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Genetic toxicity in vitro

Description of key information

In vitro genotoxicity studies performed on chlorendic anhydride returned negative results. Only an ability to interact with DNA was identified, but leading to reparable lesions.

When chlorendic acid was tested, positve results were obtained during the in vitro micronucleus study with and without metabolic activation and the in vitro gene mutation studies in mammalian cells without metabolic activation. In vitro gene mutation study in bacteria and in vitro gene mutation in mammalian cells with metabolic activation returned negative results, as well as the studies using alternative in vitro genotoxicity methods.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically undertaken and reported study but not to GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
Saccharomyces cerevisiae
Additional strain / cell type characteristics:
other: Strain: D4
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0.1, 1.0, 10, 100 and 500 µg / plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; deionised water- Justification for choice of solvent/vehicle:
Untreated negative controls:
yes
Remarks:
Methylnitrosoguanidine
Negative solvent / vehicle controls:
yes
Untreated negative controls:
yes
Remarks:
2-Nitrofluorene
Negative solvent / vehicle controls:
yes
Untreated negative controls:
yes
Remarks:
Quinacrine mustard
Negative solvent / vehicle controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 8-Aminoquinoline
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); DURATION- Preincubation period: - Exposure duration: - Expression time (cells in growth medium): Overnight- Selection time (if incubation with a selection agent): - Fixation time (start of exposure up to fixation or harvest of cells): 48 hoursSELECTION AGENT (mutation assays): SPINDLE INHIBITOR (cytogenetic assays): STAIN (for cytogenetic assays): NUMBER OF REPLICATIONS: 1NUMBER OF CELLS EVALUATED:DETERMINATION OF CYTOTOXICITY - Method: mitotic index; cloning efficiency; relative total growth; other:OTHER EXAMINATIONS: - Determination of polyploidy: - Determination of endoreplication: - Other: OTHER:
Evaluation criteria:
Evaluation Criteria for Ames AssayMost data sets are evaluated using the following criteria:1. Strains TA-1535, TA-1537, and TA-1538If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic. 2. Strains TA-98, TA-100, and D4If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and two to three times the solvent control value for strains TA-98 and D4 is considered to be mutagenic. For these strains, the dose response increase should start at approximately the solvent control value.3. PatternBecause TA-1535 and TA-100 were both derived from the same parental strain (G-46) and because TA-1538 and TA-98 were both derived from the same parental strain (D3052), there is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA-1537, responds to amutagen in nonactivation tests it will generally do so in activation tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens, they can be used to enhance the reliability of an evaluation decision.4. ReproducibilityIf a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data loses significance.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Overlay plates
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationChlorendic anhydride did not demonstrate mutagenic activity under the conditions of this study.
Executive summary:

The test substance was examined for mutagenic activity in a series of in vitro microbial assays emplyoying salmonella and saccharomyces indicator organisms. The compound was tested directly and i nthe presence of liver microsomal enzyme preparation from Aroclor-induced rats. The following results were obtained:

Toxicity test results

The compound was tested over a series of concentrations such that there was either a quantitative or qualitative evidence of some chmically-induced physiological effects at the high dose level. The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose range employed for the evaluation of this compound was from 0.1 ug to 500ug per plate.

Nonactivation test results

The results of the tests conducted on the compound in the absence of a metabolic system were all negative

Activation test results

The results of the tests conducted on the compound in the presence of the rat liver activation system were all negative

Conclusions

The test compound, chlorendic anhydride, did not demonstrate mutagenic activity in any of the assays conducted in this evaluation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only tested on 4 out of 5 recommended bacteria strains
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored as recommended by the chemical repository.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Each chemical was dissolved and diluted immediately prior to testing. The first choice of solvent was distilled water; dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water. Ethanol (95%) or acetone was used if the chemical was not soluble or stable in DMSO.
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. Bruce Ames, University of California, Berkley
- Methods for maintenance in cell culture: Cultures of each tester strain were prepared for storage essentially as described in the Supplement to the Methods Paper [Ames et al, 1975] supplied with the tester strains. Frozen cultures stored in liquid nitrogen or in a -70 °C freezer in sterile, screw-cap vials. All overnight cultures (late log phase) were obtained by incubation at 37 °C on a shaker for 12-15 hours and were routinely checked for genetic integrity.

MEDIA USED
- Type and identity of media: Columbia agar slants; Oxoid Nutrient Broth #2 (CM 67); minimal glucose medium supplemented with biotin and an excess of histidine.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 mix from male Sprague-Dawley rats and male Syrian hamsters
Test concentrations with justification for top dose:
A preliminary toxicity determination was undertaken in order to select the dose range for the mutagenesis assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solvent of choice was distilled water; DMSO was used if the chemical was insoluble or not sufficiently soluble in water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene (Tested on all strains in presence of S-9); 4-nitro-o-phenylenediamine (Tested on TA98 without S-9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: The test mixture was vortexed and allowed to incubate for 20 min at 37 °C.
- Expression time (cells in growth medium): The plates were inverted and incubated at 37°C for 48h.

NUMBER OF REPLICATIONS: At least 5 doses of each test chemical were tested on each strain in the presence of S-9 mix or buffer. Three plates were used and the experiment was repeated no less than 1 week after completion of the initial test.
Rationale for test conditions:
The preincubation procedure was selected because of evidences that it was no less sensitive than the plate test while being more effective for various chemicals.
Evaluation criteria:
Data were evaluated by the testing laboratories and NTP personnel. A "positive" response was recorded when a reproducible, dose-related increase was observed. The "inconclusive" response was applied to non-reproducible, low-level responses or to results where the investigator was not certain in making a definitive "positive" or "negative" decision.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Chlorendic acid was considered to be non-mutagenic under the conditions of the test with and without metabolic activation.
Executive summary:

The in vitro genotoxicity in bacteria of the test substance was determined using a preincubation procedure, which was a modification of the Salmonella/mammalian microsome test of Ames et al [1975] and similar to OECD Guideline for Testing of Chemicals 471 (non GLP) with deviations.

At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each bacteria strain in the presence of S-9 mix or buffer. A positive response was indicated by a reproducible, dose-related increase. Controls were valid.

Chlorendic acid was considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 28 September 2015 to 14 January 2016
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)
Deviations:
no
Remarks:
The pulse treatment group with S9-mix had to be repeated twice due to a technical error and a notable variation in cytotoxicity between the replicates
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: three human donors
- Sex, age and number of blood donors if applicable: healthy, non-smoking individuals (36, 34 and 32 years old) with no known recent exposures to genotoxic chemicals or radiation
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium (with HEPES and Glutamax), supplemented with heat inactivated (30 min, 56ºC) foetal calf serum (20%), penicillin (100 U/ml medium), streptomycin (100 μg/ml medium) and phytohaemagglutinin (2.4 μg/ml)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
2000, 1750, 1500, 1250, 1000, 500, 250, 125 and 62.5 μg/ml. 2000 ug/ml was selected as the highest dose according to OECD TG 487 as no precipitate or limiting cytotoxicity was observed.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Observed to be a suitable solvent for the test substance
Untreated negative controls:
yes
Remarks:
Vehicle
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Incubation period: 48h
- Exposure duration: 4h
- Recovery period: 20h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): acridin-orange

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED: 1000 cells per dose level for the CBPI, 2000 binucleated cells per dose level for the presence of micronuclei

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
1. The diameter of micronuclei usually varies between 1/16 and 1/3 of the diameter of the main nuclei.
2. Micronuclei are round or oval in shape.
3. Micronuclei are nonrefractile and can therefore be readily distinguished from artefacts such as staining particles.
4. Micronuclei are not linked or connected to the main nuclei.
5. Micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary.
6. Micronuclei usually have the same staining intensity as the main nuclei but occasionally staining is more intense.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)
Rationale for test conditions:
Pulse treatment with and without S9-mix was performed according to OECD TG 487. As it allowed to obtain a conclusive result, extended treatment was not considered.
Evaluation criteria:
A response was considered positive if all of the following criteria were met:
- at least one of the test concentrations exhibits a statistically significant increase compared to the concurrent solvent control.
- the increase is dose-related in at least in one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data.

A response was considered negative if all of the following criteria were met:
- none of the test concentrations exhibits a statistically significant increase compared to the concurrent solvent control.
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical solvent control data.

A test substance was considered equivocal if the response was neither positive or negative even after further investigation.
Statistics:
Chi-square test. The results were considered statistically significant when the p-value of the Chi-square test was less than 0.05.
Key result
Species / strain:
lymphocytes: peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: slight cytotoxicity observed in the group without S9-mix at the highest dose when compared to the concurrent solvent control
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: lower pH was observed with the highest concentrations but stayed within acceptable range (see attached background documents)
- Effects of osmolality: lower osmolality was observed with the highest concentration but stayed within acceptable range (see attached background documents)

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see attached background documents

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see attached background documents
- Indication whether binucleate or mononucleate where appropriate: see attached background documents

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached background documents
- Negative (solvent/vehicle) historical control data: see attached background documents

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

see attached background documents

Conclusions:
The test substance induced a dose related statistically significant increase in the number of binucleated cells containing micronuclei in the pulse treatment with and without addition of S9-mix. In addition, the number of binucleated cells containing micronuclei in the highest concentration was outside of the historical data range with and without addition of S9-mix. The substance was considered as clastogenic and/or aneugenic under the conditions of the test.
Executive summary:

The potential of chlorendic acid to induce structural chromosomal aberration in vitro was determined in accordance with OECD Guideline for Testing of Chemicals 487. A pulse treatment was performed with and without addition of S9-mix at doses up to 2000 µg/mL. The highest dose was selected as no precipitate or limiting cytotoxicity was observed. Peripheral blood lymphocytes were obtained from three different donors for the purpose of this study. Dimethylsufoxide (DMSO) was used as a solvent for the test substance. Duplicate cultures were used in all experiments. Cytotoxicity was determined from the Cytokinesis-Block Proliferation Index (CBPI). Cells were incubated for 48h with phytohaemagglutinin then treated for 4h with chlorendic acid with and without addition S9-mix. This treatment was followed with a 20h recovery in presence of Cytochalasin B then cells were harvested 72h after initiation of the cultures and prepared for microscopic analysis. Two thousand binucleated cells per concentration were examined for the presence of micronuclei. The frequencies of binucleated cells with micronuclei were used for the evaluation of micronuclei induction.

It was required to repeat the pulse treatment with S9-mix twice due to a technical error and a notable variation in cytotoxicity between the replicates.

Chlorendic acid induced a dose related statistically significant increase in the number of binucleated cells containing micronuclei in the pulse treatment with and without addition of S9-mix. In addition, the number of binucleated cells containing micronuclei in the highest concentration was outside of the historical data range with and without addition of S9-mix. The substance was considered as clastogenic and/or aneugenic under the conditions of the test.

As the pulse treatment with and without S9-mix was sufficient to obtain a clear positive result in accordance with OECD Guideline for Testing of Chemicals 487, no extended treatment was performed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
performed only without metabolic activation
GLP compliance:
no
Type of assay:
other: in vitro mammalian cell gene mutation
Specific details on test material used for the study:
no data
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no data
Metabolic activation:
without
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
1,300, 1,400, 1,500, 1,600 and 1,7000 µg/mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 6x10^5 cells/ml

DURATION
- Preincubation period: not specified
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): not specified

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate (except for the solvent tested in triplicate)

NUMBER OF CELLS EVALUATED: 600

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Rationale for test conditions:
not specified
Evaluation criteria:
Mutation frequency
Statistics:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not specified
- Definition of acceptable cells for analysis: not specified
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: not specified


Remarks on result:
other: not performed with metabolic activation
Conclusions:
There was a significant increase in mutation frequency over control counts at the highest dose tested in L5178Y mouse lymphoma cells without metabolic activation. The substance was considered as mutagenic under the conditions of the test.
Executive summary:

The capacity of chlorendic acid to induce gene mutation in mammalian cells was determined according to the method described by Clive et al. (1979) and therefore similar to OECD Testing Guideline 476 (non GLP) with deviations. L5178Y mouse lymphoma cells were tested without metabolic activation at doses ranging from 1,300 to 1,700 µg/ml. Methylmethanesulfonate was selected for the positive control.

Cells were treated for 4 hours at 37°C in medium, then washed, resuspended in medium, and incubated for 48 hours at 37°C. After expression, 3 X 10^6 cells were plated in medium supplemented with TFT for selection of cells, and 600 cells were plated in nonselective medium to determine the percentage of viable cells. Positive and solvent control were valid.

There was a significant increase in mutation frequency over control counts at the highest dose tested in L5178Y mouse lymphoma cells without metabolic activation. The substance was considered as mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
no
Type of assay:
other: mouse lymphoma forward mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
National Toxicology Program Chemical Repository, Radian Corporation (Austin, TX)
Target gene:
thymidine kinase locus (tk)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Burroughs Wellcome Co. (Research Triangle Park, NC)
- Storage: liquid nitrogen
- Methods for maintenance in cell culture: Cultures were confirmed as free from mycoplasma by cultivating or Hoechst staining techniques, and maintained in Fischer’s medium at 37°C on gyratory tables. Cultures were purged of tk+ / tk- mutants by exposure for 1 day to F10p containing THMG (thymidine, 6 pg/ml; hypoxanthine, 5 pg/ml; glycine, 7.5 pg/ml; and methotrexate, 0.1 pg/ml), then for 3 days to F10p containing THG only.

MEDIA USED
- Type and identity of media: Fischer's medium (supplemented with 2 mM L-glutamine, sodium pyruvate (110 pg/ml), 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v))
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix
Test concentrations with justification for top dose:
In the toxicity test, ten-fold differences in test compound concentrations were used, up to a maximum concentration of 5 mg/ml, unless poor solubility of the compound indicated a much lower concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium without serum; distilled water; methanol (without S9 mix only); dimethyl sulphoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density: Each exposed culture consisted of 6 x 10^6 cells

DURATION
- Preincubation period: Tubes incubated for 4 hrs on a horizontal axis roller drum rotating at 10 rpm.
- Exposure duration: Cultures were exposed to the chemicals for 4 hrs, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 ug/ml.
- Expression time (cells in growth medium): 2 days
- Selection time: Agar was solidified at 4°C for 5-10 min, then the plates were incubated for 11-14 days in 5% CO2:95% air at 37°C.

SELECTION AGENT: 3 ug trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Tested at least twice.

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth; cloning efficiency
Evaluation criteria:
Cloning efficiency, relative total growth, mutant colony count, and mutant fraction were recorded.
Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period, or a reduction in cloning efficiency.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
in two of four experiments
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity occurred at the higher doses preventing to determine the genotoxicity at these doses
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Significant mutagenic responses were obtained in two of four of these experiments (trials 2 and 4). The compound only induces genotoxicity at a narrow range of concentrations as it is too cytotoxic at higher concentrations.
Conclusions:
There was a significant increase in mutation frequency over control counts in L5178Y mouse lymphoma cells during two of the four experiment without metabolic activation. This increase only occurred in a narrow range of concentrations as chlorendic acid was too cytotoxic at higher concentrations. No significant response was obtained with metabolic activation. The substance was considered as mutagenic under the conditions of the test.
Executive summary:

The capacity of chlorendic acid to induce gene mutation in mammalian cells was determined according to the method described by Clive et al. (1979) and therefore similar to OECD Testing Guideline 476 (non GLP).

Cultures were exposed to the test substance for 4 hrs, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. Four experiments were performed without metabolic activation and two with metabolic activation at concentrations up to 2 mg/ml. Controls were valid.

There was a significant increase in mutation frequency over control counts in L5178Y mouse lymphoma cells during two of the four experiment without metabolic activation. This increase only occurred in a narrow range of concentrations as chlorendic acid was too cytotoxic at higher concentrations. No significant response was obtained with metabolic activation. The substance was considered as mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 October to 18 November 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Scientifically undertaken study and report but not to GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Unknown.
Species / strain / cell type:
mammalian cell line, other: BALB /3T3 cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0.005, 0.010, 0.020, 0.039 and 0.078 mg / ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; - Justification for choice of solvent/vehicle:
Positive controls:
yes
Positive control substance:
other: 3-Methylcholanthrene
Untreated negative controls:
yes
Remarks:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; DURATION- Preincubation period: 24 hours- Exposure duration: 72 hours- Expression time (cells in growth medium): 3 to 4 weeks.- Selection time (if incubation with a selection agent): - Fixation time (start of exposure up to fixation or harvest of cells): SELECTION AGENT (mutation assays): SPINDLE INHIBITOR (cytogenetic assays): STAIN (for cytogenetic assays): GiesmaNUMBER OF REPLICATIONS: 1NUMBER OF CELLS EVALUATED: 10000DETERMINATION OF CYTOTOXICITY - Method: mitotic index; cloning efficiency; relative total growth; other:OTHER EXAMINATIONS: - Determination of polyploidy: - Determination of endoreplication: - Other: OTHER:
Evaluation criteria:
Foci per plate.
Species / strain:
mammalian cell line, other: BALB / 3T3 cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
other: The spontaneous transformation was slightly elevated over what is normally experienced but was acceptable.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):ambiguous The test compound was evaluated for its ability to produce malignant transformation in BALB / 3T3 cells at several con centrations ranging from 0.005 mg / ml to 0.078 mg / ml. The test compound did not show toxicity over this range.Under the conditions of this study, Chlorendic Anhydride did not exhibit clear evidence of transformation ability.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay on liver, glandular stomach, duodenum, and gonadal cells was conducted using the degradation product chlorendic acid. The study was GLP-compliant and conducted according to the OECD TG 474 and 489.

A Dose-Range Finding study was conducted, which concluded test concentrations of 175, 350 and 700 mg/kg bw/day shall be used during the main study.
The study was considered as valid based on the criteria laid down in the OECD TG 474 and 489.

Micronucleus Assay
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %PCE that were similar to the vehicle control group mean and which fell within the 95% reference range of laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control. In general, individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data.
These data were indicative of a clear negative response and therefore mechanistic analysis was not required.

Comet Assay
There were no marked, dose-related, increases in hedgehogs in liver, stomach, duodenum and gonad, thus demonstrating that treatment with Chlorendic Acid did not cause excessive DNA damage that could have interfered with comet analysis.
However, it was noted that there was an increase in %hedgehogs in the duodenum at 700 mg/kg bw/day that exceeded the 95% reference range of the laboratory’s historical vehicle control range. This increase apparent in all animals in the group was considered as part of data analysis.
Overall these data in the liver, stomach, duodenum and gonad did not meet all of the evaluation criteria in the protocol and the OECD 489 test guideline for a positive result.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
Combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The rat has been selected because there is a large volume of background data in this species and has been specifically requested
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: 7-9 week
- Weight at study initiation: 175-375 g
- Assigned to test groups randomly: yes
- Housing: Wire topped, solid bottom cages
- Diet: 5LF2 EU Rodent Diet ad libitum
- Water: tap water ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C.
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: This vehicle has been used in previous in vivo studies with this test article
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were either freshly prepared prior to each dosing occasion in the Range-Finder Experiment or prepared once for the Main Experiment by formulating Chlorendic Acid in corn oil as follows:
The test article was weighed and transferred to a mortar and pestle. A small volume of vehicle was added and mixed to form smooth paste. Further vehicle was added, with mixing, to form a slurry. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume.
Duration of treatment / exposure:
Three days
Frequency of treatment:
Three administrations, at 0, 24 and 45 hours.
Post exposure period:
Not applicable. Animals are sacrificed three hours after the last administration.
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male ratsper dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl MethaneSulfonate (EMS) and Vinblastine (VIN)
- Justification for choice of positive controls: In accordance with the OECD TG 474 and 489
- Route of administration: oral gavage (EMS) or Intraperitoneal Injection (VIN)
- Doses / concentrations: 10 mL/kg (EMS), 2.5 or 5 mL/kg (VIN)
Tissues and cell types examined:
Bone marrow (from femur), liver, stomach, duodenum and gonad.
Details of tissue and slide preparation:
Micronucleus Slide Preparation
The isolated femurs were cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns. Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. Cell pellets were gently resuspended in the remaining volume and the cell suspensions from each femur combined per animal. A further 3 mL of foetal bovine serum was added to the tubes. The cells were pelleted again and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of four uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. Two slides per animal were immediately stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.

Preparation of Comet Slides
Three slides, labelled ‘A’, ‘B’ and ‘C’, were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 μL of each single cell suspension was added to 400 μL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 μL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice.
Evaluation criteria:
Micronuclei Induction
For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE (where more than two dose levels are analysed) was observed.

DNA Damage
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue
3. The increase exceeded the laboratory’s historical control data for that tissue.

The test article was considered positive in this study if all the criteria listed for either micronucleus induction and/or DNA damage were met.

The test article was considered negative in this study if for both end points, none of the following criteria were met and target tissue exposure was confirmed.

Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels and any concurrent cytotoxicity information (such as hedgehog assessment, histopathological changes and any clinical chemistry results).
Statistics:
Statistical analysis was conducted.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There were no remarkable observations for any animal dosed in the Main Experiment with the exception of mouth rubbing observed immediately after the third dose administration for 1/6 animals at 175 mg/kg/day, 3/6 animals at 350 mg/kg/day and 5/6 animals at 700 mg/kg/day. One animal at 700 mg/kg/day was also noted to exhibit paddling behaviour. There was a decrease in group mean body weight at 700 mg/kg/day between Day 1-Day 3, with percentage change values of -0.1%, compared to +7.3%, +5.3% or +5.6% for the vehicle control group, 175 mg/kg/day group or 350 mg/kg/day group, respectively.

Minimal increases in aspartate transaminase (AST) and alanine transaminase (ALT) activities were recorded for some animals administered 700 mg/kg/day. There were also small increases in chloride, urea and creatinine at 700 mg/kg/day, and small inconsistent (between animals) increases in potassium & calcium (350 and 700 mg/kg/day), phosphate (700 mg/kg/day), urea & creatinine (175 and 350 mg/kg/day).

On microscopic examination, decreased hepatocyte glycogen was noted in the liver of animals administered 700 mg/kg/day. Single cell necrosis was noted in one animal administered 700 mg/kg/day. In the stomach, inflammation and/or decreased mucous were noted in animals administered 350 or 700 mg/kg/day, with a dose relationship.
Conclusions:
Chlorendic Acid did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, nor did it induce biologically relevant increases in DNA strand breaks in the liver, stomach, duodenum or gonad of male Sprague Dawley rats when tested up to 700 mg/kg/day (an estimate of the maximum tolerated dose for this study).
Executive summary:

A combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian comet assay on liver, glandular stomach, duodenum, and gonadal cells was conducted using the degradation product chlorendic acid. The study was GLP-compliant and conducted according to the OECD TG 474 and 489.


A Dose-Range Finding study was conducted, which concluded test concentrations of 175, 350 and 700 mg/kg bw/day shall be used during the main study.
The study was considered as valid based on the criteria laid down in the OECD TG 474 and 489.


Micronucleus Assay
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %PCE that were similar to the vehicle control group mean and which fell within the 95% reference range of laboratory’s historical vehicle control data, thus confirming there was no evidence of test article related bone marrow toxicity.
Animals treated with Chlorendic Acid at 175, 350 and 700 mg/kg bw/day exhibited group mean %MN PCE frequencies that were similar to the vehicle control group mean and which fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control. In general, individual animal MN PCE frequencies were considered consistent with the laboratory's historical vehicle control animal distribution data.
These data were indicative of a clear negative response and therefore mechanistic analysis was not required.


Comet Assay
There were no marked, dose-related, increases in hedgehogs in liver, stomach, duodenum and gonad, thus demonstrating that treatment with Chlorendic Acid did not cause excessive DNA damage that could have interfered with comet analysis.
However, it was noted that there was an increase in %hedgehogs in the duodenum at 700 mg/kg bw/day that exceeded the 95% reference range of the laboratory’s historical vehicle control range. This increase apparent in all animals in the group was considered as part of data analysis.
Overall these data in the liver, stomach, duodenum and gonad did not meet all of the evaluation criteria in the protocol and the OECD 489 test guideline for a positive result.

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

Additional information

In vitro gene mutation study, 7.6.1 (4) - negative

In vitro gene mutation in mammalian cell 7.6.1 (2) - negative

Existing studies primarily address gene mutation in bacteria and mammalian cells.

In vitro studies on the acid were generally positive, but an in vivo study was negative. As the acid is regarded as an expected degradation product in both the environment and the body, this result can be applied to the anhydride.


Short description of key information:
Chlorendic anhydride did not exhibit genetic toxicity in the studies undertaken.

Chlorendic acid did not exhibit genetic toxicity in in vivo studies undertaken.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Chlorendic Anhydride is not classified as genetically toxic as all study results are negative. The impurity and degradation product, chlorendic acid, did not show positive results in in vivo studies. Therefore, altohugh positive results were seen in in vitro studies, the substance is not classified as mutagenic.