A mixture of: N-(4-chlorophenyl)-4-(2,5-dichloro-4-(dimethylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide; N-(4-chlorophenyl)-4-(2,5-dichloro-4-(methylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide

Endpoint summary

Additional information

Discussion

Pigment Orange 74 did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test Prival modification, test strains: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) with and without metabolic activation (rat liver S9 mix, and hamster liver S9 mix) at concentrations up to 5000 µg/plate.

Pigment Orange 74 didn't cause structural chromosomal aberrations when tested in the in vitro mammalian chromosome aberration test using Chinese hamster lung fibroblasts, neither with nor without metabolic activation at concentrations up to 50.2 µg/ml.

Pigments with similar chemical structure (PR 112, PR 146, PR 147, PR 170) were not genotoxic in the mammalian cell gene (HPRT) mutation test in V79 Chinese Hamster cells when tested with and without rat liver S9 metabolic activation.

In conclusion the test items did not induce gene mutations in bacteria or mammalian cells and didn't cause structural chromosomal aberrations in mammalian cells.

Read across justification

The purpose of this assessment is to provide justification for read across in order to predict the potential genetic and reproductive toxicity of the target substance Pigment Orange 74 based on available date coming from a set of source substances (Pigment Red 22, 112, 146, 147, 179).

The pigments used for read across are structurally similar and differ only by different substituents in the common core molecule. Target and source substances are solids which decompose or melt at high temperatures (>= 237°C). Solubility in water or n-octanol is very low or low (< 12 μg/L and < 3.4 mg/L, respectively). For the target substance solubility in water was determined to be below the limit of detection (<0.1 mg/L) and in n-octanol 0.16 mg/L. These values suggest poor absorption and low bioavailability for all pigments involved in this evaluation. The log n-octanol-water partition coefficients are in the range of 1.28 to 2.5 for all of the source pigments which fit together with log n-octanol-water partition coefficient of Pigment Orange 74 (>0.2). These values are far below the limit of concern considered to be critical for bio-accumulative properties. All pigments showed very limited biodegradability, which is assumed to be due to their unavailability for microorganisms. Lacking bioavailability is probably also the reason for the absence of any relevant mammalian toxicity: None of the source pigments showed any toxic effects after single oral or dermal or after repeated oral exposure including effects on reproductive performance (examined for Pigment Red 22 and 170). This pertains also for the target substance with the exception of one death in association with lung coloration observed after single oral application (2000 mg/kg bw) which might be attributed to incorrect dosing or inhaling of foamed vomit. A spontaneous not treatment related death cannot be ruled out as well. This seems not unlikely in view of the fact that no such effects were observed in an oral repeated dose study. Target and source pigments have no skin sensitising effects. All pigments are not mutagenic in the Bacterial Reverse Mutation Assay or the Mammalian Chromosomal Aberration Test. Negative results were also obtained for the source substances in the In vitro Mammalian Cell Gene Mutation Test (HPRT).

Due to the very low solubility of the pigments it can reasonably be assumed that the members of this read across approach are (nearly) not present in a dissolved form on the skin or mucous membranes after dermal, oral or inhalative exposure, i.e. they could not be absorbed via skin and mucous membranes. This conclusion is supported by the observation that the pigments evaluated do not exert any relevant toxicity.

This assessment is based on experimental data on the source pigments as compared to available data of the target pigment covering the following endpoints: Acute oral or dermal toxicity, skin and eye irritation, skin sensitisation, genotoxicity in vitro, subacute oral toxicity, toxicity to reproduction, and inherent biodegradability (for details see data matrix below).

In conclusion, the similar structure and the uniformity of physicochemical, environmental fate and toxicological properties justifies the application of read across to predict the outcome of a Developmental/Reproduction Screening Test and an In Vitro Mammalian Cell Gene Mutation Test (HPRT) of the target pigment based on available date coming from several source pigments. The minor differences in the structure do not significantly alter the basic physicochemical properties or the basic biological effects.

Data Matrix

CHEMICAL NAME	Pigment Orange 74	Pigment Red 22	Pigment Red 112	Pigment Red 146	Pigment Red 147	Pigment Red 170
Role	Target Substance	Source Substance	Source Substance	Source Substance	Source Substance	Source Substance
CAS No.	85776-14-3	6448-95-9	6535-46-2	5280-68-2	68227-78-1	2786-76-7
PHYSICAL AND CHEMICAL PROPERTIES
State of the substance at 20° C and 101,3 kPa	orange solid	red solid	red solid	red solid	red solid	red solid
Melting/freezing point	>= 278.2°C, <= 304.9°C	decomp. starting at 237°C, 534 J/g	decomp. starting at 270°C, 300 J/g	decomp. starting at 283°C, 418 J/g	decomp. starting at 278°C, 92 J/g	decomp. starting at 313°C, 80 J/g
Water solubility	< 0.1 mg/L (below the detection limit)	11.8 μg/L	9.80 μg/L	8.7 μg/L	10 μg/L	11.9 μg/L
n-Octanol solubility	0.16 mg/L   0.192 mg/100 g fat	1.80 mg/L	3.31 mg/L	0.100 mg/L	0.74 mg/l	0.225 mg/L
log Partition coefficientn-octanol/water	> 0.2 (exact value could not be determined because water solubility was below the detection limit)	2.18	2.5	1.87	1.87	1.28
Stability in organic solvents and identity of relevant degradation products	>72 h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and sesame oil	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol
TOXICOLOGICAL INFORMATION
Skin irritation	not irritating	not irritating (in vitro)	not irritating	not irritating	not irritating (read across)	not irritating
Eye irritation	not irritating	not irritating (read across) b	not irritating	not irritating	not irritating (read across)	not irritating
Skin sensitization	not skin sensitising	not skin sensitising (read across)	not skin sensitising	not skin sensitising	not skin sensitising	not skin sensitising
In vitrogene mutation study in bacteria	not mutagenic	not mutagenic	not mutagenic	not mutagenic	not mutagenic	not mutagenic
In vitrocytogenicity study in mammalian cells	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (MN in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)
In vitrogene mutation study in mammalian cells	RA-conclusion: not mutagenic (HPRT in V79 cells)	not mutagenic (read across)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)
Otherin vivomutagenicity tests	No data requirement	not mutagenic (read across)	not mutagenic (read across)	not mutagenic (read across)	not mutagenic (UDS in vivo)	not mutagenic (read across)
Acute toxicity, oral route, (LD50 mg/kg b.w., rats)	> 2000	> 2000 (read across) a	> 5000 (male/female)	> 10000 (female)	> 2000 (read across)	> 15000 (male/female)
Acute tocity, inhalation (LC50 mg/L, rats )	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available) (>1580 mg/m3/4h; RL3)
Acute toxicity dermal route (LD50 mg/kg b.w., rat)	> 2000 (male,female)	> 2000 (read across)	> 5000 (male)	> 2000 (read across)	> 2000  (read across)	> 2000 (male,female)
Short-term repeated dose toxicity study in rats (oral)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 422)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg/day (read across)	NOAEL ca. 1200 mg/kg bw (highest dose tested; OECD 407)
Sub-chronic toxicity study in rats (oral)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short -term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)
Carcinogenicity	No data requirement	No data requirement	No data requirement	No data requirement	No data requirement	No data requirement
Screening for reproduction/developmental toxicity, rats	RA-conclusion: NOAEL 1000 mg/kg bw	Applied Source Substance: NOAEL 1000 mg/kg bw (highest dose tested; OECD 422)	NOAEL 1000 mg/kg/day (read across)	NOAEL 1000mg/kg/day (read across)	NOAEL 1000 mg/kg/day (read across)	Applied Source Substance: NOAEL 1000 mg/kg bw (highest dose tested; OECD 421)
Toxicokinetic behaviour	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability
ENVIRONMENTAL FATE
Inherent biodegradability	Not readily biodegradable	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)

Justification for selection of genetic toxicity endpoint
Evaluation of genetic toxicity is based on a battery of different study protocols comprising in the case of consideration:
KEY_471_Prival_2014_Harlan_1608600, Bacterial Reverse Mutation Test Prival Modification
KEY_473_1993_HAZLETON_15576_0_437C0, In vitro Mammalian Chromosome Aberration Test
RA_PR112_KEY_476_HPRT_2008_RCC_1154200, Read Across, In vitro Mammalian Cell Gene Mutation Test, HPRT
RA_PR146_KEY_476_HPRT_2008_RCC_1136801, Read Across, In vitro Mammalian Cell Gene Mutation Test, HPRT
RA_PR147_KEY_476_HPRT_2007_RCC_1075800, Read Across, In vitro Mammalian Cell Gene Mutation Test, HPRT
RA_PR170_KEY_476_HPRT_2008_RCC_1136601, Read Across, In vitro Mammalian Cell Gene Mutation Test, HPRT

Short description of key information:
Mutagenic properties of Pigment Orange 74 were investigated in a bacterial reverse mutation assay (Prival modification; tester strains used: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2), an in vitro mammalian chromosome aberration test (Chinese hamster lung fibroblasts (V79)), and additionally in 4 in vitro Mammalian Cell Gene Mutation Tests, HPRT, which were performed with pigments of similar chemical structure (RA, Chinese hamster lung fibroblasts (V79)).

Negative results were obtained in all studies of the entire test battery.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Pigment Orange 74 did not reveal mutagenic effects in the bacterial reverse mutation assay and did not cause chromosomal aberrations in the in vitro mammalian chromosome aberration test. 4 Pigments with similar chemical structure were not genotoxic in mammalian cell gene (HPRT) mutation tests.

Therefore Pigment Orange 74 is considered not to be mutagenic and does not have to be classified for mutagenicity according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008.