m-phenylene di(acetate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli  (WP2uvrA): negative with and without metabolic activation (OECD 471, GLP)

Genetic toxicity in vitro: Chromosome aberration (in vitro mammalian cell micronucleus test): human lymphcytes: negative with and without metabolic activation (OECD 487, GLP)

Genetic toxicity in vitro: Gene mutation (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene): L5178Y mouse lymphoma cells: positive with and without metabolic activation (OECD 490, GLP)

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Resorcinol diacetate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Resorcinol diacetate
Appearance: Brown liquid
Purity/Composition: 98.52% (GC)
Test item storage: At room temperature

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-mix

Test concentrations with justification for top dose:

Experiment 1:
0, 52, 164, 512, 1600, or 5000 µg/plate
Experiment 2:
0, 187, 426, 968, 2200, or 5000 µg/plate

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Resorcinol diacetate did not precipitate on the plates at this dose level.

Vehicle / solvent:

DMSO (dimethyl sulfoxide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The dose range finding study with the two tester strains TA100 and WP2uvrA, is part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of Resorcinol diacetate used in the subsequent mutation assay was 5000 μg/plate.

Evaluation criteria:

Acceptability of the assay
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Data evaluation and statistical procedures
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

S. typhimurium, other: Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: TA98 in the presence of S9-mix (moderate reduction of the revertant colonies at the highest tested concentration) and TA100 in the absence of S9-mix (slight reduction of the revertant colonies at the highest tested concentration)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Resorcinol diacetate did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusions:

Resorcinol diacetate was negative in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) with and without metabolic activation.

Executive summary:

Resorcinol diacetate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

The test item was a brown liquid with a purity of 98.52% (GC). The test item was dissolved in dimethyl sulfoxide.

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Resorcinol diacetate did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA98 in the presence of S9-mix at the highest tested concentration.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 187 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a biologically relevant decrease in the number of revertants, was only observed in tester strain TA100 in the absence of S9-mix at the highest tested concentration.

Resorcinol diacetate did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Resorcinol diacetate is negative (not mutagenic) in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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)

Principles of method if other than guideline:

In an OECD guideline 487 (in vitro mammalian cell micronucleus test) the effect of Resorcinol diacetate on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) was examined. The possible clastogenicity and aneugenicity of Resorcinol diacetate was tested in two independent experiments.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Resorcinol diacetate
Appearance: Brown liquid
Purity/Composition: 98.52% (GC)
Test item storage: At room temperature
Purity/composition
correction factor: No correction factor required
Test item handling
Chemical name (IUPAC),
synonym or trade name: 1,3-Diacetoxybenzene
CAS Number: 108-58-7
Molecular formula: C10H10O4
Molecular weight: 194.18

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without S9-mix : 50, 500, 750, 1000, 1250, 1500 and 1600 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 50, 500, 1000, 1200, 1400, 1600 and 1800 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
Both in the absence and presence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 1250 and 1200 μg/ml not enough cytotoxicity was observed (39 and 34%), whereas the next higher concentration of 1500 and 1400 μg/ml was too toxic for scoring (97 and 80%), respectively.
The experiment was repeated in cytogenetic assay 1A.
Without and with S9-mix: 500, 1000, 1250,1300, 1350, 1400 and 1450 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).
The following dose levels were selected for scoring of micronuclei:
Without and with S9-mix: 500, 1000 and 1350 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).

Second cytogenetic assay:
To obtain more information about the possible clastogenicity and aneugenicity of Resorcinol diacetate, a second cytogenetic assay was performed in which human lymphocytes were exposed for
24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 50, 150, 200, 300, 400, 500 and 600 μg Resorcinol diacetate/ml culture medium
(24 hours exposure time, 24 hours harvest time).
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix : 50, 200 and 300 μg Resorcinol diacetate/ml culture medium
(24 hours exposure time, 24 hours harvest time).

Vehicle / solvent:

Test item preparation
No correction was made for the purity/composition of the test item.
A solubility test was performed. Resorcinol diacetate was dissolved in dimethyl sulfoxide of spectroscopic quality. Resorcinol diacetate concentrations were used within 0.5 hour after preparation.
The final concentration of the solvent in the culture medium was 1.0% (v/v). The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine, Cyclophosphamide

Details on test system and experimental conditions:

Study design
Dose range finding test
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. Resorcinol diacetate was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Resorcinol diacetate for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.

The highest tested concentration was 1942 μg/ml (= 0.01 M).

After 3 hours exposure to Resorcinol diacetate in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.

Cytotoxicity of Resorcinol diacetate in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Resorcinol diacetate for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasin B (5 μg/ml) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay. To be able to select appropriate dose levels for scoring of micronuclei a repeat assay had to be performed.

Second cytogenetic assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Resorcinol diacetate with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of slides
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol : acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene pertex and mounted with a coverslip in an automated coverslipper.

Cytotoxicity assessment
A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] : Total number of cells

t = test item or control treatment culture
c = vehicle control culture
Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

Cytogenetic assessment/scoring of micronuclei
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with WIL Research Europe study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Rationale for test conditions:

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Evaluation criteria:

Data evaluation and statistical procedures
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Resorcinol diacetate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Conclusions:

Resorcinol diacetate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.
The in vitro micronucleus asssay with Resorcinol diacetate in cultured peripheral human lymphocytes is negative.

Executive summary:

The effect of Resorcinol diacetate on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) was examined. The possible clastogenicity and aneugenicity of Resorcinol diacetate was tested in two independent experiments.

The test item Resorcinol diacetate was a brown liquid with a purity of 98.52% (GC). Resorcinol diacetate was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, Resorcinol diacetate was tested up to 1350 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, Resorcinol diacetate was tested up to 300 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Resorcinol diacetate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

Finally, it is concluded that this test is valid and that Resorcinol diacetate is negative (not clastogenic or aneugenic) in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Principles of method if other than guideline:

The effects of Resorcinol diacetate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells were examined. The test was performed in the absence and presence of S9-mix with a 3 hour treatment period (rat liver S9-mix).

GLP compliance:

yes

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene

Specific details on test material used for the study:

Identification: Resorcinol diacetate
Appearance: Brown liquid
Purity/Composition: 98.52% (GC)
Test item storage: At room temperature
Purity/composition
correction factor: No correction factor required
Chemical name (IUPAC),
synonym or trade name: 1,3-Diacetoxybenzene
CAS Number: 108-58-7
Molecular formula: C10H10O4
Molecular weight: 194.18

Target gene:

thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 10E6 cells (10E6 cells/ml for 3 hours treatment) or 6 x 10E6 cells (1.25 x 105 cells/ml for 24 hours treatment) with a number of test item concentrations increasing by approximately half log steps.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 50, 500, 750, 900, 1000, 1100, 1300 and 1500μg/ml exposure medium.
With S9-mix: 500, 600, 750, 900, 1000, 1100, 1300 and 1500 μg/ml exposure medium.

Vehicle / solvent:

The test item was dissolved in dimethyl sulfoxide. Resorcinol diacetate concentrations were used within 1 hour of preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Mutagenicity test
Eight doses of Resorcinol diacetate were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3-hour treatment period and in the absence of S9-mix with a 3-hour treatment period.
The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.

Treatment of the cells
Resorcinol diacetate was tested both in the absence and presence of S9-mix. Per culture 8 x 10E6 cells (10E6/ml) were used.
Cell cultures were exposed for 3 hours to Resorcinol diacetate in exposure medium in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0 °C and 145 spm. Solvent and positive controls were included and the solvent control was tested in duplicate.
After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 5 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.

Expression period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the mutation frequency
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10E5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

Rationale for test conditions:

L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes. The TK mutational system is able to detect base pair alterations, frame shift mutations and small deletions and clastogenic effect.

Statistics:

Data evaluation and statistical procedures
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if: no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9-mix, Resorcinol diacetate induced an up to 2.7-fold dose related increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database.
In the presence of S9-mix, Resorcinol diacetate induced an up to 7.3-fold dose related increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database.
Although the relative total growth at the highest tested concentration was too low according to the guideline (severe toxicity was observed), the increase in the mutation frequency was dose related and therefore considered biologically relevant.

Conclusions:

It is concluded that Resorcinol diacetate is positive (mutagenic) in the mouse lymphoma L5178Y TK test system under the experimental conditions described.

Executive summary:

The effects of Resorcinol diacetate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells were examined. The test was performed in the absence and presence of S9-mix with a 3 hour treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The test item of Resorcinol diacetate was a brown liquid with a purity of 98.52% (GC). The test item was dissolved in dimethyl sulfoxide.

In the first experiment, Resorcinol diacetate was tested up to a concentration of 1500 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. The relative total growth (RTG) was 27 and 5% in the absence and presence of S9-mix, respectively.

The mean spontaneous mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Resorcinol diacetate induced an up to 2.7-fold dose related increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (248 per 106 survivors).

In the presence of S9-mix, Resorcinol diacetate induced an up to 7.3-fold dose related increase in the mutation frequency. The increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (207 per 106 survivors).

Although the relative total growth at the highest tested concentration was too low according to the guideline (severe toxicity was observed), the increase in the mutation frequency was dose related and therefore considered biologically relevant.

It is concluded that Resorcinol diacetate is positive (mutagenic) in the mouse lymphoma L5178Y TK test system under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo: Gene mutation (in vitro comet assay): negative in all tissues (glandular stomach, duodenum, liver) (OECD 489, GLP)

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

Study period:

31 Jan 2022 - 28 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Physical Description: Clear pale yellow liquid
Storage Conditions: At room temperature protected from light

Species:

rat

Strain:

Wistar

Remarks:

Han

Details on species / strain selection:

The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-10 weeks
- Body weight: The body weights of the rats at the start of the treatment were within 20% of the sex mean (295.3 - 310.0 g)
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before last dosing (approximately 7 g/rat).
- Housing: Polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together. Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in, pater and/or objects for chewing, except when interrupted by study procedures/activities
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH pellets ad libitum, except during designated procedures
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

Following analyses, it was considered that there were no known contaminants in feed, water and enrichment that could have interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C (target), 21 to 22 °C (actual)
- Humidity: 40 to 70% (target), 45 to 57% (actual)
- Air changes: 10 or more air changes per hour
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 31 Jan 2022 To: 07 April 2022

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF THE DOSING SOLUTIONS:
No correction was made for the purity/composition of the test material. An adjustment was made for specific density of the test material (factor of 1.1758).
Depending on the concentration, the test material was dissolved or suspended in corn oil. The specific gravity of corn oil is 0.92 g/mL. Test material concentrations were vortexed, treated with ultra-sonic waves and stirred to obtain a homogeneous suspension or until the test material was completely dissolved.
This resulted in a yellow translucent suspension for the formulations with a concentration of 200 mg/mL and yellow solutions for the formulations with a concentration of 50 and 100 mg/mL
Test material concentrations were dosed within 3.5 hours after preparation. Any residual volumes were discarded.
The first day of dosing was designated as Day 1. The doses were given using a plastic feeding tube. The dosing volume was 10 mL/kg body weight.

DOSE-RANGE FINDING STUDY:
Selection of an adequate dose-range for the Comet main test was based on a dose-range finding study. The test procedure and conditions were similar to those applied in the main test.
In the dose-range finding study, one dose-group was used to define the MTD (Maximum Tolerated Dose) based on the toxic signs observed after dosing with different doses of the test material.
One dose group, comprising of 3 males and 3 females, was dosed for two consecutive days (once daily) with the highest concentration of test material that was used for the main study. The observation period after dosing was one to three days. During this period mortality and physical condition were recorded at least once a day.
Based on the results of the dose-range finding test, there were no substantial differences in toxicity between sexes, and as such only male animals were used in the main study. Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg bw/day were selected as appropriate doses for the main test.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

Once daily

Post exposure period:

3-4 hours after the last dose of the treatment with the test material, vehicle (negative control) or EMS (positive control), the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and, the liver, duodenum, and glandular stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

- Main study, group 4 (2000 mg/kg bw/day): 8
- Main study, group 5 (positive control): 3
- Main study, remaining treatment groups: 5
- TK study (proof of exposure): 3

Control animals:

yes, concurrent vehicle

Positive control(s):

ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline (Group 5).

Tissues and cell types examined:

liver, duodenum and glandular stomach

Details of tissue and slide preparation:

ISOLATION OF CELLS

Liver
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase dissolved in HBSS (Ca2+ - and Mg2+-free) and incubated in a shaking water bath at 37°C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells
The isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca2+, Mg2+ free). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.
 
Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study.
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodenum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded.
The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca2+, Mg2+ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

PREPARATION OF SLIDES
To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15 to 32 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.


LYSIS, ELECTROPHORESIS AND STAINING OF THE SLIDES
The cells on the slides were overnight (approximately 17-18h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator (at 4°C). After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volts/cm. The electrophoresis was performed for 20 (stomach, duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolute ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.
 
SAMPLING, FIXATION AND STORAGE OF TISSUE FOR HISTOTECHNOLOGY AND HISTOPATOLOGY
Part of the liver, glandular stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was ultimately required.

COMET SCORING
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code was placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control.


ACCEPTABILITY CRITERIA
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg bw/day

Evaluation criteria:

A test material is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is dose-related increase when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Statistics:

ToxRat Professional v 3.3.0 was used for statistical analysis of the comet assay data.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals.

Toxicity:

yes

Remarks:

Clinical observations were made in the groups treated with 1000 and 2000 mg / kg bw (tremors and lethargy). Three animals in the groups treated with 2000 mg / kg bw died on day 1 postdose.

Vehicle controls validity:

valid

Remarks:

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats within the 95% control limits of the distribution of the historical control data for the vehicle control

Positive controls validity:

valid

Remarks:

The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database

Additional information on results:

DOSE-RANGE FINDING STUDY
In the dose-range finding test, male and female animals dosed with 2000 mg test material per kilogram body weight showed treatment related clinical signs (convulsions, pinched eyes, head shaking and shivering).
The Maximum Tolerated Dose (MTD; described as the dose that will not kill the animals but will provoke signs of toxicity) was confirmed to be 2000 mg/kg bw.

MORTALITY AND TOXIC SIGNS
The animals of the groups treated with 500 mg /kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Clinical observations were made in the groups treated with 1000 and 2000 mg / kg bw (tremors and lethargy). Three animals in the groups treated with 2000 mg test material/ kg bodyweight died on day 1 postdose.

COMET SLIDE ANALYSIS
Comet slides were prepared and analyzed. No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. In addition there were no Hedgehogs observed in vehicle and test material treated groups. The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.86 ± 0.90% (mean ± SD), 4.20 ± 0.45% (mean ± SD) and 6.97 ± 2.15% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Table 25). The positive control (EMS) induced a significant increase and showed a mean Tail Intensity of 65.27 ± 2.96 (mean ± SD, p<0.001 Students t test), 35.24 ± 5.33% (mean ± SD, p<0.001 Students t test) and 51.19 ± 2.13% (mean ± SD; p<0.001 Students t test) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

BIOANALYSIS
Blood was sampled 1, 2, 4, 6 and 24 h after the second dose of satellite animals dosed with the vehicle and the highest concentration of the test material. Substance A was analyzed as indirect proof of exposure for the test substance, since the test substance was unstable in plasma. Exposure was demonstrated in plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples.

Dose Formulation Analysis

Accuracy

The concentrations analyzed in the formulations were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). No test material was detected in the vehicle.

Homogeneity

The formulations of the lowest and highest dose group were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	2.86	0.90
Test Material 500 mg/kg bw	2.74	0.80
Test Material 1000 mg/kg bw	2.21	0.32
Test Material 2000 mg/kg bw	2.06	0.41
EMS 200 mg/kg bw	65.27	2.96

 

Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.20	0.45
Test Material 500 mg/kg bw	3.70	0.39
Test Material 1000 mg/kg bw	3.86	0.90
Test Material 2000 mg/kg bw	5.42	1.60
EMS 200 mg/kg bw	35.24	5.33

       
Overview Tail Intensity in Glandular Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	6.97	2.15
Test Material 500 mg/kg bw	5.23	1.76
Test Material 1000 mg/kg bw	5.31	2.06
Test Material 2000 mg/kg bw	4.49	1.88
EMS 200 mg/kg bw	51.19	2.13

 

Historical Negative Control Data for Comet Assay

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	6.7	2.5	5.9
SD	3.2	1.4	3.4
n	26	41	30
Lower control limit (95% control limits)	0.4	-0.1	-0.7
Upper control limit (95% control limits)	13.1	5.2	12.5

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in November 2018 - November 2021

 
Historical Positive Control Data for Comet Assay

	Duodenum Tail Intensity (%) Males and Females	Liver Tail Intensity (%) Males and Females	Glandular Stomach Tail Intensity (%) Males and Females
Mean	51.1	84.2	57.9
SD	10.0	6.3	9.7
n	26	42	30
Lower control limit (95% control limits)	31.4	71.9	38.9
Upper control limit (95% control limits)	70.7	96.5	76.8

SD = Standard deviation

n = Number of observations

 

Historical control data from experiments performed in November 2018 - November 2021

 

 

Conclusions:

The comet assay is valid and the test substance is not genotoxic in the Comet assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing of male rats, exposed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw/day (the maximum tolerated dose).

Executive summary:

A comet assay was performed in male rats according to OECD guideline 489 and in accordance with GLP principles. 

 

The objective of this study was to obtain information on the potential genotoxicity of  the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Study design

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The test material was a clear pale yellow liquid. The test material was dissolved or suspended in corn oil, depending on the concentration.

 

No test material was detected in the vehicle. The concentrations analyzed in the formulations were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). The formulations of the lowest and highest dose group were homogeneous (i.e. coefficient of variation ≤ 10%).

 

Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg bw/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes in a dose range finding study only males were used in the main study.

 

In the main study male animals were dosed with vehicle (corn oil), test material (at 500, 1000 and 2000 mg/kg body weight/day) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.

 

Clinical signs of toxicity were limited to the middle and high dose group and included tremors and lethargy.

 

In addition, blood for bioanalysis of the test substance in plasma was collected from satellite animals for the 2000 mg/kg bw/day group (highest dose group) and from satellite animals for the vehicle control group. Substance A was analyzed as indirect proof of exposure for the test substance, since the test substance was unstable in plasma.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

 

Results

No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals.

 

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.86 ± 0.90% (mean ± SD), 4.20 ± 0.45% (mean ± SD) and 6.97 ± 2.15%
(mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 65.27 ± 2.96%
(mean ± SD), 35.24 ± 5.33% (mean ± SD) and 51.19 ± 2.13% (mean ± SD) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Exposure was demonstrated in plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples. Hence, all criteria for an acceptable assay were met.

 

Conclusion

In conclusion, the test is valid and the test material is not genotoxic in the Comet assay, as shown in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions used in this study.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6.1/1 : Summary of genotoxicity data:

Test n°	Test Guideline / Reliability	Focus	Strains / cells tested	Metabolic activation	Test concentration	Statement
1 (2016)	Ames Test (OECD 471, GLP) Key, rel.1	Gene mutation (in vitro)	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100 E.Coli WP2uvrA	-S9 +S9	Tested up to 5,000 µg/plate	-S9: not mutagenic + S9: not mutagenic
2 (2016)	MNT (OECD 487, GLP) Key, rel. 1	Cytogenicity (in vitro)	Human lymphocytes	-S9 +S9	Tested up to 1,942 µg/ml	-S9: not mutagenic + S9: not mutagenic
3 (2016)	Gene mutation (TK) assay (OECD 490) Key, rel. 1	Gene mutation (in vitro)	L5178Y/TK+/--3.7.2C mouse lymphoma cells.	-S9 +S9	Tested up to 1,500 µg/ml	-S9: not mutagenic + S9: not mutagenic
4 (2022)	Gene mutation (Comet) assay (OECD 489) Key, rel.1	Gene mutation (in vivo)	Wistar-Han (male): liver, stomach and duodenum cells	N/A	Tested up to 2,000 mg/kg bw (2x daily exposures)	Not genotoxic

 

Gene mutation Assay (Test n° 1)

In an OECD guideline 471 study (Bacterial Reverse Mutation Assay) Resorcinol diacetate was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). 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. Based on the results of this study it is concluded that Resorcinol diacetate is negative (not mutagenic) in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Chromosome aberration Assay (Test n° 2)

In an OECD guideline 487 study (in vitro mammalian cell micronucleus test) the effect of Resorcinol diacetate on the number of micronuclei formed in cultured peripheral human lymphocytes at concentrations of up to 1,942 µg/ml in the presence and absence of a metabolic activation system was examined (3h exposure time ±S9). The possible clastogenicity and aneugenicity of Resorcinol diacetate was tested in two independent experiments.

Resorcinol diacetate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. It is concluded that Resorcinol diacetate is negative (not clastogenic or aneugenic) in human lymphocytes. 

 

Gene mutation assay in mammalian cells (Test n° 3)

In an OECD guideline 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) the effects of Resorcinol diacetate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells were examined. The test was performed at concentrations of up to 1,500 µg/ml in the absence and presence of S9-mix.

In both the presence and absence of S9, Resorcinol diacetate induced an increase in mutation frequency compared to background which was out with the 96% confidence interval of the historical control database. It was concluded that Resorcinol diacetate is positive (mutagenic) in the mouse lymphoma L5178Y TK test system under the experimental conditions.

 

Gene mutation assay in vivo (Test n° 4)

In an OECD 489 study (In Vivo Mammalian Alkaline Comet Assay), the effects of Resorcinol diacetate on the induction of DNA strand breaks in cells isolated from multiple tissues (glandular stomach, duodenum and liver) of male Wistar-Han rats was investigated. Male animals were dosed with vehicle (corn oil), test material (at 500, 1000 and 2000 mg/kg bw/day – limit dose) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.

Clinical signs of toxicity were limited to the middle and high dose group and included tremors and lethargy. No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. The mean positive and vehicle control tail intensities were within the 95% control limits of the distribution of the historical control databases. Adequate numbers of cells and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Exposure was demonstrated in plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples. Hence, all criteria for an acceptable assay were met.

In conclusion, Resorcinol diacetate is not genotoxic in the Comet assay, as shown in liver, duodenum and glandular stomach cells from male rats dosed via oral gavage for two consecutive days up to the limit dose under the experimental conditions.

 

Conclusion

Four (Annex VII - IX) genetic toxicity studies are available (three in vitro studies, and one in vivo study) of sufficient quality and reliability to assess the respective endpoints. All studies are on the registered substance itself. The gene mutation in bacteria and micronucleus induction in human lymphocytes consistently report that Resorcinol diacetate is negative for genetic toxicity. The in vitro gene mutation study in mammalian cells (TK) reported a positive result.

 

According to ECHA REACH Annex VIII, as “Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII”, a follow-up in vivo alkaline comet assay (OECD 489) was conducted (following ECHA approval of a Testing Proposal). The results of this study were negative in all cell types evaluated.

 

It is concluded that Resorcinol diacetate does not meet the criteria for classification as a mutagen, in accordance with Regulation (EC) No 1272/2008 (CLP).

Justification for classification or non-classification

Four (Annex VII - IX) genetic toxicity studies are available (three in vitro studies, and one in vivo study) of sufficient quality and reliability to assess the respective endpoints.

 

The available Ames test according to OECD 471 (Bacterial Reverse Mutation Assay) and the MNT according to OECD 487 (in vitro mammalian cell micronucleus test) with Resorcinol diacetate were negative.

 

The available HPRT test according to OECD 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) with Resorcinol diacetate was positive.

 

A follow-up in vivo alkaline comet assay (OECD 489) was negative in all cell types evaluated.

 

It is concluded that Resorcinol diacetate does not meet the criteria for classification as a mutagen, in accordance with Regulation (EC) No 1272/2008 (CLP).