N,N'-(2,5-dimethyl-1,4-phenylene)bis[4-[(5-chloro-2-methylphenyl)azo]-3-hydroxynaphthalene-2-carboxamide

Endpoint summary

Additional information

All members of the disazocondensation red pigment category non mutagenic in the standard ames test. Pigment Red 144 and 166 were in addition tested with the Prival modification for azo compounds and also found to be non mutagenic. The Ames test is considered the most relevant ones for poorly soluble inert substances such as pigments. Bacteria are much more resistant to mechanical stress from particles in the incubation medium and they can be treated at higher doses compared to mammalian cell culture. For two substances, a weak mutagenic effect was reported at highly precipitating concentrations which indicates the problem of genotoxic impurities. One of these two substances is Pigment Brown 23 for which two other Ames tests are available showing absence of mutagenicity, although in a plate-incorporation set-up and not in a pre-incubation set-up. More importantly, the batch used in the positive Ames test was used for the in-vivo micronucleus assay and the in-vivo test for unscheduled DNA synthesis and both studies showed absence of genotoxicity. The other is Pigment Red 166 which in one of four valid assays showed a borderline positive result at highly precipitating concentrations in strain TA 98 with metabolic activation (rat liver S9).

 

Data on clastogenicity is available for four of the nine members of the category. Pigment Red 166 is one of the pigments of the low molecular weight side (795 g/mol), it was found to be not clastogenic in a valid micronucleus study in vivo (Ciba-Geigy Ltd 1981). Also Pigment Brown 23 and Pigment Red 221 were not clastogenic in a valid micronucleus study in vivo (BASF 2010a, Ciba-Geigy Ltd 1995). Pigment Red 242 was not clastogenic in a valid in-vitro study for clastogenicity (RCC 1992). It is considered adequate to conclude from this data set to the other five members because the data set includes the two substances for which the highest water solubility was measured and which in theory would be the most likely to be taken up by cells.

 

Data on mutagenicity in mammalian cells is available in form of a valid hprt test in V79 cells and a MLA assay without metabolic activation for Pigment Red 166 and an in-vivo study for undeschuled DNA synthesis for Pigment Brown 23. Both showed absence of mutagenicity. In-vitro testing of mammalian cells in vitro with organic pigments is generally hindered by the sensitivity of the cultivated cells to precipitates. Considering that the pigments are too large and bulky for significant uptake, this information on mutagenicity in vitro is considered to be sufficient.

 

	PR 262	PR 166	PR 144	PBr 41	PBr 23	PR 214	PR 220	PR 221	PR 242
	79665-24-0	3905-19-9	5280-78-4	68516-75-6	35869-64-8	40618-31-3	68259-05-2	71566-54-6	52238-92-3
Mol. weight	781.7	794.5	828.9	844.5	850.0	863.4	925.8	925.8	930.5
Water solubility (μg /L)	16.4	<6.5	11.2	0.5 – 1	<20	6.1	14.2	91	18.9
n-octanol solubility (μg /L)	55.4	<6.5	21.9	56	<20	17.8	<10	24	41
Log Pow  (calculated from solubilities)	0.53	n.a.	0.29	1.7 - 2.1	n.a.	0.47	<-0.15	-0.57	0.33
Ames	not mutagenic K1 Non Prival	not mutagenic* (K1, Prival and non-Prival)	not mutagenic K1 Prival and Non Prival	not mutagenic K1 Non Prival	not mutagenic* K1 non-Prival	not mutagenic K1 Non Prival	not mutagenic K2, non Prival	not mutagenic K1, non Prival	not mutagenic K1 Non Prival
Clastogenicity in vitro									Not clastogenic K1
Mutagenicity in mammalian cells in vitro		not mutagenic (hprt + MLA) K1
Clastogenic-ity in vivo (MN)		Non clastogenic K2			Non clastogenic K1
Mutagenicity in mammalian cells in vivo (UDS)					not mutagenic K1
CAS of amine A	95-79-4	95-82-9	95-82-9	608-27-5	89-63-4	95-82-9	none	none	121-50-6
CAS amine B	6393-01-7	106-50-3	615-66-7	2243-62-1	615-66-7	20103-09-7	6393-01-7	20103-09-7	20103-09-7

As discussed in the toxicokinetics section, disazocondensation red pigments are highly conjugated aromatic ring systems that are linked with azo and amide bonds. They do not carry ionisable groups, are poorly soluble and unreactive and are not susceptle to pH-dependent hydrolysis. Available toxicity data indicates that they are too large and to insoluble for uptake after ingestion. This is important because if azo reduction occurred, primary aromatic amines would be generated. As summarized in the overview table on the properties of the amine components in the toxicokinetic section (Table 3), a number of amine components give positive results in the Ames test. There is however no indication of this in the available data on the disazocondensation red pigments.

 

Pigment Red 262 (CAS 79665-24-0, 782 g/mol)

A GLP compliant study following OECD testing guideline 471 with one restriction was performed (CCR 1988). The tester strain S. typhimurium TA 1538 was used instead of an E.coli strain. The substance is a commercial product described as having approximately 100% purity. The plate incorporation method was used to test doses of 10; 100; 333.3; 1000; and 5000 µg/plate.Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in some of the test groups with and without metabolic activation at the highest investigated dose.

 

 

Pigment Red 166 (CAS 3905-19-9, 794.5 g/mol)

Four GLP compliant Ames tests following OECD testing guideline 471 were performed (BSL 2006a,b,c,d); two studies used the Prival modification for azo compounds, and two used the standard design. The following strains were tested: TA 98, TA 100, TA 1535, TA 1537, and E. coli WP2 uvrA. Test concentrations were based on a pretest (cytotoxicity) and ranged from 31.6 to 5000 µg/ plate. There were no deviations to the OECD guidelines and these studies are valid without restriction. No mutagenicity was observed in three assays and a borderline positive result was observed in one assay. Specifically, the mutant frequency was increased 3.1 and 3.2 fold in strain TA98 using the classical rat S9 for metabolic activation and an increase above 3 is the threshold for a positive result. The increase occurred at doses in the precipitating range and was only above the threshold at the highest dose of 5000

µg/ plate.

A positive result in older Ames test was obtained at highly precipitating concentrations with an older product containing this pigment: A dose-dependent increase was observed in the presence of metabolic activation in strains TA 98 and TA 1538 (Ciba-Geigy Ltd 1980, 1981). It is concluded that the effects observed in the older studies were caused by mutagenic impurities and that Pigment Red 166 itself is not mutagenic in bacteria.

 

Pigment Red 166 was investigated in GLP and OECD guideline 476 compliant study for mutagenicity in V79 cells (Ciba-Geigy Ltd 1989). Mutagenicity tests were performed at concentrations of 900, 720, 360, 180, 90,45µg/mL and the concentrations were chosen based on pre-tests for cytotoxicity. The pigment was dissolved or suspended inDMSO at room temperature. Due to the intense red colour of the test substance, the complete solubilisation of the test substance could not be observed and therefore no filtration of this solution was performed. Lower concentrations were obtained by dilution of this solution with DMSO. In both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10(exp 6) cells plated was not detected at any concentration of the test substance and there was also no indication of any concentration- mutant frequency relation in either experiment.

 

In addition, a point mutation test with mouse lymphoma cells (L58147Y) with the single dose of 1 mg/mL test substance was performed (Ciba-Geigy Ltd 1982). No increased number of mutations could be detected compared to control. Under the conditions with the given restrictions (test substance without metabolic activation, no positive control, single test substance concentration) the test substance showed no mutagenic activity.

A nucleus anomaly study is available for Pigment Red 166 (Ciba-Geigy Ltd 1981) and this study design is comparable to that of a micronucleus assay (OECD guideline 474). More parameters than micronuclei in erythroblasts are recorded, but only 1000 bone marrow cells were evaluated without giving the actual number of erythroblasts. This is considered

acceptable because the extra dose of 5000 mg/kg bw would not have been included in a modern micronucleus study.The study was performed prior to the introduction of GLP and OECD testing guidelines, but contains adequate reporting details and is considered valid.

Pigment Red 166 was administered by gavage. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg on two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses were examined. The following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells and e) polyploid cells. The bone marrow smears from animals treated with various doses showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 10.02% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

 

Pigment Red 166 was tested in an in vitro test for transformation-inducing properties in mouse fibroblasts (Ciba-Geigy Ltd 1981b). BALB/3T3 cells were treated with concentrations of 2.1, 1.05, 0.53, 0.27 and 0.14µg/ml in DMSO. The highest concentration caused a 25% reduction in colony forming ability as determined in a pre-test for cytotoxicity. This test system permits the morphological detection of changes due to transformations in mammalian cells induced by chemical substances. BALB/3T3 cells multiply in culture until a monolayer is formed, after which no further division and growth occurs. Treatment of the cells with chemical carcinogens gives rise to cellular transformations and subsequently to the development of visible colonies superimposed on the customary cell monolayer. In both negative controls, i.e. the vehicle-treated control and the untreated control, no colonies of transformed cells were detected. Also no transformed colonies were detected in cultures treated with Pigment Red 166. In the positive controls treated with methylcholanthrene (2.0 and 1.0µg/ml), the transformation-frequency values ((= number of transformed cells/10000 surviving cells) were 6.88 and 3,57, respectively. The study is assigned a validity of 2 because it was not performed according to GLP and did not take into account a metabolizing system.

 

 

Pigment Red 166 was tested for mutagenic effects on mouse lymphoma cells (L5178Y) in a host-mediated assay system (Ciba-Geigy Ltd 1981c). The study was not performed under GLP and does not follow a standard OECD genotoxicity test design, although it uses in part the design of the mouse lymphoma assay (OECD 476). Mutagenic effects of the substance are demonstrable on comparing the number of colonies in cultures of target cells harvested from treated mice with that from control animals. Since the intact animal possesses the ability to detoxify and eliminate the potentially mutagenic compound, or alternatively the compound may only produce mutagenic effects after metabolic activation, the host-mediated assay is conducted to allow inferences regarding the effects of the compound in vivo. L5178Y-cells were inoculated intraperitoneally (1 000 000 cells/animal). Three days after inoculation of the target cells the substance was given orally to four animals in the stated dose (5000 mg/kg). A further group of four animals served as control. Three days after the administration of the substance, the cells were removed from the peritoneal cavity under aseptic precautions and mutants were selected by treatment with antimetabolites. As the mutant frequency did not increase above a factor of 2.5, Pigment Red 166 was found to be non mutagenic. The study did not include a positive control experiment, which is why it is not considered reliable.

 

Pigment Red 166 was tested for its ability to induce sister chromatide exchange in bone marrow cells of Chinese hamsters according to EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay) guideline with the deviations that only four animals per sex and dose group were used (Ciba-Geigy Ltd 1981d). Animals received a single gavage dose of 1250, 2500, 5000 mg/kg in PEG400. The animals were sacrificed 24 h after the application

and 2 h after an intraperitoneal injection of colcemide at 10 mg/kg. From the bone marrow drop-preparations were made and stained according to a modified fluorochrome plus Giemsa technique. The number of SCEs in animals from the control group and in those treated with the various doses was not significantly different. A "positive control" experiment with DMBA (100 mg/kg) yielded a mean value of 11.1 SCEs per cell. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

 

 

Pigment Red 144 (CAS 5280-78-4, 829 g/mol)

A GLP and OECD 471 compliant study with modification for azo compounds was performed with Pigment Red 144 (Clariant 2007). The first experiment used the plate incorporation assay with rat liver S9 and the second experiment the pre-incubation test with hamster liver S9. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were tested. The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000mg/plate. The plates incubated with the test item showed normal background growth up to 5000mg/plate with and without metabolic activation in both experiments. No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and proved the validity of the experiment.

A GLP compliant standard Ames test was performed in 2009 (VUOS). The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471. Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in DMSO and assayed in doses of 15-5000 mg/plate which were applied to plates in volume of 0.1 mL. Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors. Pigment Red 144 was non mutagenic for all the used bacterial strains with as well as without metabolic activation.Appropriate reference mutagens were used as positive controls and proved the validity of the experiment.

 

A non GLP compliant Ames test with four tester strains also showed absence of mutagenicity (BASF 1974).

 

Pigment Brown 41 (CAS 68516-75-6, 844 g/mol)

 

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (RCC 1983). The test was performed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 158,500,1580 and 5000 micrograms per plate. No toxic effect of the test material was observed. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. The presence of microsomal activation did not influence these findings. Positive control

incubations confirmed the validity of the study. Pigment Brown was found to be non mutagenic.

 

Another Ames test was performed using tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Escherichia coli WP2 uvrA (HRC 1993). DMSO was used as vehicle; tested doses were between 10 and 5000 micrograms per plate. The S9 fraction of liver homogenate from rats induced with polychlorinated biphenyl was used to simulate metabolic activation. An independent repeat experiment was not included. Positive control incubations confirmed the validity of the study. Pigment Brown 41 was found to be non mutagenic.

 

Pigment Brown 23 (CAS 35869-64-8, 850 g/mol)

 

Pigment Brown 23 was found to be non mutagenic in two Ames tests, one of them being a GLP and OECD 471 compliant study with five tester strains (Ciba-Geigy 1985). Pigment Brown 23 was not genotoxic in a micronucleus assay in vivo and a test for unscheduled DNA repair in liver in vivo.

Borderline mutagenic effects were observed in a third Ames test which is in the ownership of JHOSPA and of which the lead registrant was informed (JHOSPA 2010). As this occurred at precipitating concentrations, it may be due to an impurity. The assay was a standard assay using rat liver S9 for metabolic activation and the pre-incubation method.

 

In the GLP and OECD 471 compliant Ames test using S.typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, the compound was tested as a suspension in DMSO in the presence and absence of S9 metabolic enzymes and at doses of 20 to 5000μg/plate (Ciba-Geigy 1985). In the experiments performed without and with microsomal activation, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Owing to a growth-inhibiting effect of the substance in the experiments without and with microsomal activation a reduction in the colony count was observed at the highest concentration. At the concentrations of 78μg/0.1 ml and above the substance precipitated in soft agar. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

The older Ames test was performed in the presence and absence of rat liver S-9 microsomal activation system and at doses of 0,2 to 2000μg/plate (Batelle 1979). S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 did not show an increased number of revertant colonies. DMSO was used as solvent. No independent repeat experiment was included. Positive and negative control substances gave the expected results and confirmed the validity of the study.

 

In order to determine mutagenicity in vivo, the substance was assessed for its potential to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in hepatocytes of Wistar rats in vivo at 3-hour and 14-hour sampling time (BASF 2010a). The study was performed under GLP and followed OECD testing guideline 486. For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. No increase in unscheduled DNA repair activity compared to vehicle treated animals was observed. The positive control substance 2-acetylaminofluorene administered once orally in a dose of 50 mg/kg body weight led to a relevant increase in unscheduled DNA synthesis activity.No signs of toxicity were observed at both sacrifice intervals.Discolored feces was observed at the 3 and 14 hours sacrifice interval in both test groups. No reduced viability of hepatocytes as indication for test substance induced toxicity was observed.

 

The substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (BASF 2010b). For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulphate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

 

Pigment Red 214 (CAS 40618-31-3, 863 g/mol)

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (RCC 1989). Liver homogenate from rats induced with Arochlor 1254 was used for metabolic activation. The test material was tested at six concentrations ranging from 10 to 5000 micrograms per plate with DMSO as vehicle. Toxic effects, evidenced by a reduction in the spontaneous revertants, occurred in some of the test and without metabolic activation at the highest concentration. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. Positive control incubations confirmed the validity of the study. Pigment Red 214 was found to be non mutagenic.

 

Pigment Red 220 (CAS 68259-05-2, 926 g/mol)

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (Ciba-Geigy 1980).

The test was performed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 158,500,1580 and 5000 micrograms per plate. No toxic effect of the test material was observed.

Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. The presence of microsomal activation did not influence these findings. Positive control incubations confirmed the validity of the study. Pigment Brown was found to be non mutagenic.

 

 

 

Pigment Red 221 (CAS 71566-54-6, 926 g/mol)

Pigment Red 221 was tested for mutagenic effects in vitro in five histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli (Ciba-Geigy Ltd 1994). The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The study was performed under GLP and according to OECD

testing guideline 471. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in ethanol and tested at five concentrations in the range of 312.5 to 5000.0 mg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the concentrations of 61.7 to 5000.0 mg /plate. In the first and second mutagenicity test, it was tested as a suspension at all concentrations. In order to clarify if solutions of the test material yield different results, a further repetition was performed at concentrations in the range of 1.6 to 5000.0mg/plate. In this repetition the lowest substance concentration was completely dissolved in ethanol. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In all three experiments, performed with and without metabolic activation, none of the tested concentrations led to an increase in the incidence of either histidine- or tryptophan prototrophic mutants by comparison with the negative control.

 

Pigment Red 221 was investigated for clastogenic (and/or aneugenic) effects on mouse bone marrow cells in vivo in a GLP compliant study following OECD testing guideline 474 (Ciba-Geigy Ltd 1995). The test substance was administered once by gavage to groups of 5 male and 5 female Tif MAGf (SPF) mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamide (64 mg/kg). From the high dose group and from the negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. The high dose applied (limit dose) was toxic as manifested in the tolerability test by various symptoms. In all dosage groups assessed at the different periods post treatment, no biologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group.

 

 

Pigment Red 242 (CAS 52238-92-3, 931 g/mol)

 

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (CCR 1991). Liver homogenate from rats induced with Arochlor 1254 was used for metabolic activation. The test material was tested at five concentrations ranging from 10 to 5000 micrograms per plate with DMSO as vehicle. Toxic effects were not observed. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. Positive control incubations confirmed the validity of the study. Pigment Red 242 was found to be non mutagenic.

 

A non GLP compliant Ames test with four tester strains also showed absence of mutagenicity (Sandoz 1979).

 

Pigment Red 242 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in a GLP compliant study following OECD testing guideline 473 (CCR 1992).

Preparation of chromosomes was done 18 h (low, medium and high concentration range), and 28 h (high concentration range) after start of treatment with the test article which was dissolved in a mixture of DMS0/Ethanol (15:85). The treatment interval was 4 h.

In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control culture where 25

metaphases were scored.The concentrations of the test article were chosen according to

the pre-experiment for toxicity and the solubility of the test article. Concentrations of 1.0 mg/Land higher could not be dissolved totally either in aqueous or organic solutions. Without S9 mix concentrations were 3, 6 and 15mg/ml (18h) or 15mg/ml (28h). With S9 mix, concentrations were 1, 3 and 10mg/ml (18h) or 10mg/ml (28h). In the main experiment at least slight reductions of mitotic index could be observed after treatment with the respective highest concentration scored at fixation interval 28 h. There was no biologically relevant increase in cells with structural aberrations after treatment with any concentration of the

test article either with or without metabolic activation by 89 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Justification for selection of genetic toxicity endpoint
Valid in-vivo study for genotoxicity. But more than one study needed for this endpoint.

Short description of key information:
Adequate and reliable experimental data on mutagenicity in bacteria is available for all disazocondensation red pigments. The Ames tests for Pigment Red 144 and 166 were also performed using the Prival modification for azo compounds.
Adequate and reliable experimental data on clastogenicity is available for four disazocondensation red pigments (PR 221, PR 242, PR 166, PB 23)
Adequate and reliable experimental data on mutagenicity in mammalian cells is available for two disazocondensation red pigments (PR 166 (hprt), PB 23 (UDS in vivo)) .

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

 

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. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.