4-(4-nitrophenylazo)-2,6-di-sec-butyl-phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2 in Vitro Genotoxicity studies are available for Mortrace SB - In vitro gene mutation study in bacteria and in vitro cytogenicity / chromosome aberration study in mammalian cells. The test material showed positive results in the in vitro gene mutation study in bacteria and negative results in the in vitro cytogenicity / chromosome aberration study in mammalian cells.

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

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study did not include testing on Escherichia coli.

Principles of method if other than guideline:

Test Cultures:
Fresh cultures for mutagenesis testing were prepared by quickly thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2. After growth for approximately 10 hours at 37°C in an orbital shaking incubator, samples of each culture were diluted 1:4 in distilled water and optical densities were determined at 650 nm. Cultures with optical densities of 0.40 to 0.60 (representative of cells in late exponential or early stationary phase) were utilized for this study.

Control Articles:
Triplicate cultures of each strain were evaluated with the appropriate solvent in the presence and absence of S9 to serve as negative solvent controls. In order to validate the integrity of the test system, triplicate cultures of each tester strain were also evaluated with known positive control chemicals.

TOXICITY PRESCREEN:
Toxicity of the test article was determined in a preliminary toxicity prescreen by evaluating the growth of the background lawn and/or frequency of
spontaneous revertants.

Treatment with Test and Control Articles: Treatments were performed by combining 0.1 ml tester strain, 0.5 ml 0.2M NaH P04 (pH 7.4) and 0.1 ml test article or solvent in sterile glass tubes preheated to 30°C. The tubes were vortexed and incubated at 30°C for 30 minutes. Following the 30-minute incubation, 2 ml top agar (supplemented with 0.5mM histidine/0.5mM biotin) was added, and the mixture was poured onto minimal glucose plates, evenly distributed, and allowed to solidify. Within an hour the plates were inverted and incubated in the dark at 37°C for 48 hours.

Scoring: Following the 48-hour incubation, the background lawn and spontaneous revertants were scored for normal, inhibited or no growth. Inhibited growth was characterized by the absence of a confluent bacterial lawn and/or the presence of pindot colonies.

MUTATION ASSAY:
Salmonella which have undergone reversion to his+ form colonies in the absence of histidine. In contrast, his Salmonella can only undergo a limited number of doublings (due to the histidine supplement in the top agar) and form the typical background lawn. Following incubation for 48 hours, revertant colonies are enumerated with an automated colony counter. All mutation assays are performed in triplicate cultures in all five tester strains for each test article dose, as well as positive and solvent controls.

Treatment with Test and Control Articles: Treatment for the mutation assay was performed exactly as described in the toxicity pre screen, except that the test and control articles were evaluated in triplicate cultures in all five strains in the presence and absence of an exogenous metabolic activation system (S9). For cultures treated in the presence of S9, 0.5 ml of the S9 mixture replaced the 0.5 ml phosphate buffer. The S9 mixture contained 8mM MgCI , 33mM KCl, 4mM NADP, 20mM glucose-6- phosphate, 2.8 U/ml G6PDH, 2mM NADH, 2mM FMN, 100mM NaH2 Po4 (pH 7.4) and 30% (v/v) uninduced male Syrian Golden hamster liver homogenate.

Bacterial Contaminant Evaluation: To ensure the sterility of solvents, compounds and equipment, standard contamination evaluations were performed with the assay. The solvent, top agar, S9 mix, and top dose of the test article were evaluated at the same volumes used in the assay. The test article, solvent and S9 mix were evaluated as in the mutation assay, but in the absence of added Salmonella. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for bacterial growth.

Scoring: Revertant colonies were enumerated on an Artek electronic colony counter interfaced with an Apple computer for data acquisition. Solvent and positive controls were scored first, and test article treated cultures were scored only if the average negative control values were within historical ranges (see below). A summary of the results is presented in the Summary Data Tables (pages 8-10).

Deviations:
Working Cultures:
Original Statement: Fresh cultures for mutagenesis testing are prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2 for tester strains TA1537, TA1538 and TA98 and minimal glucose medium for strains TA1535 and TA100 in 125 ml screw-capped Erlenmeyer flasks.

Corrected Statement: Fresh cultures for mutagenesis testing are prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Specific histidine loci.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not appplicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mixture includiing 30% (v/v) uninduced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors.

Test concentrations with justification for top dose:

Marker SB was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0 and 167 ug/plate. Based on these results, Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard solvent

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-anthramine, benzidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Three

Evaluation criteria:

Evaluation Criteria:
A positive result is defined as a reproducible, statistically significant two-fold dose-dependent increase in the number of histidine-independent colonies, with at least one dose point inducing an average revertant frequency that is two-fold that of the solvent control. Significance is determined
using the program developed by Snee and Irr (1981). This program applies a linear regression analysis to the data points and any p value less than 0.05 is considered significant. Alternatively, if the test article produces an increase greater than or equal to three times the solvent control value (in the absence of a dose-dependent increase), the test chemical is considered positive. A negative result is defined as the absence of a dose-dependent two-fold increase in the number of histidine-independent revertants (or three-fold increase in the absence of any dose-dependency).

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Marker SB (non-volatile) was evaluated in a toxicity pre screen by treating duplicate cultures of strains TA1538 and TAI00 with five doses of Marker SB in the absence of S9. Results of the pre screen indicated Marker SB produced inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) at doses of 50'.0 and 167 ug/plate in strain TA1538, and at a dose of 50.0 ug/plate in strain TAlOO. Complete toxicity was observed at doses of 500, 1670 and 5000 ug/plate in strain TA1538, and at doses of 167, 500, 1670 and 5000 ug/plate in strain TAlOO. In addition, the test article was found to precipitate from solution at the 1670 and 5000 ug/plate dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9. Similar increases in revertant frequencies of approximately two- to five-fold were again observed in strains TA1538, TA98 and TAl00 in the retest. Although the revertant frequencies generally decreased at the highest dose level in each assay (150 or 167 ug/plate; due to toxicity), the increases observed over the remainder of the dose range were linear. All positive and negative control values in both assays were within acceptable limits.

Conclusions:

Interpretation of results (migrated information):
positive

The results for Marker SB (non-volatile) were positive in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

Executive summary:

Marker SB (non-volatile) was evaluated in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of Marker SB was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Marker SB in the absence of S9. Results of the prescreen indicated Marker SB produced inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) at doses of 50.0 and 167 ug/plate in strain TA1538, and at a dose of 50.0 ug/plate in strain TA100. Complete toxicity was observed at doses of 500, 1670 and 5000 ug/plate in strain TA1538, and at doses of 167, 500, 1670 and 5000 ug/plate in strain TA100. In addition, the test article was found to precipitate from solution at the 1670 and 5000 ug/plate dose levels.

Based upon these findings, Marker SB was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0 and 167 ug/plate. Three extra dose levels of Marker SB were evaluated with and without S9 in the event of unacceptably high toxicity at the highest dose levels. The S9 mixture included 30% (v/v) uninduced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors.

Revertant frequencies for all doses of Marker SB in all strains without S9, and strain TA1535 with S9, approximated or were less than those observed in the concurrent negative control cultures. However, increased revertant frequencies of approximately two- to four-fold were observed in strains TA1537, TA1538, TA98 and TA100 in the presence of S9. Therefore, Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9. Similar increases in revertant frequencies of approximately two- to five-fold were again observed in strains TA1538, TA98 and TA100 in the retest.

Although the revertant frequencies generally decreased at the highest dose levels in each assay (150 or 167 ug/plate; due to toxicity), the increases observed over the remainder of the dose range were linear. All positive and negative control values in both assays were within acceptable limits.

Therefore, the results for Marker SB (non-volatile) were positive in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

Reference 2

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:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This lest was according to the method described in SEPA (States Environmental Protection Administrat'ion of China) The Guidelines for the Testing of Chemicals, 473 in vitro Chromosome Aberration Test. The original report was written in Chinese and was translated to English. The full report is not available at this time. Information entered is from a condensed report for this study.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster cells

Details on mammalian cell type (if applicable):

- Type and identity of media: 1640 culture with 10% calf serum filtrated with 0.22 µm filter before use.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 metabolic activation system

Test concentrations with justification for top dose:

Four test levels were tested. Both in the non metabolic activation system (-S9 mix) and in the metabolic activation system (+S9 mix) for the 24h, the doses were 20, 10, 5, 2.5µg/ml. For 48h exposure in the non metabolic activation system, the doses were 12, 6, 3, 1.5 µg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard vehicle used.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION:
CHL cell treatment with test article - Test dish (diameter 60mm) with 5ml culture capacity was inoculated with test cells at 1.6x10E-4/ml for 24h exposure and 1.0x10E-4 /ml for 48h exposure.

DURATION
- Preincubation period: not applicable
- Exposure duration: 24 hrs both in the non metabolic activation system (-S9 mix) and in the metabolic activation system (+S9 mix), 48h exposure in the non metabolic activation system.
- Expression time (cells in growth medium): 24 hr with and without metabolic activation system, 48 hr without metabolic activation system.
- Fixation time (start of exposure up to fixation or harvest of cells): When preparing the slide, the solution was centrifuged and the sediment was smeared on the slide. After dryness, it was stained with Giemsa solution (PH 6.89) for 16-20 minutes. Then the slides were examined with microscope.

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): Giemsa solution (PH 6.89)

OTHER: Structure aberration examination was included chromatid-type aberration: single chromatid exchange and breakage. Chromosome-type aberration: sister chromosome exchange and breakage. Polyploids were also recorded. 100 metaphases for each test group were examined.

Evaluation criteria:

Microscopic examination:
Structure aberration examination was included chromatid-type aberration: single chromatid exchange and breakage. Chromosome-type aberration: sister chromosome exchange and breakage. Polyploids were also recorded. 100 metaphases for each test group were examined.

Criteria for aberration determination:
Aberration rate: <5% negative -
Aberration rate: 5-9% possible ±
Aberration rate: 10-19% positive +
Aberration rate: > 20% positive ++

Also repeated positive findings in one test point and dose-response relation were needed to judge a positive result.

Statistics:

Not available.

Species / strain:

mammalian cell line, other: Chinese hamster 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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
IC50 Testing:
IC50 was tested with non-metabolic activation (-S9mix, 24h and 48h exposure) system and metabolic activation (+S9mix, 24h exposure) system. 1 vehicle control and 3 test concentrations were performed with duplicate treatment. The results showed the IC50 were 15 µg/ml for 24h exposure in the non metabolic activation system and 10 µg/ml for 48h exposure in the non-metabolic activation system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Microscopic examination result (-S9 mix) with 24 h exposure to test article

Group		Doseµg/ml		Metaphase		Gap		ctb		csb		cte		cse		Struct rate		Pol %		Result
vehicle		0		100		1		1		0		0		0		1.0		1		-
Test Article		20		100		0		1		0		0		0		1.0		1		-
		10		100		2		0		0		0		0		0.0		0		-
		5		100		3		1		0		1		0		2.0		2		-
		2.5		100		1		0		0		0		0		0.0		0		-
MMC		0.10		100		3		5		2		17		4		28.0		0		++

Gap was not used to judge final result, ctb means chromatid break, cte means chromatid exchange,

csb means chromosome break, cse means chromosome exchange, pol means polyploid, struct rate means structural aberration rate.

 

Table 2. Microscopic examination result (+S9 mix) with 24 h exposure to test article

Group		Doseµg/ml		Metaphase		Gap		ctb		csb		cte		cse		Struct rate		Pol %		Result
vehicle		0		100		0		1		0		0		0		1.0		1.0		-
Test Article		20		100		0		0		0		0		0		0.0		0.0		-
		10		100		0		1		0		1		0		2.0		1.0		-
		5		100		0		1		0		0		0		1.0		0.0		-
		2.5		100		1		0		0		1		0		1.0		0.0		-
CP		20		100		5		6		3		16		5		30.0		0.0		++

 

Table 3. Microscopic examination result (-S9 mix) with 48 h exposure to test article

Group	Doseµg/ml	Metaphase	Gap	ctb	csb	cte	cse	Struct rate	Pol %	Result
vehicle	0	100	0	0	0	0	0	0.0	0.0	-
Test Article	12	100	0	1	0	0	0	1.0	0.0	-
	6	100	1	0	0	1	0	1.0	1.0	-
	3	100	0	0	0	0	0	0.0	0.0	-
	1.5	100	1	0	0	1	0	1.0	1.0	-
MMC	0.05	100	5	4	2	17	3	26.0	0.0	++

 

Conclusions:

Interpretation of results (migrated information):
negative

The test article, MARKER 1, showed negative results for chromosome aberration potential in the test system.

Executive summary:

CHL (Chinese hamster cells) Chromosome Aberration Test of MARKER 1 was conducted to determine if the test article would cause chromosome aberrations in CHL cultured mammalian cells.

Metaphase analysis of the CHL was perfonned. Four test levels were tested. The doses were selected by reference with the IC50. The highest concentration of test substance distributed in the culture medium was higher than the IC50, both in the non metabolic activation system (-S9 mix) and in the metabolic activation system (+S9 mix) for the 24h, the doses were 20, 10, 5, 2.5 µg/ml. For 48h exposure in the non metabolic activation system, the doses were 12, 6, 3, 1.5 µg/ml. Meanwhile control treatments were performed at same time, that is, vehicle control, positive controls (mitomycin C, MMC) for non-metabolic activation system (-S9 mix) and positive control (cyclophosphamide, CP) for metabolic activation system (+S9 mix).

Microscopic examination was performed to determine the chromosome aberration potential of the test article.

The final findings showed that chromosome aberration rate in all treatment doses was <5%, that is, a negative result for the test article. On the other hand, the two positive control treatments (MMC and CP) showed chromosome aberration rates >20% and indicated the test system condition was reliable. So the conclusion is that the CHL test showed negative results for the chromosome aberration potential of the test article MARKER 1.

Genetic toxicity in vivo

Description of key information

4 Genotoxicity studies are available for Mortrace SB - Ames assay (Prival modification); in vitro Chromosome Aberration assay, in vivo mouse micronucleus and a Pig A in vivo Gene mutation assay (addon to the OECD 421 reproscreen study).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 474, EEC Directive 84/449, L 251, B.12 and in accordance with the Principles of Good Laboratory Practices (GLP)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Enviromuental Protection Agency, Code of Federal Regulations, Title 40, Subpart F—Genetic Toxicity, Revision July 1, 1986 “In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.”

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 grams
- Assigned to test groups randomly: yes
- Fasting period before study: Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum
- Housing: individually housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: recommeded by various regulatory agencies
- Amount of vehicle (if gavage or dermal): 20 ml/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in corn oil. The vehicle was chosen to its relative non—toxicity for the animals. All animals received a single standard volume of 20 ml/kg body weight orally.

Duration of treatment / exposure:

single

Frequency of treatment:

single

Post exposure period:

At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

Twelve animals, six males and six females, were treated per dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): recommended by various regulatory agencies
- Route of administration: orally, once
- Doses / concentrations: 30 mg/kg body weight

Tissues and cell types examined:

not applicable

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Gieznsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test article, is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.

Statistics:

Standard statistical methods were employed

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY - In pre-experiments 4 animals (2 males, 2 females) received orally a single dose of 500, 1000, 2000 or 5000 mg/kg b.w., respectively, Mortrace SB Conc. formulated in corn oil and the volume administered was 20 ml/kg b.w.
Clinical signs of toxicity such as reduction of spontaneous activity, eyelid closure, apathy and abdominal position were noted in the 500 and 1000 mg/kg dose group. The above symptoms along with mortality (50%) was noted in the higher doses of 2000 and 5000 mg/kg bw.. On the basis of these results 1000 mg/kg b.w. Mortrace SB Conc. was estimated to be close to the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY -
In the micronucleus assay 7 out of 36 animals treated with Mortrace SB Conc. died. The mean number of normochromatic erythrocytes was not increased aftertreatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that Mortrace SB Conc. had no cytotoxic properties.
In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Mortrace SB conc. were in the same range as compared to the negative control groups.
30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

None

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

The test article Mortrace SB Conc. was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered orally was 20 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Twelve animals per test group were treated and if available (lethalities were observed after treatment with the test article), ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for inicronuclei.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 1000 mg/kg b.w..

In pre-experiments this dose level was estimated to be close to the maximum tolerated dose. The animals expressed toxic reactions. However, in the micronucleus assay 7 out of 36 animals treated with Mortrace SB Conc. died.

The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that Mortrace SB conc. had no cytotoxic properties.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Mortrace SB Conc. were in the same range as compared to the negative control groups.

30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Bacterial Mutagenicity:

Marker SB (non-volatile) was evaluated in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of Marker SB was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Marker SB in the absence of S9. Results of the prescreen indicated Marker SB produced inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) at doses of 50.0 and 167 ug/plate in strain TA1538, and at a dose of 50.0 ug/plate in strain TA100. Complete toxicity was observed at doses of 500, 1670 and 5000 ug/plate in strain TA1538, and at doses of 167, 500, 1670 and 5000 ug/plate in strain TA100. In addition, the test article was found to precipitate from solution at the 1670 and 5000 ug/plate dose levels. Based upon these findings, Marker SB was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0 and 167 ug/plate. Three extra dose levels of Marker SB were evaluated with and without S9 in the event of unacceptably high toxicity at the highest dose levels. The S9 mixture included 30% (v/v) uninduced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors. Revertant frequencies for all doses of Marker SB in all strains without S9, and strain TA1535 with S9, approximated or were less than those observed in the concurrent negative control cultures. However, increased revertant frequencies of approximately two- to four-fold were observed in strains TA1537, TA1538, TA98 and TA100 in the presence of S9. Therefore, Marker SB was re-evaluated in all five strains at doses of 1.00, 5.00, 10.0, 25.0, 50.0, 75.0, 100 and 150 ug/plate with S9. Similar increases in revertant frequencies of approximately two- to five-fold were again observed in strains TA1538, TA98 and TA100 in the retest. Although the revertant frequencies generally decreased at the highest dose levels in each assay (150 or 167 ug/plate; due to toxicity), the increases observed over the remainder of the dose range were linear. All positive and negative control values in both assays were within acceptable limits. Therefore, the results for Marker SB (non-volatile) were positive in the Prival Modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

In vitro Chromosomal Aberration CHL (Chinese hamster cells)

Chromosome Aberration Test of Mortrace SB was conducted to determine if the test article would cause chromosome aberrations in CHL cultured mammalian cells. Metaphase analysis of the CHL was perfonned. Four test levels were tested. The doses were selected by reference with the IC50. The highest concentration of test substance distributed in the culture medium was higher than the IC50, both in the non metabolic activation system (-S9 mix) and in the metabolic activation system (+S9 mix) for the 24h, the doses were 20, 10, 5, 2.5µg/ml. For 48h exposure in the non metabolic activation system, the doses were 12, 6, 3, 1.5µg/ml. Meanwhile control treatments were performed at the same time, that is, vehicle control, positive controls (mitomycin C, MMC) for non-metabolic activation system (-S9 mix) and positive control (cyclophosphamide, CP) for metabolic activation system (+S9 mix). Microscopic examination was performed to determine the chromosome aberration potential of the test article. The final findings showed that chromosome aberration rate in all treatment doses was <5%, that is, a negative result for the test article. On the other hand, the two positive control treatments (MMC and CP) showed chromosome aberration rates >20% and indicated the test system condition was reliable. Based on these findings, it is concluded that Mortrace SB was not clastogenic in CHL cells.

In Vivo mouse micronucleus

Mortrace SB Conc. was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. This vehicle was used as negative control. Based on a prelimenary toxicity screen a dose level of 1000 mg/kg bw was selected. Per treatment period, 12 animals (6 per sex) were dosed with vehicle control, test material or positive control. Micronuclei were assessed at 24 hours, 48 hours and 72 hours. Consequently a total of 36 animals (18 per sex) were treated with test material, vehicle or positive controls. Where possible, 10 animals per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. During the assay 7 animals in the treatment groups died, 1 male in the 24 hours treatment grou, 3 males and 1 female in the 48 hours group and 2 males in the 72 hours group.

The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that Mortrace SB conc. had no cytotoxic properties.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Mortrace SB Conc. were in the same range as compared to the negative control groups. 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

In conclusion, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Consideration of the validity of the study:

While the PCE/NCE ratio was not altered in the mouse MNT study, the MTD was clearly reached at 1000 mg/kg bw, as mortality was observed in 7/36 mice treated with the dose. Additionally, the 28d repeated dose toxicity study showed effects on the hematopoietic system still at doses of 50 mg/kg bw, so that it can be reasoned that the substance is sufficiently bioavailable to blood and the bone marrow to make the negative outcome of the in vivo MNT relevant.

In vivo mutagencicity: PigA assay in rats

Due to the positive findings in the Ames assay it was considered appropriate to perform a follow up in vivo assessment of mutagenicity to identify whether classification for mutagenicity is appropriate and whether further assessments of mutagenicity in vivo ar needed. The negative micronucleus assay indicates that the substance is not clastogenic in vivo, but this is no longer considered as sufficient follow up to a positive bacterial mutagenicity assay. Consequently an in vivo assay was added into the conduct of the reproductive screening study required for REACH registration.

The Pig-a assay is an in vivo gene mutation assay. It was first reported in 2008 [1-4] and since then has received extensive interest as a potential assay for regulatory safety assessment [5-9]. A Workgroup, made up of experts from academic, regulatory, and industrial laboratories, was formed in 2012 under the auspices of the International Workshop on Genotoxicity Testing (IWGT) to review the development of the Pig-a assay in the context of safety assessment strategies. The Workgroup consensus on the underlying science of the Pig-a assay, technical considerations for the assay protocol, a view to assay acceptance in a regulatory context, and recommendations on where and how further progress on developing the assay have been published [8]. An OECD test guideline development for the Pig-a assay has been initiated.

Principle of the assay

The Pig-a assay is based on the identification of mutant cells that have an altered repertoire of cell surface markers. The assay was developed from an understanding of the molecular nature of a rare human acquired genetic disorder, paroxysmal nocturnal hemoglobinuria (PNH). The Pig-a gene (phosphatidylinositol glycan, class A gene) codes for the catalytic subunit of an N-acetyl glucosamine transferase that is involved in an early step of glycosylphosphatidylinositol (GPI) biosynthesis. GPI anchors an assortment of protein markers (e.g., CD59, CD24, and CD55) to the exterior surface of the cytoplasmic membranes of higher eukaryotes. In mammals, Pig-a is an X-linked gene present as a single functional copy in cells from both males and females. Other genes involved in GPI biosynthesis are autosomal and have two functional copies. A single inactivating mutation in the Pig-a gene is sufficient to make a cell deficient in GPI anchors and, as a consequence, deficient in surface-bound GPI-anchored markers. Since it is exceedingly unlikely that anchor deficiency would occur due to inactivating mutations in both copies of the autosomal genes involved in GPI synthesis, measuring GPI deficiency is considered ‘virtually equivalent’ to measuring Pig-a mutation [3, 10-12].

Validation of the assay

Multi-laboratory trials initiated by Litron Laboratories [13], the Japanese Research Group [14], and the HESI initiative [7] have contributed to establishing protocols for the assay, testing the inter-laboratory reproducibility of the assay, and in expanding the number of agents tested.

The assay has been investigated extensively in peripheral blood erythrocytes of rats and has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans. Currently the IWGT Workgroup recommends measuring CD59-deficient erythrocytes in the peripheral blood of rats for use in safety assessment.

Though no studies to date have specifically tested the intra-laboratory reproducibility of the assay beyond assaying technical replicates, there is good evidence for a high degree of inter-laboratory transferability and reproducibility based on the results with several potent and weak mutagens (and low doses of potent mutagens) that were tested in a systematic manner as part of the Litron trial [15-20]. Results from the Japanese trial, using a different staining protocol, have also demonstrate a similar degree of inter-laboratory reproducibility [14]. Therefore, the available data show that the assay is both robust and demonstrably reproducible within and across laboratories for test agents with a range of mutagenic potency.

The IWGT Workgroup compiled a list of 41 agents tested in the rat Pig-a assay in the report [8]. Most of the agents were genotoxic in one or more tests, including 26 that were Ames' test positive. The Pig-a assay identified most of the agents expected to be positive as positive in the assay. Of the 8 agents tested that generally are considered non-genotoxicants, all were negative in the assay with 5 of those tested to the maximum tolerated dose (MTD) or the limit dose using the most sensitive protocols (i.e., using immunomagnetic seperation).

The Pig-a assay is based on the identification of the phenotype alteration resulting from gene mutation. Establishing the mutational basis of the response is a challenge because the assay measures the mutant phenotype in enucleated erythrocytes; however, there is good evidence from nucleated bone marrow and spleen cells from rodents, and extensive experience from human PNH patients, that supports the conclusion that GPI-anchored protein-deficient cells, in fact, have mutations in the Pig-a gene. The IWGT Workgroup concluded that the weight of evidence was consistent with a direct association between Pig-a mutation (rather than gene silencing or enzymatic inactivation) and the phenotype measured in the assay, and indicated that lack of absolute proof should not preclude use of the Pig-a assay for regulatory purposes [8].

Placement of the assay within regulatory genotoxicity testing strategies

Therefore, the IWGT Workgroup recommended a placement of the Pig-a assay within regulatory genotoxicity testing strategies [8]: “The Pig-a assay should be considered an appropriate in vivo follow-up to positive results in bacterial and in vitro mammalian cell gene mutation assays. …... the Workgroup noted the potential of using the Pig-a assay, as a measure of gene mutation, to complement the micronucleus (MN) assay, which measures clastogenicity/aneugenicity. Both can be readily included in routine in vivo safety evaluations, especially when the assays are integrated into subchronic (28 or 90 day) treatment protocols.”

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has accepted the Pig-a assay as one of tests to investigate the in vivo relevance of in vitro mutagens (positive bacterial mutagenicity) in DNA reactive impurities in pharmaceuticals [21].

Scientific guideline for the assay

Detailed technical considerations for the assay protocol have been provided by the IWGT Workgroup [8], e.g., test animals/sex, number of doses, maximum doses, and age and number of animals, prescreening animals, treatment and sampling schedules, sample analysis, data analysis and interpretation.

The IWGT Workgroup also recognized that one of the most attractive features of the Pig-a assay is its potential for integration into repeat dose toxicology studies and with other genetic toxicology assays. The combination of several assays in one set of test animals is also consistent with the 3Rs principles for animal welfare. The assay requires only microliter samples of peripheral blood, which can be obtained in a minimally invasive manner without disturbing the assessment of other endpoints. Unlike the TGR gene mutation test, the Pig-a assay is not limited to specific strains of animals, and the timing of sample collection is not as critical as it is for the in vivo Comet assay. An official OECD test guideline development for the Pig-a assay has been initiated.

In vivo mutagenicity assessment of MORTRACE SB using the Pig-a assay

The present Pig-a assay was integrated into a Reproductive/Developmental Toxicity Screening Test to promote the 3Rs principles. All the study parameters were compliant to the protocol provided by the IWGT Workgroup. The animals were prescreened (as described in the IWGT guidance) to maximize the assay sensitivity and specificity. The frequencies (×10-6) of CD59-deficient reticulocytes (RETCD59-) and CD59-deficient red blood cells (RBCCD59-) in the vehicle control animals were within the reported background range(≤5 × 10-6) [8]. The mutant frequencies of RETCD59- and RBCCD59- in the positive control group were significantly higher than those in the vehicle control group. The percentage of reticulocytes (% RET) in the 200 mg/kg/day test material group was significantly higher than that in the vehicle control group and the increase was dose-dependent, indicative of bone marrow exposure to the test material or metabolites. Therefore, this study met the requirement for a valid assay according to the IWGT Workgroup criteria and, under the experimental conditions of this study, indicated that MORTRACE SB does not have in vivo mutagenic potential.

References

1. Bryce, S.M., J.C. Bemis, and S.D. Dertinger, In vivo mutation assay based on the endogenous Pig-a locus. Environ Mol Mutagen, 2008. 49(4): p. 256-64.

2. Miura, D., et al., Development of an in vivo gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat. Environ Mol Mutagen, 2008. 49(8): p. 614-21.

3. Miura, D., et al., Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants. Environ Mol Mutagen, 2008. 49(8): p. 622-30.

4. Phonethepswath, S., et al., Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. Mutat Res, 2008. 657(2): p. 122-6.

5. Dobrovolsky, V.N., et al., The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment. Environ Mol Mutagen, 2010. 51(8-9): p. 825-35.

6. Lynch, A.M., et al., New and emerging technologies for genetic toxicity testing. Environ Mol Mutagen, 2011. 52(3): p. 205-23.

7. Schuler, M., et al., Need and potential value of the Pig-a in vivo mutation assay-a HESI perspective. Environ Mol Mutagen, 2011. 52(9): p. 685-9.

8. Gollapudi, B.B., et al., The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup. Mutat Res Genet Toxicol Environ Mutagen, 2015. 783: p. 23-35.

9. Godin-Ethier, J., et al., Characterisation of an in vivo Pig-a gene mutation assay for use in regulatory toxicology studies. Mutagenesis, 2015. 30(3): p. 359-63.

10. Kimoto, T., et al., Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression. Mutat Res, 2011. 723(1): p. 36-42.

11. Miura, D., et al., Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated fisher 344 rats. Environ Mol Mutagen, 2011. 52(5): p. 419-23.

12. Mortazavi, Y., et al., The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot. Blood, 2003. 101(7): p. 2833-41.

13. Dertinger, S.D. and R.H. Heflich, In vivo assessment of Pig-a gene mutation-recent developments and assay validation. Environ Mol Mutagen, 2011. 52(9): p. 681-4.

14. Kimoto, T., et al., Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody. Mutat Res, 2013. 755(2): p. 126-34.

15. Bhalli, J.A., et al., Report on stage III Pig-a mutation assays using benzo[a]pyrene. Environ Mol Mutagen, 2011. 52(9): p. 731-7.

16. Cammerer, Z., et al., Report on stage III Pig-a mutation assays using N-ethyl-N-nitrosourea-comparison with other in vivo genotoxicity endpoints. Environ Mol Mutagen, 2011. 52(9): p. 721-30.

17. Dertinger, S.D., et al., International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories. Environ Mol Mutagen, 2011. 52(9): p. 690-8.

18. Lynch, A.M., et al., International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea. Environ Mol Mutagen, 2011. 52(9): p. 699-710.

19. Shi, J., et al., Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies. Environ Mol Mutagen, 2011. 52(9): p. 711-20.

20. Stankowski, L.F., Jr., et al., Integration of Pig-a, micronucleus, chromosome aberration, and Comet assay endpoints in a 28-day rodent toxicity study with 4-nitroquinoline-1-oxide. Environ Mol Mutagen, 2011. 52(9): p. 738-47.

21. ICH, Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk M7. ICH Harmonised Tripartite Guideline, 2014.

Justification for selection of genetic toxicity endpoint
In vivo study, most relevant for assessment

Justification for classification or non-classification

According to CLP, evidence from in vivo studies is decisive for classification of mutagenicity endpoints. An in vivo mouse MNT and in vivo mutagenicity assay (Pig A) were negative with the substance, so that classification is not warranted.