Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 2-methyl-1,3-benzenediamine and sodium methoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria - non-guideline study (equivalent or similar to OECD TG 471, pre-GLP): negative;

Mutagenicity in mammalian cells - study according to OECD TG 476 (GLP) with a structural analogue (CAS 106276-80-6): negative;

In vitro cytogenicity/ micronucleus study - study according to the proposal for the OECD TG for the in vitro MNT (GLP) with a structural analogue (CAS 386254-45-1): negative

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

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP non-guideline study, available as unpublished report, predates implementation of GLP and/or development of study guidelines, notable limitations in design and/or reporting.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

according to:
- AMES, B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sci. USA 10_, 782-786.
- AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection. Proc. Natl. Acad. Sci. USA 70, 2281-2285.
- AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ ammaliannyiicrosome Mutagenicity Test. Mut. Res. 31, 347-364.

GLP compliance:

no

Remarks:

prior to GLP implementation

Type of assay:

bacterial reverse mutation assay

Target gene:

his-locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg/0.1 mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: Daunorubicin-HCl; N-methyl-N'-nitro-N-nitrosoguanidine; 9(5)aminoacridine hydrochloride monohydrate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Wth metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: The plates were incubated for about 48 hours at 37°C in darkness.

NUMBER OF REPLICATIONS: three petri dishes were prepared per strain and per group (i.e. per concentration or per control group).

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

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 specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations 225, 675, 2025 µg/0.1 mL the substance precipitated in soft agar.

COMPARISON WITH CONTROL DATA:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with the test substance revealed no marked differences.

No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

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:

other: Well documented, according to national standards with GLP

Principles of method if other than guideline:

The procedure and experimental design are in accordance with the following guideline/ references:
- A proposal for a new OECD Guideline for the in vitro micronucleus test, 1998;
- Mut. Res., 439, 183 - 190, 1999;
- Mut. Res., 303, 163 - 169, 1993;
- Toxicol. in Vitro, 7, 185 - 193, 1993

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (with Earle's salt)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S9 mix

Test concentrations with justification for top dose:

Mixed Population Method (MP):
- 24 hours exposure, 24 hours harvest time, without S-9 mix: 0, 6.25, 12.5, 25, 50, 100 µg/ml
- 4 hours exposure, 24 hours harvest time, with S-9 mix: 0, 31.25, 62.5, 125, 250, 500 µg/ml
Mitotic Shake 0ff Method (MSO) (24 hours mitotic shake off):
- 24 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, without S-9 mix: 0, 6.25, 12.5, 25, 50, 100 µg/mI
- 4 hours exposure, 24 hours mitotic shake off, 27 hours harvest time, with S-9 mix: 0, 125, 250, 500, 1000, 1500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data are available.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

PROCEDURE:
- Mixed Population Method (MP): The experimental procedure was carried out based on the method of KALWEIT, et al. (Mut. Res., 439, 183-196, 1999).
- Mitotic Shake off Method (MSO): The experimental procedure was carried out based on the method of SEELBACH, A. et al. (Mut. Res., 303, 163 - 169, 1993; and Toxicol. in Vitro, 7, 185 - 193, 1993).
- 1000 cells were analyzed for micronuclei for each culture, i.e. 2000 cells for each test group.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei;
- The proportion of micronucleus-containing cells exceeded both the concurrent negative control range and the negative historical control range

A test substance is generally considered nongenotoxic in this test system if:
- There was no significant increase in the number of micronucleus-containing cells at any dose above concurrent negative control frequencies and within the historical control data.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was observed macroscopically depending on the test conditions and on the time of observation from about 200 µg/mI culture onward.


RANGE-FINDING/SCREENING STUDIES: The doses for the Mixed Population Method (24 hours sampling time) were determined from an appropriate range-finding cytotoxicity test with cultures exposed to a wide dose range of the test article, i.e. 1.0 µg/ml - 1500 µg/ml culture medium both without S-9 mix (continuous treatment of 24 hours) and after adding a metabolizing system (pulse treatment of 4 hours). In this pretest, various parameters were checked for all or at least some selected doses. As a rule, the highest dose/concentration for nontoxic test substances should not exceed a limit of 5 mg/ml or 10 mM, as recommended by the EEC, OECD and by an ICPEMC Task Group for in vitro studies using mammalian cells. Doses > 1500 µg/ml led to an inhomogeneous suspension which could not administered any longer. On the basis of the findings from the pretest, 100 µg/ml (0.136 mM) without S-9 mix and 1000 µg/ml (1.362 mM) with metabolic activation were selected as the top doses. This selection was based on the cell count, cell attachment, solubility of the test substance and quality of the cells.


RESULTS:
- The negative controls (vehicle controls) gave frequencies within the range expected for the V79 cell line.
- Both of the positive control chemicals for clastogenicity, i.e. ethyl methane sulphonate and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei both in the MP and MSO.
- Micronucleus Frequency: An increase in the number of micronucleated cells was not found either without S9 mix or after the addition of a metabolizing system.
- Mitotic Index (MI): no suppression of the mitotic activity was observed under any of the experimental conditions.
- Proliferation Index (PI): a weak cytotoxic response was observed only without S9 mix at 100 µg/ml.
- Cell Count: a dose-dependent growth inhibition was observed under all experimental conditions from about 100 µg/ml - 125 µg/ml onward.
- Micronucleus Frequency: An increase in the number of micronucleated cells was not observed either without S-9 mix or after the addition of a metabolizing system.

The micronucleus frequency in MP method based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation

Hours		Test groups	S9 mix	Mean (% absolute)
Exposure	Harvest
24	24	Vehicle control, DMSO	-	0.5
24	24	6.25 µg/ml	-	1.0
24	24	12.50 µg/ml	-	0.6
24	24	25.00 µg/ml	-	0.7
24	24	50.00 µg/ml	-	0.9
24	24	100.00 µg/ml	-	1.2
24	24	350.00 µg/ml	-	5.6
4	24	Vehicle control, DMSO	+	0.6
4	24	31.25 µg/ml	+	0.5
4	24	62.50 µg/ml	+	1.0
4	24	125.00 µg/ml	+	0.4
4	24	250.00 µg/ml	+	0.9
4	24	500.00 µg/ml	+	0.9
4	24	2.5 µg/ml CPP	+	18.9

 

The micronucleus frequency in 24-h Mitotic Shake Off method based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation

Hours			Test groups	S9 mix	Mean (% absolute)
Exposure	Mitotic Shake Off	Harvest
24	24	27	Vehicle control, DMSO	-	0.6
24	24	27	6.25 µg/ml	-	0.8
24	24	27	12.50 µg/ml	-	0.5
24	24	27	25.00 µg/ml	-	0.7
24	24	27	50.00 µg/ml	-	0.9
24	24	27	100.00 µg/ml	-	0.7
24	24	27	350.00 µg/ml	-	6.1
4	24	27	Vehicle control, DMSO	+	0.5
4	24	27	125.00 µg/ml	+	0.9
4	24	27	250.00 µg/ml	+	1.0
4	24	27	500.00 µg/ml	+	1.0
4	24	27	1000.00 µg/ml	+	1.3
4	24	27	1500.00 µg/ml	+	1.3
4	24	27	2.5 µg/ml CPP	+	15.4

 

Conclusion:

On the basis of the results of the present study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S-9 mix or after adding a metabolizing system. These findings were confirmed in two modified versions of the in vitro micronucleus assay carried out independently of each other. Thus, under the experimental conditions of this assay, the test substance is considered not to be a chromosome-damaging (clastogenic) agent nor does it induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - Jun 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21st July 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI National Institute of Pharmacy

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- The CAS number represents the old CAS number of the test substance.
- Batch No.of test material: D1003111P2
- Appearance: Yellow powder
- Expiry date: 30 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: The solubility and behaviour of the test item and its solutions - suspensions in the applied test system was determined in the preliminary solubility test. The RPMI 5 Medium (the treatment medium) was taken into consideration as vehicle at the 3-hour and 24-hour treatments.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: mouse lymphoma L5178Y TK+/- cell line (clone 3.7.2c)
- doubling time: 9 - 10 hours
- Methods for maintenance in cell culture if applicable: Stocks of the mouse lymphoma L5178Y TK+/- cell line (1-mL portions) in culture medium supplemented with 7% (v/v) DMSO are stored in liquid nitrogen (about -196°C). Each batch used for mutagenicity testing was checked for mycoplasma contamination previously. For cell cultivation, deep-frozen stock cultures were thawed at 37°C in a water bath, and volumes of 1 mL were transferred into 25 cm² flasks containing 10 mL RPMI-10 medium. After incubation for about one day, the cells were centrifuged at 1000 rpm (173xg) for 5 minutes. Subsequently, the medium was removed and the cells were resuspended in 20 mL RPMI-10 medium, transferred to 75 cm² flasks and incubated until use. The cells were subcultured twice weekly (routine passage in 75 cm² flasks). All incubations occurred with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity.
- Modal number of chromosomes: stable karyotype with a near diploid number of 40 ± 1 chromosomes

MEDIA USED
- Type and identity of media:
* RPMI-0 = RPMI 1640 medium including stable glutamine supplemented with: 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) sodium pyruvate (10 mM)
* RPMI-5 = Treatment medium (with S9 mix): RPMI-0 supplemented with 5% (v/v) fetal calf serum (FCS)
* RPMI-10 = Treatment medium (without S9 mix) and subculturing cells: RPMI-0 is supplemented with 10% (v/v) fetal calf serum
* RPMI-20 = Cloning efficiency and selection medium: RPMI-0 supplemented with 20% (v/v) fetal calf serum
* "THMG" medium = Pretreatment medium A: RPMI-10 supplemented with thymidine 3.0 μg/mL, hypoxanthine 5.0 μg/mL, methotrexate 0.1 μg/mL and glycine 7.5 μg/mL
* "THG" medium = Pretreatment medium B: RPMI-10 supplemented with thymidine 3.0 μg/mL, hypoxanthine 5.0 μg/mL and glycine 7.5 μg/mL
* "TFT" medium = RPMI-20 supplemented with trifluorothymidine (TFT) 4.0 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced rats

Test concentrations with justification for top dose:

The following concentrations were investigated in the Assay 1:
3-hour treatment (with and without S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL

The following concentrations were investigated in the Assay 2:
24-hour treatment (without S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL
3-hour treatment (with S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL

The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 5 Medium (treatment medium)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

PRELIMINARY SOLUBILITY AND TOXICITY TESTS
A preliminary solubility test was performed for selection of the appropriate vehicle and treatment concentrations to be used in the dose-range finding test. In the preliminary solubility test the test item direct addition and the use of the aqueous RPMI 5 medium as vehicle was considered. In the preliminary toxicity test a 3-hour treatment in the presence and absence of S9 mix and a 24-hour treatment in the absence of S9 mix were performed to determine the test item toxicity. The treatment procedure of the cell cultures was the same as described below for the main mutation assays.
In the preliminary toxicity test, single cultures were used (with exception of the RPMI 5 medium controls at the 3-hour treatment (+ S9) and at the 24-hour treatment (- S9)) and positive controls were not included. The concentrations of 5000, 2000, 1000, 316, 100, 31.6, 10 and 3.2 µg/mL were investigated at the 3-hour treatments in the absence and also in the presence of S9 mix and at the 24-hour treatment in the absence of S9 mix. The investigation of the 5000 and 2000 µg/mL concentrations was possible, however these treatments were strongly precipitated at the end of the incubation periods. The colour and the thickness of the obtained suspension at > 1 mg/mL concentration highly disturbed the scoring. The treated cells were washed following treatment with RPMI 10 medium and resuspended in 10 mL RPMI 10 culture medium. Cell concentrations were adjusted to 8 cells/mL and, for each dose, 0.2 mL was plated into each well of a 96-well microtiter plate. The microtiter plates were incubated at 37 °C +/- 1 °C in a humidified incubator gassed with 5 % (v/v) CO2 in air for 9 - 10 days (at the RPMI 5 medium controls 15 - 16 days). Wells containing viable clones were identified by eye and counted. At the end of this test the harmonized relative survival of each culture was determined. The obtained cell growth data were used to choose the dose levels for the main assays. Six concentrations were selected for the main mutation experiments.

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10^7 cells per 75 cm² flask (3-hour treatment) and 4 x 10^6 cells per 25 cm² flask (24-hour treatment)

DURATION
- Preparation of test cultures: For the experiments, 3-3 vials were thawed rapidly, cells were diluted in RPMI 10 medium and incubated at 37 °C +/- 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Well growing cells, subcultures were established in an appropriate number of flasks.
- Exposure duration: 3-hour treatment and 24-hour treatment
- Expression time: 2 days
- Selection time: 2 weeks

SELECTION AGENT: trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- relative survival (% RS)
- relative suspension growth (% RSG)
- relative viability (% RV)
- relative total growth (% RTG)

Evaluation criteria:

The assay is considered valid if all of the following criteria are met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (above 50 - 170 mutants per 10^6 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency of the negative controls is within the range of 65 % to 120 % on Day 3 (at the end of the expression period).
4. At least four test concentrations are present, where the highest concentration produces 80 - 90 % toxicity, precipitation, or is 5 mg/mL, or the highest practical concentration.

The test item is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Results, which only partially satisfied the criteria, should be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity should require careful interpretation when assessing their biological significance. Indeed, extreme caution should be exercised with positive results obtained at levels of survival lower than 10 %. There is no requirement for verification of a clear positive response. Equivocal or negative results need to be verified in a follow-up experiment. Modification of study parameters in the follow-up experiment should be considered.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- pH and osmolality values were within acceptable ranges.

MUTANT FREQUENCY
- Assay 1: The obtained mutation frequencies remained nearly in the same (vehicle control) range far below the threshold (GEF criterion) for positive call and statistically significant differences in comparison with the negative control were not obtained in any case (Dunnett’s Test, α=0.05).
- Assay 2, 24 h treatment: The calculated mutation frequencies of the treated concentrations (3.2-1000 μg/mL) were in the same range, did not show dose-related tendencies, and did not differ statistically significantly from the mutation frequency of the RPMI 5 Medium (vehicle) control (Dunnett’s Test, α = 0.05).
- Assay 2, 3 h treatment: The mutation frequencies in the treated concentrations did not differ statistically significantly from the mutation frequency of the vehicle control (Dunnett’s Test, α=0.05).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Assay 1: No toxicity was observed in the whole examined concentration range based on the harmonised relative survival (harmonised RS) and based on the relative total growth values (RTG). The 23 % inhibition obtained at the concentration of 1000 μg/mL based on the harmonised RS in absence of S9 Mix was evaluated as reflecting the biological variability of the test system. The viability data did not show any test item effect in the function of the increasing concentration in the whole concentration range.
- Assay 2, 24 h treatment: No toxicity was observed in the whole examined concentration range based on the harmonised relative survival (harmonised RS) values and 46 % inhibition was obtained at the concentration of 316 μg/mL and 63% at 1000 μg/mL based on the relative total growth values (RTG) indicating the inhibitory effect of the test item. The viability data did not show any test item effect in the function of the increasing concentration in the whole concentration range.
- Assay 2, 3 h treatment: Differently from the Assay 1 results slight cytotoxicity was obtained based on the harmonised relative survival (harmonised RS) values. 39 % was obtained at 1000 μg/mL, 30 % at the concentration of 316 μg/mL. Based on the relative total growth 21 % inhibition was obtained at 316 μg/mL and 43 % at 1000 μg/mL.
In conclusion the test item showed a slight inhibitory effect that manifested at the higher concentration levels in absence and also in presence of metabolic activation. The obtained corresponding harmonised RS and RTG value changes (were caused –supposedly- by not observable inhomogeneities of the treatment suspensions) remained mostly within a biological variability range of the applied system.

Table 1: Results of the preliminary toxicity test

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
3.16 10 31.6 100 316 1000 2000 5000	29/192 31/192 36/192 29/192 26/192 29/192 30/192 32/192	1.2001 1.1426 1.0501 1.1847 1.2571 1.1817 1.1658 1.1199	100.00 117.88 94.79 114.78 98.10 104.33 87.12 68.13
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.16 10 31.6 100 316 1000 2000 5000	77/384 22/192 38/192 21/192 45/192 32/192 38/192 35/192 35/192	1.0062 1.3984 1.0203 1.4199 0.9107 1.1199 1.0133 1.0641 1.0641	100.00 138.39 123.75 121.39 83.22 97.15 80.65 77.53 63.19
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.16 10 31.6 100 316 1000 2000 5000	66/384 40/192 50/192 58/192 37/192 47/192 53/192 29/192 46/192	1.1110 0.9875 0.8414 0.7485 1.0312 0.8831 0.8100 1.1817 0.9082	100.00 119.41 115.19 110.15 111.63 90.81 67.17 44.54 26.84

Table 2: Summarized results of the survival data of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	56/384 68/384 57/384 69/384 48/384 65/384 57/384 140/384	1.2334 1.0886 1.1925 1.1187 1.3051 1.1159 1.2091 0.6347	100.00 88.51 84.30 89.79 94.24 82.40 77.01 43.09
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	65/384 57/384 54/384 51/384 56/384 56/384 56/384 306/384	1.1568 1.2198 1.2407 1.2871 1.2250 1.2306 1.2211 0.1447	100.00 105.62 105.18 102.48 100.43 105.53 93.65 9.78

Table 3: Summarized results of the survival data of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	74/384 93/384 117/384 88/384 95/384 63/384 84/384 218/384	1.0352 0.9183 0.7563 0.9622 0.8927 1.1497 0.9571 0.3596	100.00 85.34 83.06 98.96 91.81 104.72 80.85 23.51
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	64/384 69/384 84/384 69/384 69/384 64/384 77/384 143/384	1.1279 1.0865 0.9510 1.0730 1.0962 1.1261 1.0349 0.6229	100.00 99.06 81.66 87.33 79.41 69.60 61.07 59.41

Table 4: Summarized results of the viability data of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Relative Viability [% RV]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	66/384 99/384 57/384 69/384 59/384 60/384 63/384 92/384	1.1182 0.8500 1.1957 1.0938 1.1762 1.1695 1.1460 0.9061	100.00 76.02 106.94 97.82 105.19 104.59 102.49 81.03
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	74/384 60/384 69/384 67/384 65/384 61/384 67/384 314/384	1.0494 1.1654 1.0826 1.1216 1.1115 1.1588 1.1016 0.1284	100.00 111.06 103.16 106.89 105.92 110.43 104.98 12.24

Table 5: Summarized results of the viability data of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Relative Viability [% RV]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	60/384 65/384 57/384 59/384 81/384 57/384 64/384 114/384	1.1784 1.1604 1.2133 1.1749 0.9798 1.2194 1.1704 0.7752	100.00 98.47 102.96 99.70 83.14 103.48 99.32 65.78
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	59/384 56/384 54/384 51/384 56/384 53/384 58/384 117/384	1.1773 1.2153 1.2701 1.2848 1.2163 1.2433 1.1965 0.7440	100.00 103.23 107.88 109.13 103.31 105.60 101.63 63.19

Table 6: SG, RSG and RTG data of the first assay

Concentration [µg/mL]	Suspension growth (SG)	Relative Suspension Growth [% RSG]	Relative Total Growth [% RTG]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	15.19 18.68 16.30 14.72 15.29 14.39 12.32 9.01	100.00 122.99 107.29 96.92 100.66 94.76 81.11 59.30	100.00 93.49 114.73 94.81 105.88 99.11 83.12 48.05
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	17.27 17.70 17.97 17.37 16.28 13.08 16.65 2.65	100.00 102.50 104.05 100.56 94.27 75.74 96.43 15.35	100.00 113.84 107.34 107.49 99.86 83.64 101.24 1.88

Table 7: SG, RSG and RTG data of the second assay

Concentration [µg/mL]	Suspension growth (SG)	Relative Suspension Growth [% RSG]	Relative Total Growth [% RTG]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	32.07 25.64 25.64 30.74 31.65 16.85 11.99 5.31	100.00 79.97 79.96 95.88 98.72 52.56 37.38 16.56	100.00 78.75 82.33 95.59 82.08 54.39 37.12 10.89
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	14.92 14.47 13.76 13.23 14.55 11.23 8.34 9.22	100.00 96.98 92.17 88.62 97.48 75.26 55.88 61.78	100.00 100.11 99.43 96.71 100.70 79.47 56.79 39.04

Table 8: Summarized mutagenicity results of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Mutation frequency
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	625/768 671/768 635/768 641/768 619/768 634/768 640/768 241/768	114/768 53/768 108/768 78/768 106/768 80/768 91/768 338/768	29/768 44/768 25/768 49/768 43/768 54/768 37/768 189/768	92.45 79.78 79.86 82.89 91.96 82.13 79.98 654.29
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	631/768 636/768 645/768 639/768 650/768 644/768 626/768 487/768	95/768 92/768 69/768 92/768 86/768 90/768 102/768 118/768	42/768 40/768 54/768 37/768 32/768 34/768 40/768 163/768	93.73 81.12 80.78 82.36 75.28 76.17 93.11 1781.18

Table 9: Summarized mutagenicity results of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Mutation frequency
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	587/768 594/768 609/768 581/768 618/768 596/768 599/768 140/768	107/768 115/768 102/768 124/768 96/768 85/768 87/768 421/768	74/768 59/768 57/768 63/768 54/768 87/768 82/768 207/768	114.73 110.98 96.01 118.91 111.30 104.25 106.72 1113.90
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	628/768 637/768 616/768 599/768 634/768 600/768 632/768 174/768	91/768 92/768 104/768 104/768 84/768 95/768 91/768 338/768	49/768 39/768 48/768 65/768 50/768 73/768 45/768 256/768	85.79 77.47 87.10 97.32 79.54 99.72 81.63 1010.51

Conclusions:

Under the conditions of this study, the test item did not induce gene mutations in the presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the test item handling, the test item was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control.

The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions. The following concentrations were investigated in the Assay 1 and Assay 2:

The following concentrations were investigated in the Assay 1:

3-hour treatment (±S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL;

The following concentrations were investigated in the Assay 2:

24-hour treatment (-S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL;

3-hour treatment (+S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) and diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

In the main assays, the relative harmonised survival, the relative total growth of the cells, the viability (colony-forming ability at the end of the 2 day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined.

The result of Assay 1 was considered to be negative, hence in the second Assay was performed with the originally planned concentration levels chosen based on the preliminary cytotoxicity test with the treatment periods of 3 (in presence of metabolic activation) and 24 hours (in absence of metabolic activation).

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments as well as in connection with the number of analyzable concentration levels (at least four). The test item concentrations were chosen based on the test item solubility. In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

In the performed assays the obtained mutation frequencies (in the absence and also in the presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control throughout the study (Sections 10.2 and 10.3).

Under the conditions of this study, the test item did not induce gene mutations in the presence and the absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Mutagenicity in bacteria:

In the Ames test (similar to OECD Guideline 471; pre-GLP) the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (strain TA 98, TA 100, TA 1535 and TA 1537). The investigations were performed with the following concentrations of the test substance: 25, 75, 225, 675 and 2025 µg/0.1 mL/plate, with and without microsomal activation (Aroclor 1254 induced rat liver S9 mix). At the concentrations 225, 675, 2025 µg/0.1 mL/plate, with and without metabolic activation, the substance precipitated in soft agar. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Mutagenicity in mammalian cells:

Mutagenicity of the actual substance in mammalian cells was not evaluated. However, there are reliable data of a structural analogue available, that can be used to fill this data gap.

An in vitro mammalian cell assay (OECD TG 476, GLP) was performed with CAS 106276-80-6 in mouse lymphoma L5178Y TK+/- 3.7.2 C cells. Treatments were carried out for 3 hours with and without metabolic activation (+/- S9 Mix) and for 24 hours without metabolic activation (- S9 Mix). The concentrations applied in the assays were chosen according to the solubility and cytotoxicity results of a pre-experiments. The test item was considered as relatively insoluble. Therefore, it was investigated beyond its limit of solubility under culture conditions and a concentration range from 3.2 – 1000 µg/mL was chosen. The relative harmonized survival, the relative total growth of the cells, the viability (colony-forming ability at the end of the 2-day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. The performed assays fulfilled the validity criteria in connection with the negative control and positive control treatments as well as in connection with the number of analyzable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. In the performed assays the obtained mutation frequencies (in the absence and also in the presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control throughout the study. Under the conditions of this study, the test item did not induce gene mutations in the presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Based on the similar properties of both substances, this is assumed for the actual substance as well.

In vitro cytogenicity/ micronucleus study:

There is no in vitro cytogenicity or micronucleus study for the actual substance. However, there are reliable data from a structural analogue available, that can be used as read-across.

CAS 386254-45-1 was assessed in V79 cells in vitro for possible clastogenic or aneugenic activity leading to inducement of micronuclei both in the presence and in the absence of a metabolizing system at doses of 6.25 to 1500 µg/mL according to a proposal for a new OECD Guideline for the in vitro micronucleus test (1998) with the mixed population method (MP) and the mitotic shake off method (MSO). The study was conducted in compliance with GLP. Test substance precipitation was observed macroscopically depending on the test conditions and the time of observation from about 200 µg/mL culture onward. 1000 cells were analyzed for micronuclei for each culture, i.e. 2000 cells for each test group. The negative controls (vehicle controls) gave frequencies within the range expected for the V79 cell line. Both positive control chemicals for clastogenicity, i.e. ethyl methane sulphonate and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei both in the MP and MSO. On the basis of the results of the study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. These findings were confirmed in two modified versions of the in vitro micronucleus assay carried out independently of each other. Under the experimental conditions of this assay, the test item is considered not to be a chromosome-damaging (clastogenic) agent nor does it induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.

Due to the similar chemical structure as well as properties, this result can be transferred to the actual substance and no cytogenetic toxicity in mammalian cells in vitro is assumed for the actual substance as well.

This is confirmed by the results of a chromosome aberration study with 3,4,5,6-tetrachlorophthalimide (CAS 1571-13-7), which is a structural component of the actual substance. Herein, CHL/IU cells were treated for 6 h with and without metabolic activation (S9 mix from phenobarbital and 5,6-benzoflavone induced rats) and 24 h without metabolic activation with the test substance suspended in 0.5 % carboxymethylcellulose-sodium solution. Based on the results of a preliminary examination (cell growth inhibition test), the highest test concentration in each test series was set as 10 mM or IC50 or higher, and 4 to 6 concentrations in total were then set by applying the common ratio of 2. As positive controls 3,4-benzopyrene (with metabolic activation) and mitomycin C (without metabolic activation) were used. As a result of the main test, the frequencies of appearance of structural and numerical aberrations of chromosomes were less than 5 % in any concentration of each test series, and the result was thus negative. Therefore, it was concluded that 3,4,5,6-tetrachlorophthalimide does not possess any clastogenic or aneugenic potential.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.