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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Rationale for reliability of the study report related to the source substance: Guideline study (OECD 476), GLP compliant (original reliability: 1) - Read across hypothesis: The similar chemical 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimum essential medium containing 10% fetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (phenobarbital/beta-naphthoflavone-induced rat liver protein with cofactors)

Test concentrations with justification for top dose:

3.1, 6.3, 12.5, 25.0, 50.0 and 400 μg/ml with and without metabolic activation

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: w/o metabolic activation; 7,12-Dimethylbenz(a)anthracene with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (4h-incubation without serum, 24 h incubations with serum)


DURATION
- Preincubation period: none
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution


NUMBER OF REPLICATIONS: 5/experiment, 2 experiments


NUMBER OF CELLS EVALUATED: colonies >50 cells


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

test item classified as positive for mutagenicity if induces either concentration-related increase of mutant frequency or reproducible and positive response at one of the test points (at least three times above the spontaneous mutation frequency)

Statistics:

linear regerssion on dose-dependeny of mutant frequencies using SYSTAT statistics software

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at 12.5 µg/ml and higher (Exp. I without S9-mix), at 25.0 µg/ml and higher (Exp. I with S9-mix and Exp. II)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: negligible decrease (0.1 pH-units) at highest dose
- Effects of osmolality: negligible increase (+3 mOsm) at highest dose

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results of V79/HPRT assay with test item

Concentration [microgram/mL]	mutant colonies per 10^-6 cells**	mutant colonies per 10^6 cells**	Cytotoxicity (yes/no)	Remarks
	— MA	+ MA
0*	7.6, 6.2	5.2, 21.2	no	Exp. I
3.1	13.6, 8.5	11.7, 13.4	no
6.3	7.8, 14.7	8.6, 26.8	no
12.5	12.3, 8.6	13.4, 19.5	no
25.0	12.3, 10.9	7.4, 22.3	no
400	15.9, 10.6	13.1, 12.5	no
Positive control***	151.7, 87.5	1239.9, 1528.8	-MA: no; +MA: yes
0*	13.9, 11.3	-	no	Exp. II
3.1	11.4, 6.1	-	no
6.3	12.2, 7.7	-	no
12.5	17.1, 12.9	-	no
25.0	11.0, 9.6	-	no
400	5.5, 7.7	-	no
Positive control***	186.6,. 136.9	-	yes

*solvent control with DMSO

** mean of 5 plates each in culture I and culture II

***without MA: EMS, with MA: DMBA

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).

In conclusion, 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)
Reproductive Toxicity – Fertility	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)
Reproductive Toxicity – Developmental Toxicity	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)
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)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test item was 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 at concentrations up to 400 µg/mL.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments using duplicate cultures. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h.

The highest applied concentration (400 µg/mL) was limited by the solubility properties of the test item in DMSO and aqueous media.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies confirming the sensitivity of the test system and the activity of the S9 mix.