Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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). 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.

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 in the presence and absence of a metabolic activation system was examined. 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.

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 in the absence and presence of S9-mix.

Resorcinol diacetate was positive (mutagenic) in the mouse lymphoma L5178Y TK test system under the experimental conditions.

According to ECHA REACH Annex VIII, “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.”

https://echa.europa.eu/documents/10162/21650280/oecd_test_guidelines_genotoxicity_en.pdf/56ab5788-0103-4716-8903-59ab0c942efe

Based on the results of the in vitro genotoxicity results (positve HPRT test) a Comet assay according to OECD guideline 489 is proposed.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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.

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:
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.

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:
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

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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

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

According to ECHA REACH Annex VIII, “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.”

Therefore a Comet assay according to OECD guideline 489 is proposed.

A classification for genotoxicity of Resorcinol diacetate is postphoned until the results of the Comet assay are available.