1-[(2-chloro-4-nitrophenyl)azo]-2-naphthol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I (including the additional experiment with strain TA 98): 3; 10; 33; 100; 333; 1000; 2500; and 5000 ug/plate.
Experiment II: 33; 100; 333; 1000; 2500; and 5000 ug/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
positive with metabolic activation

Under the experimental conditions reported, the test item induced gene mutations by frameshifts in the genome of the strain TA 98 in the presence of metabolic activation. Therefore, C.I Pigment Red 4 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of C.I. Pigment Red 4 to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two complete experiments with and without liver microsomal activation and one additional experiment solely with strain TA 98 in the presence of metabolic activation. The results of the additional experiment are reported as part of experiment I. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I (including the additional experiment with strain TA 98): 3; 10; 33; 100; 333; 1000; 2500; and 5000mg/plate.

Experiment II: 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 bath independent experiments.

 

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers was observed following treatment with C.I Pigment Red 4 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) in strains TA 1535, TA 1537, TA 100, and WP2 uvrA. However, a substantial increase was observed in strain TA 98 in the presence of metabolic activation in both independent experiments. The required threshold of two times the mutation frequency of the corresponding solvent control was well exceeded at concentrations as low as 3mg/plate in experiment I and 33mg/plate in experiment II. To verify the results of experiment I, an additional plate incorporation test was performed with strain TA 98 in the presence of metabolic activation. In this additional experiment the result of the first experiment was confirmed.

 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat S9-mix

Test concentrations with justification for top dose:

First main experiment:
without S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7 and 3277.3* ug/ml
with S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7 and 3277.3* ug/ml

Second main experiment:
without S9-mix: 78.1, 156.3, 312.5, 625 ,937.5, 1250.0, 1875, 2500 and 3277.3* ug/ml
with S9-mix: 78.1, 156.3, 3 l2.5, 625, 937.5, 1250.0, 1875, 2500 and 3277.3* ug/ml

*= 10 mM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

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:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

C.I Pigment Red 4 is not mutagenic in this in vitro HPRT assay with V79 Chinese hamster lung cells

Executive summary:

In this study the potential of C.I. Pigment Red 4 (Batch No. BRAB 036744) to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster lung in vitro was investigated.

The test item was suspended in cell culture medium and tested at the following concentrations:

First main experiment:

without S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7 and 3277.3* ug/ml

with S9-mix: 6.4, 12.9,25.7, 51.3, 102.5,204.9,409.7, 819.4, 1638.7 and 3277.3* ug/ml

Second main experiment:

without S9-mix: 78.1, 156.3, 312.5, 625 ,937.5, 1250.0, 1875, 2500 and 3277.3* ug/ml

with S9-mix: 78.1, 156.3, 3 l2.5, 625, 937.5, 1250.0, 1875, 2500 and 3277.3* ug/ml

*= 10 mM

at 6.4 = no selection was performed because higher concentrations were used

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced no severe toxic effects with and without metabolic activation in both main experiments.

Higher concentrations were not applied because of the 10 mM limitation (OECD guideline).

The test item produced microscopic precipitation down to a concentration of 25.7 ug/ml after treatment time. Macroscopic precipitation of the test item was observed at 204.9 ug/ml after treatment time and higher concentrations.

This GLP study was experimentally initiated on 23-May-2005.

Up to the highest investigated dose the test item induced no relevant of dose-dependent increase in mutant colony numbers in two independent experiments. Appropriate reference mutagens used as positive controls showed a significant increase in mutant colonies, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

In conclusion, C.I Pigment Red 4 did not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

C.I. Pigment Red 4 is considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction.

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 44012008 of 30 May 2008 and the USA EPA OPPTS guideline 40 CFR 799.9537. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Test item (ug/ml) 25, 50, 100, 200, 400, 800

Results.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The maximum dose tested was limited by formulation difficulties and was also limited by precipitate of the test item occurring on the slides at the maximum dose and restricting the ability to accurately Score metaphases.

Conclusion.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.11. 2019 - 16.01.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.62 In Vivo Mammalian Alkaline Comet Assay, Commission Regulation (EU) No 2017/735

Version / remarks:

adopted 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld , Germany
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation:
M: 220 - 419 g
F: 203 - 262 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were kept in groups of 2 - 3 by sex in MAKROLON cages (type III plus). Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
- Diet: ad libitum
- Water: ad libitum
Certificates of analysis of diet, drinking water and bedding material are QAU archived.
- Acclimation period: 6 - 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 10%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.8% aqueous hydroxypropylmethylcellulose - Methocel, batch no. 13D03-N03, Fagron GmbH & Co. KG, 22885 Barsbüttel, Germany

- Justification for choice of solvent/vehicle: 0.8% aqueous hydroxypropylmethylcellulose was chosen as vehicle as it is known not to produce any toxic effects according to known available information.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VERSAL RED 2GC was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The administration volume was 20 mL/kg b.w..

In line with normal practice in this type of in vivo study, no analysis of the dose form is required. A test on stability and homogeneity was not necessary as the test item formulation was prepared immediately before administration.

Duration of treatment / exposure:

45 hours

Frequency of treatment:

Three times at 0, 24 and 45 hours

Post exposure period:

3 hours

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s): not specified, it is an example of positive control substance from OECD 489 for any tissue
- Route of administration: oral by gavage, administered two times at 24 and 45 hours
- Doses / concentrations: 125 mg/kg b.w./day

Tissues and cell types examined:

M: liver, glandural stomach, duodenum cells, testicle
F: ovaries

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels were selected based on the results of a Limit preliminary experiment to determine the maximum-tolerated-dose (MTD) using two animals per dose. Based on the results the maximum-tolerated dose level (100%; 2000 mg/kg b.w./day, p.o), a medium-tolerated dose level (50% MTD) and a lower dose (20% MTD) were tested. No signs of systemic toxicity were noted at the highest dose level of 2000 mg/kg b.w./day, p.o.
In line with normal practice in this type of in vivo study, no analysis of the dose formulation is required.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were treated with the test item at three intervals of 0, 24 and 45 hours and liver tissues were sampled at 48 hours (3 hours after the last dose) in order to accommodate the sampling requirements of the comet assay. Treatment with the positive reference item was performed twice: at 24 and 45 hours (3 hours before sampling).

DETAILS OF SLIDE PREPARATION:
For the preparation of slides the CometAssay® HT Kit (Trevigen) was used according to the manufacturer´s instructions. Each cell suspension was mixed 1:10 with low-melting agarose. An adequate amount of the cell-agarose mixture was applied to two wells of the 20-well microscope slide. The preparation of the slides was completed within two (2) hours after sacrifice.
The prepared slides were incubated in a lysis buffer overnight at +2°C to +8°C. After incubation, the slides were incubated in freshly prepared alkaline unwinding solution (pH>13) for 20 minutes at room temperature.

METHOD OF ANALYSIS:
The slides were analysed by epifluorescence microscopy at 200 x magnification. At least 150 liver cells per animal and tissue were evaluated using the Comet Assay IV software (Perceptive Instruments Ltd).

Evaluation criteria:

Percent tail DNA (% tail intensity) was determined to assess DNA damage. The DNA fragment intensity in the tail was expressed as a percentage of the cell's total intensity. The median % tail DNA for each animal was calculated. The mean of the individual animal’s median was then determined.

Only cells of good morphology (clearly defined head and tail with no interference with neighbouring cells) were scored for % tail DNA. Artefacts were avoided. The occurrence of hedgehogs was determined based on the visual scoring and tabulated but not evaluated and reported.

Statistics:

Generally, means and standard deviations were calculated. Intergroup comparisons with the control group were made by an analysis of variance (ANOVA) followed by the DUNNETT multiple comparison test (p ≤ 0.05 and p ≤ 0.01) for the cells of liver and testis. A square root transformation of the data was needed in the case of stomach cells because of the high standard deviation. In addition, a nonparametric ANOVA test (Kruskal-Wallis Test) was needed in the case of duodenum and ovary cells, due to the failing of the normality test. The positive control data were compared to the values of the vehicle control group values by unpaired Student's t-test (t) (normal distribution) (WILCOXON MANN-WHITNEY Test with StatXact 4 (not normal distributed). Significantly different data were indicated in the tables of the report. A possible dose-response-relationship would have been examined by linear regression analysis employing PEARSON's correlation coefficient.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

File with data on study results is attached (PigmentRed4_CometAssay_result tables.pdf)

Conclusions:

In conclusion, under the present test conditions VERSAL RED 2GC tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration to male and female rats for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing cells of the liver, glandular stomach, duodenum and testis (males) or ovaries (females).
In the same system, ethyl methane sulfonate (positive reference item) induced significant damage.

Executive summary:

VERSAL RED 2GC was assayed in an in vivo alkaline comet (single cell gel electrophoresis) assay for the detection of DNA strand breaks in cells or nuclei isolated from liver, glandular stomach, duodenum and testis tissue of male CD rats, as well as ovary tissue from female CD rats. The test item was administered orally to rats once daily for three days at 0, 24 and 45 hours, and the tissues were sampled at 48 hours (3 hours after the last dose).

VERSAL RED 2GC was suspended in 0.8% aqueous hydroxypropylmethylcellulose. The dose levels for the main study had been selected based on the result of a preliminary Limit test of 2000 mg/kg b.w./day, p.o employing two animals per dose. No signs of systemic toxicity were noted.

Three ascending dose levels of 400, 1000 or 2000 mg VERSAL RED 2GC/kg b.w./day, p.o. and the vehicle (0.8% aqueous hydroxypropylmethylcellulose) were administered three times at 0, 24 and 45 hours. The positive reference item (125 mg ethyl methane sulfonate/kg b.w./day, p.o.) was administered two times at 24 and 45 hours. Each group consisted of 5 male and 5 female rats. The administration volume was 20 mL/kg b.w./day. The test was divided in 4 different preparation groups containing one or two animals of each group which were tested in parallel at each stage of the experiment.

No signs of systemic toxicity were noted up to the highest dose level of 2000 mg VERSAL RED 2GC/kg b.w./day, p.o.

150 cells of liver, glandular stomach, duodenum and testis (males) or ovaries (females) per animal and tissue collected 48 hours post 1st administration were evaluated. The grade of DNA fragmentation is expressed in percent tail DNA (% tail intensity).

Treatment with VERSAL RED 2GC did not cause any test item-related increase in the tail intensity for the cells of liver, glandular stomach, duodenum and testis (all males) or ovaries (females) at any of the three tested dose levels of 400, 1000 and 2000 mg test item/kg b.w./day compared to the vehicle control data.

The group mean values of the median tail intensities of the test item treatment groups were between 6.325% and 7.83%, 9.58% and 11.806% or 11.554% and 12.856% for liver, stomach and duodenum cells, respectively. The corresponding values for vehicle control were 4.726%, 8.152% or 12.554% for liver, stomach and duodenum cells, respectively. The mean values of the median tail intensity were between 5.341% and 6.220% or 1.082% and 1.681% for the cells of testis and ovaries, respectively. The corresponding values for vehicle control were 5.326% or 1.782% for the cells of testis and ovaries, respectively.

All values obtained were within the upper limits of the historical control data, except for the vehicle control group of stomach cells and test item treatment groups of liver and stomach cells which were slightly above the background data. As the slightly higher data are no extreme outliers, were within published historical control data and older rats at an age of 9 – 12 weeks were used, to assure maternity of the animals for evaluation of testis and ovary cells, the results are considered acceptable for the inclusion into the historical control data and the test is considered valid.

Administration of ethyl methane sulfonate (positive reference) significantly increased the % tail intensity for the cells of liver, glandular stomach, duodenum and testis (all males) or ovaries (females).

All values were well within the historical background data, except the positive control for the stomach and duodenum cells which were above the historical background data. As the positive control is significantly increased, the data are considered acceptable for the inclusion into the historical control data and the test is considered valid.

 

In conclusion, under the present test conditions VERSAL RED 2GC tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration to male and female rats for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay employing cells of the liver, glandular stomach, duodenum and testis (males) or ovaries (females).

In the same system, ethyl methane sulfonate (positive reference item) induced significant damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No adverse effects due to the test item C.I. Pigment Red 4 have been seen in vitro in OECD 473 and OECD 476 studies while ambiguous, positive and negative test results have been seen in OECD 471 study with and without metabolic activation.

Additional information from genetic toxicity in vivo:

The test item C.I. Pigment Red 4 tested up to the highest reasonable dose level of 2000 mg/kg b.w. per day by repeated oral administration to male and female rats for three days showed no genotoxic properties in the in vivo alkaline comet (single cell gel electrophoresis) assay (OECD 489) employing cells of the liver, glandular stomach, duodenum and testis (males) or ovaries (females).

Justification for classification or non-classification

Overall conclusion: the test item C.I. Pigment Red 4 is not considered to be mutagenic based on overall information from available studies.