of the experiment are archived by the laboratory. The lymphocyte`s cell cycle length was ca 16 hours.

Criterion 2
The cytotoxicity of the highest three concentrations selected for genotoxicity evaluation was not higher than 55±5%. Concurrently, at least 2000 binucleated cells were scored in each concentration of genotoxicity evaluation. The three highest test item concentrations were evaluated for genotoxicity.

Criterion 3
Results of negative and positive controls - specifically numbers of binucleated cells with micronuclei were in actual ranges of historical controls.
Statistics:
In evaluation of results the Two-fold increase rule is used for the main. In border-line cases, when decision is difficult, the statistical methods are used for the support. For increasing of number of cells with micronuclei comparison to the negative control is used Fisher´s exact test and for dependence of increasing number of cells with micronuclei on dose is used trend test.
Key result
Species / strain:
lymphocytes: peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The lymphocyte`s cell cycle length during the experiments was 16 hours. This is suitable to experimental design described above according to OECD TG.

pH of the medium RPMI 1640 without test item was 7.2 (Sigma Aldrich Lot. No. RNBG 4755). In the highest final concentration (2000 mg/mL) the pH was measured and it was 7.5.

This minor shift could not lead to artifactual positive results which do not reflect the genotoxicity of the test chemical.

 

Concentrations evaluated in analysis of cytotoxicity:

In the 1stexperiment (genotoxicity testwith and without metabolic activation – 3 hours),the concentrations 62.5,125, 250, 500, 1000 and 2000 mg/mL have been analysed for cytotoxic effect.

In the 2ndexperiment (genotoxicity test with and without metabolic activation – 3 hours),the concentrations 0.488, 0.977, 1.953, 3.906, 7.813, 15.625 and 31.25 mg/mL have been analysed for cytotoxic effect.

In the 3rdexperiment (genotoxicity test with and without metabolic activation – 23 hours),the concentrations 15.625 and 31.25 mg/mL have been analysed for cytotoxic effect.

 

Concentrations evaluated in analysis of genotoxic effect:

In the 1stexperiment (genotoxicity testwith and without metabolic activation – 3 hours) there were not analysable concentrations.

In the 2ndexperiment (genotoxicity test without metabolic activation – 3 hours), the concentrations7.813, 15.625 and 31.25 mg/mL have been analysed for genotoxic effect.

In the 3rdexperiment (genotoxicity test without metabolic activation – 23 hours), the concentrations7.813, 15.625 and 31.25 mg/mL have been analysed for genotoxic effect.

Conclusions:
In a micronucleus test according to OECD Test No. 487 the test item methylhydroquinone had no genotoxic effects in the human peripheral blood lymphocytes with as well as without metabolic activation.
Executive summary:

Genotoxicity testing according to OECD Test No. 487: In Vitro Mammalian Cell Micronucleus Test:

No genotoxicity effect was found in any concentration and any treatment (treatment with or without metabolic activation in short and prolonged time of exposure).

On the basis of the chapter 3.10 – Evaluation and interpretation of results (and OECD TG 487 recommendations), the test item:

·        did not exhibit a statistically significant increase (two-fold increase rule) compared with concurrently solvent negative control

·        did not show evidently the dependence ofincreasing number of cells with micronuclei on concentration (dose-response relationship)

·        results of test item from the 3rdexperiment (23h treatment without metabolic activation) show slightly increased number of binucleated cells with micronucleus relative to historical negative control data. The results of negative control from the 3rdexperiment (23h treatment without metabolic activation) was near the upper limit of historical negative control data. So increasing number of the binucleated cells with micronucleus was caused by increasing number of micronucleus in donor´s blood.

On the basis of informations above, the test item is considered as clearly negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo study, tumor initiation and tumor promotion were investigated using methylhydroquinone as test item in the pyloric mucosa of male F344 rats. Methylhydroquinone showed very weak tumor-promoting activities and some tumor-initiating activities in the pyloric mucosa of rat stomach.

Ornithine decarboxylase (ODC) and replicative DNA synthesis (RDS) were used as markers of tumor promotion and DNA single strand scission and unscheduled DNA synthesis (UDS) as markers of tumor initiation.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Unscheduled DNA synthesis via induction ornithin decarboxylase
Single strand scission determined by an alkaline elution method.
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male
Route of administration:
oral: gavage
Vehicle:
99% methylhydroquinone (no vehicle)
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
48 h
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
90 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
not given
Control animals:
yes
Positive control(s):
MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)
Tissues and cell types examined:
pyloric mucosa
Evaluation criteria:
Ornithine decarboxylase (ODC) and replicative DNA synthesis (RDS) as markers of tumor promotion,
DNA single strand scission and unscheduled DNA synthesis (UDS) as markers of tumor initiation.
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Remarks:
low effects at 200 mg/kg bw
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Methylhydroquinone showed very weak tumor-promoting activities and some tumor-initiating activities in the pyloric mucosa of rat stomach.
Executive summary:

In an in vivo study, tumor initiation and tumor promotion were investigated using methylhydroquinone as test item in the pyloric mucosa of male F344 rats. Methylhydroquinone showed very weak tumor-promoting activities and some tumor-initiating activities in the pyloric mucosa of rat stomach.

Ornithine decarboxylase (ODC) and replicative DNA synthesis (RDS) were used as markers of tumor promotion and DNA single strand scission and unscheduled DNA synthesis (UDS) as markers of tumor initiation.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification