Calcium bis[4-[[1-[[(2-chlorophenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, similar to OECD TG 471, GLP-compliant, S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2, with and without metabolic activation, 1997, K1

Gene mutation (TK): negative, similar to OECD TG 476, GLP-compliant, mouse lymphoma L5178Y cells, with and without metabolic activation, 1997, K1

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:

April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his and trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from aroclor induced rats and syrien hamster

Test concentrations with justification for top dose:

Range finder and first experiment 2.4, 12, 60, 300, 1500 micro g per plate, with and without S9
second experiment 93.75, 187.5, 375, 750, 1500 micro g per plate, with and without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene: one strain, for standard S9 mix, with S9, 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), second experiment with pre-incubation 30 min at 30°C

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: in triplicate

OTHER: The S-9 used for the 'standard' plate-incorporation treatments was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 used for the 'Prival' treatments was prepared from uninduced male Golden Syrian hamsters.

range finder: tested in TA 100, in triplicate +/- S9 mix, with positive and solvent control, incorporation method

counting: Seescan Colony Counter (Seescan pic), Manual counts were performed for all treatments performed at 1500 /micro g/plate, as precipitation of the test agent on these plates prevented an accurate automated count being obtained.

Evaluation criteria:

The test article was considered to be mutagenic if:
1) the assay was valid
2) Dunnett's test gave a significant response (p < 0.01) and the data set showed a significant dose correlation
3) the positive responses described in 2) were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis

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:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1500 micrgram/plate

RANGE-FINDING/SCREENING STUDIES: yes, see above under study details

COMPARISON WITH HISTORICAL CONTROL DATA:

the second experiment was done twice due to an error in the first trial (induced instead of uninduced hamster liver)

treatments of strain TA100 in Experiment 1 produced a small increase in revertant numbers (1.3 fold over the negative solvent control counts) in the presence of metabolic activation, that was however statistically significant at the 1% levelwhen the data were analysed using Dunnett's test. However, this increase was not reproduced in the second experiment (either with 'standard' or 'Prival' S-9 mix) and was therefore not attributed to mutagenic activity, but was considered to have been a chance occurrence. This study was therefore considered to have provided no clear evidence of mutagenic activity.

Conclusions:

It was concluded that the test item did not induce mutation under the conditions employed for this study, both in the absence and in the presence of metabolic activation systems (both 'standard' and 'reductive' S-9 mixes).

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations using the S. typhimurium strains TA1535, TA 1537, TA 1538, TA 98, and TA 100, and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and with and without preval modification. Each concentration, including the controls, was tested in triplicate. The test item was tested at five concentrations in the range of 2.4 and 1500 µg/plate (experiment I) and 93.75 to 1500 µg/plate (experiment II).

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) or by preval modification.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

expiry date of test item not given, mutant frequency of one of the negative control replicates was below 60 mutants/10^6 viable cells

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Target gene:

thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 10
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor induced rats, MOLTOX

Test concentrations with justification for top dose:

Experiment 1 and 2: 0, 11.25, 22.5, 45, 90, 180

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

diluted 100-fold in the treatment medium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h
- Expression time (cells in growth medium): 2d

SELECTION AGENT (mutation assays): resistance tested with TFT

NUMBER OF REPLICATIONS: 2 independent experiments

Evaluation criteria:

The test article was considered to be mutagenic if all the following criteria were met:
1) die assay was valid
2) the inutant frequency at one or more doses was significantly greater than that
of the negative confrol
3) there was a significant dose-relationship as indicated by the linear trend
analysis
4) the effects were reproducible.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each freatment dose based on Dunnett's test for multiple comparisons, and secondly the data was checked for a linear trend in mutant frequency widi freatment dose using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no changes in pH after treatment
- Precipitation: at 2 top dose levels

RANGE-FINDING/SCREENING STUDIES: yes, single cultures, no positive controls included, concentration 5.625 to 180 micro g/mL

COMPARISON WITH HISTORICAL CONTROL DATA: yes

pH

pH measurements were assessed on post-treatment media from the cytotoxicity rangefinder. The negative controls and cultures treated with the top dose gave the following results: 7.54 and 7.25 in the absence and presence of S-9 respectively for the negative control cultures and 7.53 and 7.22 for 180 µg/mL. Therefore there was no significant change in pH in cultures treated with the test item at the top dose of 180 µg/mL.

Toxicity

In the cytotoxicity range-finder, six doses were tested separated by two fold intervals and ranging from 5.625 to 180 µg/mL. Upon addition of the test article to the cultures and after the freatment incubation period, precipitate was observed at the top dose tested in the absence and presence of S-9 (180 µg/mL). No marked toxicity was observed at any dose level tested in the absence or presence of S-9. Accordingly, in both Experiments 1 and 2, five doses were tested ranging from 11.25 to 180 µg/mL. In Experiment 1, upon addition of the test article to the cultures, precipitate was observed at the top two doses tested in the absence and presence of S-9 (90 and 180 µg/mL). Furthermore, after the treatment incubation period, precipitate was observed at these doses and also at 45 µg/mL in the presence of S-9 only. In Experiment 2, upon addition of the test article to the cultures, precipitate was observed at the top dose tested in die absence and presence of S-9 (180 µg/mL). Furthermore, after the treatment incubation period, precipitate was observed at this dose and also at 90 micro g/mL in the absence of S-9 and at 45 and 90 µg/mL in the presence of S-9. No marked toxicity was observed at any dose level tested in Experiment 1 or 2, in the absence or presence of S-9. Two days after treatment, all doses tested in both Experiments 1 and 2, were selected to determine viability and 5-trifluorothymidine resistance.

Conclusions:

When tested up to its solubility limit in tissue culture medium, the test article did not induce mutation at the tk locus of L5178Y mouse lymphoma cells in two independent experiments in the absence or presence of S-9. It is concluded that, under the conditions employed in this study, the substance is not mutagenic in this test system.

Executive summary:

In this study, mouse lymphoma cells (L5178Ytk^+/-) were treated with 11.25 to 180 µg/ml of the test item in presence or absence of S9-mix from Aroclor induced rat liver. The assay was performed in two independent experiments; each concentration was tested in triplicate. Two days after treatment, all doses tested, were selected to determine viability and 5-trifluorothymidine resistance. Precipitation was observed at the top two dose levels in each experiment, toxicity did not occur. No significant increases in mutant frequency were observed following treatment with the test item at any dose level in the absence or presence of S-9, in both experiments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test: negative, similar to EU Method B.12, GLP-compliant, mouse, 1997, K1

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 36-43 days
- Weight at study initiation: 25-33g male, 20-28g female
- Assigned to test groups randomly: yes
- Fasting period before study: not starved before dosing
- Housing: groups of no more than three animals of the same sex
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 52-59
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 1996-10-23 To: 1996-12-31

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) 1%
- Concentration of test material in vehicle: 5, 10 and 20 mg/ml
- dose volume: 20 ml/kg
- The test article preparations were protected from light and used within approximately 2 hours of initial formulation

Duration of treatment / exposure:

24h and 48h respectively

Frequency of treatment:

once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

actually injected

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

actually injected

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

actually injected

No. of animals per sex per dose:

5 per sex and dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamid. dissolved in saline, 40 mg/kg bw single dose, intraperitonally, on the second day of dosing

Tissues and cell types examined:

both femours removed, bone marrow smear examined

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: range finder

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Test article and vehicle treated mice were killed in groups, 24 or 48 hours after the second administration; CPA-treated mice were killed 24 hours after the single dose. Mice were killed by asphyxiation with carbon dioxide in the same order as they were dosed.

DETAILS OF SLIDE PREPARATION: The tubes containing bone marrow-serum-mix were centrifuged and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube using a Pasteur pipette and from each tube one drop of
suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and tag number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide. Slides were allowed to air-dry and were fixed for 5 minutes in absolute methanol before being stained. One slide from each set of two was then taken, the other was kept in reserve. After rinsing several times in water, slides were stained for 10 minutes in filtered Giemsa stain diluted 1:6 (v/v) in distilled water. Stained slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

METHOD OF ANALYSIS: ratio of PCE/NCE for each animal and the mean for each group was calculated, compared with historical negative control ranges, compared with the numbers in vehicle control groups, further statistical test (for linear trend) was used to evaluate possible dose-response relationships

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.

Statistics:

- inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity x2 test
- numbers of micronucleated PCE in each treated group (males and females, separately and combined) were compared with the numbers in vehicle
control groups by using a 2 x 2 contingency table to determine x2 (Improbability values of p < 0.05 were to be accepted as significant
- statistical test (for linear trend) was used to evaluate possible dose-response relationships

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

decrease in PCE/NCE ratio

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: lethargy, hunched posture, tremors, prostration, twisting, coldness, yellow feces, eye closure
- mortalities: no mortalities at 250 mg/kg bw, 1-2 animals per sex 500 - 1600 mg/kg bw, no survivors at 1800 and 2000 mg/kg bw

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): frequencies of micronucleated PCE were similar to the values for vehicle control groups at both sampling times
- Ratio of PCE/NCE (for Micronucleus assay): Mice treated with the test substance generally exhibited group mean ratios of PCE to NCE which were lower than those in concurrent controls indicating exposure of the target tissue to the test article
- Statistical evaluation: no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article at either sampling time

Eye closure was apparent at 100 and 200 mg/kg/day. Prostration, hunched appearance, piloerection, coldness, eye closure and twisting were seen among animals dosed at 400 mg/kg/day. Three males and two females receiving this dose were either found dead or were killed in extremis prior to sampling. Loss of body weight was seen at all dose levels.

Conclusions:

It is concluded that the test item did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 400 mg/kg/day, a dose at which limited mortality and cytotoxicity were apparent.

Executive summary:

Test item was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which the test article, made up in 1% (w/v) methyl cellulose, was administered to mice intraperitoneally. The test article was administered once daily on two consecutive days to groups of three male and three female mice at doses covering the range 250 to 2000 mg/kg/day. Observations were made over a 4 day period following the second administration and signs of toxicity recorded. For the micronucleus test, test substance was made up as described and administered at 100, 200 and 400 mg/kg/day to groups of five male and five female mice killed 24 or 48 hours after the second administration. Several animals receiving the highest dose died prior to the scheduled sampling times indicating that it would not have been possible to administer the test article at an appreciably higher dose.

The negative (vehicle) control in the study was 1 % (w/v) methyl cellulose also administered intraperitoneally once daily on two consecutive days. Groups of five male and five female mice treated with this were killed and sampled 24 or 48 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in physiological saline and administered intraperitoneally as a single dose at 40 mg/kg to groups of five male and five female mice which were killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2x2 contingency x2 test).

Slides from all dose groups were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control ranges. Mice treated with the test substance exhibited group mean ratios of PCE to NCE which were generally lower dian those in concurrent controls indicating exposure of the target tissue to the test article. Frequencies of micronucleated PCE, however, were similar to the values for vehicle control groups at both sampling times. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article at either sampling time.

It is concluded that the test article did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 400 mg/kg/day, a dose at which limited mortality and cytotoxicity were apparent.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genotoxicity in vitro:

There are three reliable, GLP-conform studies available to assess the potential of the test articel for gene mutations in bacteria and mammalian cells and for formation of micronuclei in bone marrow derived erythrocytes in mice.

The first study similar to OECD TG 471 was performed to investigate the potential of the test item to induce gene mutations using the S. typhimurium strains TA1535, TA 1537, TA 1538, TA 98, and TA 100, and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and with and without preval modification. Each concentration, including the controls, was tested in triplicate. The test item was tested at five concentrations in the range of 2.4 and 1500 µg/plate (experiment I) and 93.75 to 1500 µg/plate (experiment II).

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) or by preval modification.

 

In the second study similar to OECD TG 476, mouse lymphoma cells (L5178Ytk^+/-) were treated with 11.25 to 180 µg/ml of the test item in presence or absence of S9-mix from Aroclor induced rat liver. The assay was performed in two independent experiments; each concentration was tested in triplicate. Two days after treatment, all doses tested, were selected to determine viability and 5-trifluorothymidine resistance. Precipitation was observed at the top two dose levels in each experiment, toxicity did not occur. No significant increases in mutant frequency were observed following treatment with the test item at any dose level in the absence or presence of S-9, in both experiments.

 

Genotoxicity in vivo: 

In the course of the last study, the test article was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels similar to the EU Method B.12. The choice of dose levels was based on an initial toxicity range-finding study. The test substance was administered intraperitoneally at 100, 200 and 400 mg/kg/day to groups of five male and five female mice killed 24 or 48 hours after the second administration. Cyclophosphamide (CPA) served as positive control and was administered as a single dose at 40 mg/kg to groups of five male and five female mice which were killed after 24 hours. Both femurs of each animal which was exposed were removed and bone marrow extracted to prepare slides with air dried bone marrow smear. Slides from all dose groups were analysed.

Clinical signs of toxicity were present in all treated animals. Eye closure was apparent at 100 and 200 mg/kg/day. Prostration, hunched appearance, piloerection, coldness, eye closure and twisting were seen among animals dosed at 400 mg/kg/day. Three males and two females receiving this dose were either found dead or were killed in extremis prior to sampling. Loss of body weight was seen at all dose levels.

Microscopic analysis of the slides revealed normal group mean ratios of PCE (polychromatic erythrocytes) to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE at the control group. Mice treated with the test articel exhibited mean ratios of PCE to NCE which were generally lower than those in concurrent controls indicating exposure of the target tissue to the test article. Frequencies of micronucleated PCE were similar to control groups at both sampling times.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.