p-benzoquinone dioxime

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence: positive, within the in vitro bacterial reverse mutation assay, with and without S-9 activation, 2018

1. positive, in vitro bacterial reverse mutation in strains: TA98, TA100, TA1535 and/or TA1537 (with and without S-9 activation), eq. or similar to OECD TG 471, Haworth, 1983

2. positive, in vitro bacterial reverse mutation in strains: TA98, TA100, TA1535, TA1537 and WP2 uvrA (with and without S-9 activation), eq. or similar to OECD TG 471, Dunkel, 1985

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:

weight of evidence

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Main test (plate incorporation method): All strains: series of half log doses up to 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Not reported. It can be presumed by applicant assessment that a standard Ames test vehicle would be used, water or DMSO are typical.
- Justification for choice of solvent/vehicle: Not reported

Untreated negative controls:

yes

Remarks:

Not specified. Concurrent Negative Controls were run.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48h at 37 degrees Celsius

NUMBER OF REPLICATIONS: 3

Statistics:

A statistical analysis was not performed or reported.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

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:

positive

Cytotoxicity / choice of top concentrations:

not specified

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:

positive

Cytotoxicity / choice of top concentrations:

not specified

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:

positive

Cytotoxicity / choice of top concentrations:

not specified

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:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The precise strain/strains which yielded positive results: within the standard Ames et al. (1975) protocol was not indicated per se in the report. Merely that positive with one of either TA98, TA100, TA1535 and/or TA1537 was observed; i.e. "Conclusion based on all Salmonella strains and activation systems." and/or "Conclusions based on E coli responses.", respectively.

Conclusions:

Interpretation of results:
positive
Under the conditions of this study the test item was considered to be mutagenic in the presence and absence of S9 activation. The test item induced gene mutations in the strains of S. typhimurium and E. coli utilzed.

Executive summary:

The study was performed using methods equivalent or similar to the requirements of OECD Guideline 471, to evaluate the potential mutagenicity of sixty substances in an inter laboratory study which included the test item. The method used was the plate-incubation method bacterial reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in both the presence and absence of S-9 mix. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower. The vehicle was not specified. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a positive, i.e. mutagenic response in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in either the presence or absence of S-9 mix.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Tested strains: Salmonella: TA98, TA100, TA1535 and TA1537 only with and without metabolic activation

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Tested strains: Salmonella: TA98, TA100, TA1535 and TA1537 only with and without metabolic activation

GLP compliance:

no

Remarks:

Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (the precise activation system varied by laboratory, however trends in activation to different chemicals was one focus of the study)

Test concentrations with justification for top dose:

Range finding test (pre-incubation method): TA100 strain: 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).
- If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate

Main test (pre-incubation method): All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
- At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer.
Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

Confirmatory test: All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:choice was distilled water; dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water. Ethanol (95%) or acetone was used if the chemical was not soluble or stable in DMSO.
- Justification for choice of solvent/vehicle: Depending on solubility (determined prior to testing).

Untreated negative controls:

yes

Remarks:

choline chloride, glycerol, glycine, mannitol, and sodium phosphate, tested at random concurrently

Negative solvent / vehicle controls:

yes

Remarks:

concurrent Solvent Controls

True negative controls:

not specified

Positive controls:

yes

Remarks:

concurrent Positive Controls

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: with metabolic activation: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation in agar (pre-incubation method)
0.5 ml of S-9 mix or 0.1M PO4 buffer was dispensed into an appropriate number of 13 x 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate with shaking in the early tests or standing (dependent on laboratory) in line with earlier tests, for 20 min at 37°C.
The protocol was later changed to eliminate the shaking procedure, because the commercial shakers available would not fit in the Class 11 Type B hoods and, for the purposes of laboratory safety, it was inadvisable to incubate the chemicals at 37°C in the open laboratory.
Following the preincubation period, 2.5 ml (EGG) or 2.0 ml (CWR, SRI) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1951] in a 15 x 100 mm plastic petri dish. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h

DURATION
- Expression time (cells in growth medium): 48h at 37 degrees Celsius

NUMBER OF REPLICATIONS: 3

Range finding test (pre-incubation method): TA100 strain: 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).
- If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate

Main test (pre-incubation method): All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
- At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer.
Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

Confirmatory test: All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be two-fold over background.

Statistics:

A statistical analysis was performed. As a result of statistical analyses, a number of calls were changed from the original “negative” to “equivocal.” The statistical analysis did not result in any “positive” or “equivocal” calls being called “negative, under the conditions of the study (dated, 1983).

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

No negative control substances yielded a positive

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

No negative control substances yielded a positive

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

No negative control substances yielded a positive

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

No negative control substances yielded a positive

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

The precise strain/strains which yielded positive results: within the standard Ames et al. (1975) protocol was not indicated per se in the report. Merely that positive with one of either TA98, TA100, TA1535 and/or TA1537 was observed.

Conclusions:

Interpretation of results:
positive
Under the conditions of this study the test item was considered to be mutagenic in the presence and absence of S9 activation. The test item induced gene mutations in strains of S. typhimurium utilized.

Executive summary:

The study was performed using methods equivalent or similar to the requirements of OECD Guideline 471, to evaluate the potential mutagenicity of two hundred and fifty substances in an inter laboratory study which included the test item. The method used was the pre-incubation method bacterial reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, in both the presence and absence of S-9 mix. The test item was an 88% purity grade sample obtained from a commercial source. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower using distilled water or dimethyl sulfoxide (DMSO). If the chemical was insoluble or not sufficiently soluble in water. Alternatively, ethanol (95%) or acetone was typically used if the chemical was not soluble or stable in DMSO. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a positive, i.e. mutagenic response in Salmonella typhimurium strains TA98, TA100, TA1535 and/or TA1537 in either the presence or absence of S-9 mix.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4.2 the study does not need to be conducted if there is adequate data from an in vivo cytogenicity test available. There is (1) an available in vivo mammalian erythorocyte micronucleus test concerning in vivo cytogenicity (in somatic cells) and (2) an available unscheduled DNA synthesis (UDS) with mammalian liver cells test concerning in vivo mammalian gene mutation (in somatic cells) and (3) an available in vivo carcinogenicity study. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.7, July 2017) the study does not need to be conducted.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4.3 the study does not need to be conducted if there is adequate data from an in vivo gene mutation test available. There is (1) an available in vivo mammalian erythorocyte micronucleus test concerning in vivo cytogenicity (in somatic cells) and (2) an available unscheduled DNA synthesis (UDS) with mammalian liver cells test concerning in vivo mammalian gene mutation (in somatic cells) and (3) an available in vivo carcinogenicity study. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.7, July 2017) the study does not need to be conducted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

1. negative, in vivo unscheduled DNA synthesis (UDS) up to 250 mg/kg bw (lethality at 500 mg/kg bw) in PVG rats, eq. or similar to OECD TG 486, Westmoreland, 1992

2. negative, in vivo mammalian bone marrow erythrocyte micronucleus up to 250 mg/kg bw (lethality at 500 mg/kg bw) in F344 rats, eq. or similar to OECD TG 474, Westmoreland, 1992.

Weight of evidence: based upon presence > 1%wt of impurity: 4-nitrosophenol (CAS 104-91-6) with harmonised classification: CLP Regulation (EC) 1272/2008: germ cell mutagen category 2: H341: Suspected of causing genetic defects, effects upon germ cells cannot be excluded in the substance. Therefore a precautionary hazard classification is adopted with a conclusion of potential: evidence adverse effects to germ cells.

In line with the above classification criteria definition.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

no

Remarks:

Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Historically utilised species and strains were used that were commonly associated with genetic toxicity and carcinogenicity assays

Micronucleus assay: female rat F344 species/strain

For parallel in vivo Liver UDS and related testing:
rat PVG strain - female: Used in Liver UDS Test.
rat PVG strain - male/female: Used in hepatic function and Liver S-phase assay

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 7 - 11 weeks
- Weight at study initiation: Not reported.
- Assigned to test groups randomly: Not reported.
- Fasting period before study: Not reported.
- Housing: Individually in makrolon cages before and after treatment
- Diet (e.g. ad libitum): No. 1 SDS (Special Diets Services) diet ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: Not repoted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported (guideline specifies)22 ± 3 °C)
- Humidity (%): Not repoted (guideline recommends 30-75%)
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): Not reported (guideline specifies 12 hrs dark / 12 hrs light)

IN-LIFE DATES: Not reported.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium
- Justification for choice of solvent/vehicle: A solubility test was not performed within the study although applicant assessment indicates that the test item should be fully miscible in the solvent/vehicle employed.
- Concentration of test material in vehicle: (i) Bone marrow micronucleus Assay: Female F344 rats: 125, 250, and 500 mg/kg. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for assessment.
(ii) Liver UDS Assay: Female PVG rats were dosed orally with 125 or 250 mg/kg ; (iii) Liver S-phase Assay: Male/Female PVG rats were given a single oral dose of 250 mg/kg (iv) Hepatic function Assay: Female PVG rats were given a single oral dose of 250 mg/kg – which was confirmed using female F344 rats as part of the micronucleus assay at 250 mg/kg bw and negative control.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A solubility test was not performed within the study although applicant assessment indicates that the test item should be fully miscible in the solvent/vehicle employed. The vehicle was 0.3% [w/v] test item in METHOCEL™ cellulose ether K15M Premium. Concentrations were as follows:
(i) Bone marrow micronucleus Assay: Female F344 rats: 125, 250, and 500 mg/kg. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for assessment. Parallel assays: (ii) Liver UDS Assay: Female PVG rats were dosed orally with 125 or 250 mg/kg ; (iii) Liver S-phase Assay: Male/Female PVG rats were given a single oral dose of 250 mg/kg (iv) Hepatic function Assay: Female PVG rats were given a single oral dose of 250 mg/kg. Results were confirmed using female F344 rats as part of the micronucleus assay at 250 mg/kg bw and negative control.

Animals were treated with the selected doses of the test item, with the vehicle alone or with the positive control by oral application. The treatment regimen by applicant assessment appears in accordance with published standard guidelines. For determining potential effects in blood marrow erythrocytes, a period of 24 and 48 hours was selected for the groups treated with the test item and with the vehicle alone. With the positive control substance (Cyclophosphamide, 10 mg/kg at 10 mL/Kg dose volume) a time point of 24 hours was selected.

The top dose was limited by lethality (observed at 500 mg/kg bw in F344 female rat where there were 6 of 8 mortalities).

Duration of treatment / exposure:

24 and 48 hours exposure time, main study group and negative control; 24 hours for positive control (Cyclophosphamide, 10 mg/kg at 10 mL/Kg dose volume)

Frequency of treatment:

Single treatment oral dose

Post exposure period:

Not applicable. Bone marrow smears were made immediately post termination at 24 and 48 hours respectively and slides immediately stained according to procedure of literature [Pascoe and Gatehouse, 1986]. Hepatic function assay groups were assessed for up to 1 week post exposure.

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
125 mg/kg bw
Basis:
nominal conc.

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.

Excluded from assessment due to lethality

No. of animals per sex per dose:

5 to 9 females per dose.

Control animals:

yes, concurrent vehicle

other: Positive Control

Positive control(s):

Cyclophosphamide [CAS 6055-19-2], 10 mg/kg at 10 mL/kg dose volume) dissolved in vehicle
- Justification for choice of positive control(s): Positive control substance is listed in OECD TG 474
- Route of administration: Oral gavage
- Doses / concentrations: 10 mg/kg mg/kg dissolved in sterile water (10 mL/kg)

Tissues and cell types examined:

Bone marrow was extracted and smear preparations were made and 2000 Polychromatic, (PCE) erythrocytes were scored for the presence of micronuclei.
Five hundred erythrocytes per animal were scored to determine the percentage of PE among all erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose selection was not documented. Applicant assessment indicates that doses > 250 mg/kg bw may yield toxicity (lethality) based on data reported in the study which reported mortalities in F344 rats at 500 mg/kg bw. Therefore it can be presumed that the authors based the maximum dose on the maximum tolerated dose (MTD) that did not yield lethality.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
For determining micronucleus assay effects, following a single dose, the respective treatment groups were terminated at 24 and/or 48 hours, bone marrow smears were made according to standard procedures.

DETAILS OF SLIDE PREPARATION:
The procedure was as follows: All slides were coded prior to analysis and stained with haematoxylin and eosin according to literature procedure [Pascoe and Gatehouse, 1986].

METHOD OF ANALYSIS:
2,000 polychromatic erythrocytes (PE) were analysed for the presence of micronuclei. Five hundred erythrocytes per animal were scored to determine the percentage of PE among all erythrocytes. Statistical analysis was performed using likelihood ratio test.

OTHER:
To confirm that the animals were actually exposed to the intended test concentrations an assessment of hepatic function was conducted. Female PVG rats were given a single oral dose of 250 mg/kg test item, serum samples were analysed after 24 hr, 48 hr, and 1 week. In addition, serum samples were taken 1 week prior to dosing for comparative purposes. Samples were analysed for ALP, ALT, AST, ALB, TP, T-Bil, and BA. It is considered that this spectrum of measurements would provide an accurate profile of any test item-related hepatotoxicity.

Evaluation criteria:

Under OECD TG 474 guideline:
Comparison would be made between the number of micronucleated polychromatic erythrocytes (PCE) occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes (PCE) observed at the 24 and/or 48-hour termination time compared with the vehicle control group

- A negative response would be fulfilled if the positive response criteria were not fulfile (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.

Data is subject to statistical analysis, where appropriate.

Statistics:

Statistical analyses of the micronucleus frequencies were performed using the likelihood ratio test [Amphlett and Delow, 1984]

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 500 mg/kg bw lethality was observed

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Not applicable. The dose range was selected based on available data from historical acute toxicity studies in the rat (LD50 oral = 464 mg/kg bw).
- Solubility: The test item was fully soluble in the vehicle employed.
- Clinical signs of toxicity in test animals: Not reported.
- Evidence of cytotoxicity in tissue analyzed: Not reported.
- Rationale for exposure: According to standard guideline and to compare with other higher tier studies.
- Harvest times: 24 and 48 hours in accordance with standard guidelines.
- High dose with and without activation: Not applicable.
- Other: Not applicable.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No effect observed.
- Induction of micronuclei (for Micronucleus assay): No effect observed.
- Ratio of PCE/NCE (for Micronucleus assay): Not reported.
- Appropriateness of dose levels and route: The maximum dose level was up to that which yielded mortality (6/8), which was excluded from further MN assessment.
- Statistical evaluation: No statistically significant effects reported.

1. The test item did not induce micronucleus formation at either time point, nor was there any reduction in the percentage of PE in the bone marrow of treated animals.

2. In the parallel hepatic function assay (female PVG rats, 250 mg/kg): No significant increases were seen in levels of AST, ALB, TP, and BA at any of the sample times. Significant increases were seen in T-BiI (24 hr; 2.6 ± 0.3 µmol/L), ALP (48 hr; 366 ± 24 iU/L), and ALT (1 week; 70.5 ± 6.6 iU/L) compared to concurrent controls. The authors considered these increases small, not time-related, and all of the measured values were within historical control ranges. It was therefore assumed that the statistical significance achieved for these results is due to normal physiological variation and that they are of no toxicological significance. The results of liver function tests presented show no evidence of functional impairment or damage to the livers of female PVG rats 1, 2, or 7 days after receiving a single oral dose of 250 mg/kg.

3. In the parallel S-phase assay (female and male PVG rats, 250 mg/kg): it was considered there was a time-related increase in the incidence of cells undergoing semiconservative DNA synthesis with maximum S-phase induction occurring 24 h after dosing. This was also confirmed in the present micronucleus assay and testing in female F344 rats. Male PVG rats were dosed orally with the same dose of test item and an increase in S-phase synthesis was observable both 24 and 48 hr after dosing. In males, induction of DNA synthesis was greater 48 hr after dosing. The authors indicated the results suggest the test item induces S-phase synthesis in the livers of female rats 14, 24, and 48 hrs after a single oral dose of 250 mg/kg. However, is not a male/female hepatocarcinogen.

Table 1.0 – Table of results

Treatment	Dose / mg/kg	Sample time / hours	MN PE / 1000 PE x ± SD
Vehicle control	0	24	1.6	± 1.0
Test item	125	24	0.8	± 0.9
Test item	250	24	1.0	± 0.8
Test item	500	24	1.4	± 0.6
Vehicle control	0	48	1.0	± 0.3
Test item	125	48	0.6	± 0.6
Test item	250	48	0.8	± 0.7
Test item	500	48	1.0 #1	-
Positive control #2	10 mg/kg	24	13.2	± 4.2

#1: Only 2 out of 8 animals survived, 48 hr after treatment

#2: PC = Cyclophosphamide, 10 mg/kg

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study, no evidence of test item induction of micronucleated erythrocytes and genotoxic properties.

Executive summary:

The test item was investigated in a method equivalent or similar to OECD TG 474, for mammalian bone marrow erythrocyte micronucleus assessment for genotoxicity in F344 female rats. The test item was administered orally at single doses to 5 to 9 females within 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium at doses of 125, 250 and 500 mg/kg bw and a dose volume of 10 mL/kg. The vehicle was used as the negative control and the positive control was cyclophosphamide at 10 mg/kg in sterile water at 10 mL/kg. At 24 and 48 hours exposure time, main study group and negative control and 24 hours for positive control the exposure was terminated, and bone marrow smears were made immediately. The slides were immediately stained with haematoxylin and eosin according to procedure of literature. 2,000 polychromatic erythrocytes (PE) were analysed for the presence of micronuclei. Five hundred erythrocytes per animal were scored to determine the percentage of PE among all erythrocytes. Statistical analysis was performed using likelihood ratio test. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for further assessment. The test item did not induce micronucleus formation at either time point, nor was there any reduction in the percentage of PE in the bone marrow of treated animals. Under the conditions of this study, there was no evidence of test item induction of micronucleated erythrocytes and genotoxic properties.

Reference 2

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

Principles of method if other than guideline:

- Principle of test:
(i) According to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo) indicates DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents. The test is usually based on the incorporation of 3H-TdR into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle.
Test substances are generally administered as a single treatment administered orally by gavage. The number of animals should be at least 3 analysable animals per group. Where a significant historical database has been accumulated, only 1 or 2 animals are required for the concurrent negative and positive control groups. At least two dose levels are used, the highest dose as defined as the dose producing signs of toxicity. The highest dose may also be defined as a dose that produces some indication of toxicity in the liver (e.g. pyknotic nuclei).
(ii) A limit test may be performed if no adverse effects are observed < 2000 mg/kg unless human data/exposure indicate a need for a higher limit dose level.
(iii) Liver cells are prepared from treated animals normally 12-16 hours after dosing. An additional earlier sampling time (normally 2-4 hours post-treatment) is generally necessary unless there is a clear positive response at 12-16 hours. However, alternative sampling times may be used when justified on the basis of toxicokinetic data.
(iv) Freshly isolated mammalian liver cells are incubated usually with medium containing 3HTdR for an appropriate length of time, e.g. 3 - 8 hours are then developed and slides developed and analysed for morphology and signs of overt cytotoxicity.
(v) Slides should be coded before grain counting. Normally 100 cells are scored from each animal from at least two slides; the scoring of less than 100 cells/animal should be justified. Grain counts are not scored for S-phase nuclei, but the proportion of S-phase cells may be recorded.
(vi) The amount of 3H-TdR incorporation in the nuclei and the cytoplasm of morphologically normal cells, as evidenced by the deposition of silver grains, should be determined by suitable methods.
(vii) Grain counts are determined over the nuclei (nuclear grains, NG) and nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, CG). CG counts are measured by either taking the most heavily labelled area of cytoplasm, or by taking an average of two to three random cytoplasmic grain counts adjacent to the nucleus. Other counting methods (e.g. whole cell counting) may be used if justified.
(vii) Examples of positive and negative response criteria are given in OECD Guideline 486. A positive result from the UDS test with mammalian liver cells in vivo indicates that a substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A negative result indicates that, under the test conditions, the test substance does not induce DNA damage that is detectable by this test.

- Short description of test conditions:
(a)(i) Liver UDS: The test utilised methodology consistent with the above, sampling was conducted at 2 and 14 hours after dosing justified by the authors, and slides were prepared and scored. Either 25 or 50 cells assessed per slide and 2 or 3 slides per animal (therefore total: 50 to 150 cells and ca. 100 typically). Assessment was including criteria of: Ashby et al. (1985) An assessment of the in vivo rat hepatocyte DNA-repair assay. Mutat. Res. 156 : 1-18.
(ii) S-Phase synthesis measurement: Measurement of S-phase synthesis included a random sample of 1000 cells from each slide and 2 or 3 slides per animal. Sampling at 2, 14, 24, and 48 hours after dosing.
(b) Other: The test assay also included by extension by examination of (i) hepatic function and (ii) Liver S-phase assay (to determine S-phase induction after 24 hours after dosing) and (iii) parallel bone micronucelus test: in female F344 rats: at 125, 250, and 500 mg/kg doses and 24 and 48 hours sampling. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for assessment.
- Parameters analysed / observed: See above.

GLP compliance:

no

Remarks:

Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

other: PVG (inbred hooded)

Details on species / strain selection:

Historically utilised species and strains were used that were commonly associated with genetic toxicity and carcinogenicity assays

rat PVG strain - female: Used in Liver UDS Test.
rat PVG strain - male/female: Used in hepatic function and Liver S-phase assay

The rat F344 species/strain was utilised for parallel in vivo micronucleus assay assessment.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 7 - 11 weeks
- Weight at study initiation: Not reported.
- Assigned to test groups randomly: Not reported.
- Fasting period before study: Not reported.
- Housing: Individually in makrolon cages before and after treatment
- Diet (e.g. ad libitum): No. 1 SDS (Special Diets Services) diet ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: Not repoted.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported (guideline specifies)22 ± 3 °C)
- Humidity (%): Not repoted (guideline recommends 30-75%)
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): Not reported (guideline specifies 12 hrs dark / 12 hrs light)

IN-LIFE DATES: Not reported.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium
- Justification for choice of solvent/vehicle: A solubility test was not performed within the study although applicant assessment indicates that the test item should be fully miscible in the solvent/vehicle employed.
- Concentration of test material in vehicle: (i) Liver UDS Assay: Female PVG rats were dosed orally with 125 or 250 mg/kg ; (ii) Liver S-phase Assay: Male/Female PVG rats were given a single oral dose of 250 mg/kg (iii) Hepatic function Assay: Female PVG rats were given a single oral dose of 250 mg/kg
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A solubility test was not performed within the study although applicant assessment indicates that the test item should be fully miscible in the solvent/vehicle employed. The vehicle was 0.3% [w/v] test item in METHOCEL™ cellulose ether K15M Premium. Concentrations were as follows:
(i) Liver UDS Assay: Female PVG rats were dosed orally with 125 or 250 mg/kg ; (ii) Liver S-phase Assay: Male/Female PVG rats were given a single oral dose of 250 mg/kg (iii) Hepatic function Assay: Female PVG rats were given a single oral dose of 250 mg/kg
(iv) Parallel - Bone marrow micronucleus Assay: Female F344 rats: 125, 250, and 500 mg/kg. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for assessment.

Animals were treated with the selected doses of the test item, with the vehicle alone or with the positive control by oral application. The treatment regimen by applicant assessment appears in accordance with published standard guidelines. For determining UDS, a period of 2 and 14 hours was selected for the groups treated with the test item and with the vehicle alone. With the positive control substance (2-acetylaminofluourene, 75 mg/kg at 10 mL/Kg dose volume) a time point of 14 hours was selected.

The top dose was limited by lethality (observed at 500 mg/kg bw in F344 female rat)

Duration of treatment / exposure:

2 and 14 hours exposure time, main study group and negative control; 14 hours for positive control (2-acetylaminofluourene, 75 mg/kg at 10 mL/Kg dose volume)

Frequency of treatment:

Single treatment oral dose

Post exposure period:

Not applicable. Primary hepatocytes were freshly isolated by in situ-collagenase perfusion referenced from Butterworth et al. [1987]. Hepatic function assay groups were assessed for up to 1 week post exposure.

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
125 mg/kg bw
Basis:
nominal conc.

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
250 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

(i) 3 female per groups (liver UDS Test) and 3 females (S-Phase Synthesis Measurement)
(ii) Five to nine animals (male/female) in parallel bone marrow micronucleus assay

Control animals:

yes, concurrent vehicle

other: positive control received the promutagen 2-acetylaminofluourene [CAS 56-96-3], 75 mg/kg at 10 mL/Kg dose volume) dissolved in vehicle

Positive control(s):

2-acetylaminofluourene [CAS 56-96-3], 75 mg/kg at 10 mL/kg dose volume) dissolved in vehicle
- Justification for choice of positive control(s): Positive control substance is listed in OECD TG 486
- Route of administration: Oral gavage
- Doses / concentrations: 75 mg/kg mg/kg dissolved in corn oil (10 mL/kg)

It was noted by the authors: The positive control data for this study were unexpectedly low. However, the mean net grain for this group was > 5 (5.29 +/- 0.53) and >20% of cells (55%) were in repair. This could therefore be judged as a positive response citing Butterworth et al. which is a primary reference cited by OECD TG 486.
[Reference: Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G. and Williams, G. (1987). A Protocol and Guide for the In Vivo Rat Hepatocyte DNA-Repair Assay. Mutation Res., 189, 123-133]

Tissues and cell types examined:

Primary hepatocytes freshly isolated from rats previously treated with the test substance

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose selection was not documented. Applicant assessment indicates that doses > 250 mg/kg bw may yield toxicity (lethality) based on data reported in the study which reported mortalities in F344 rats at 500 mg/kg bw. Therefore it can be presumed that the authors based the maximum dose on the maximum tolerated dose (MTD) that did not yield lethality.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
For determining UDS, a period of 2 to 14 hours was selected for the groups treated with the test item and with the vehicle alone. With the positive control substance a time point resulting in an optimal UDS response is usually selected, this was 14 hours.
Primary hepatocytes were freshly isolated by in situ-collagenase perfusion.

DETAILS OF SLIDE PREPARATION:
The procedure was as follows: Hepatocytes were isolated, cultured, and labelled with 3H-(methyl)thymidine according to the method of Ashby et al. [1985] with the inclusion of a 0.5 mM EGTA buffer before perfusion with buffers 1 and 2 as described by Butterworth et al. [1987]. Perfusion buffers were not gassed and antibiotics were omitted from these buffers. Slides were prepared, coded, and analysed using an AMS 40-10 image analyser. Slides were scored according to the criteria of Ashby et al. [1985] for UDS induction.

METHOD OF ANALYSIS:
For measurement of S-phase synthesis induction, the number of S-phase and non-S-phase cells were counted in a random sample of 1,000 cells from each slide, usually from 2 to 3 slides per animal. Hepatocytes in S-phase, with nuclei very densely labelled with silver grains, were clearly distinguishable from cells in repair.

OTHER:
To confirm that the animals were actually exposed to the intended test concentrations an assessment of hepatic function was conducted. Female PVG rats were given a single oral dose of 250 mg/kg test item, serum samples were analysed after 24 hr, 48 hr, and 1 week. In addition, serum samples were taken 1 week prior to dosing for comparative purposes. Samples were analysed for ALP, ALT, AST, ALB, TP, T-Bil, and BA. It is considered that this spectrum of measurements would provide an accurate profile of any test item-related hepatotoxicity.

Evaluation criteria:

Criteria for a positive response
The test item is generally considered to be active in the DNA repair test if one of the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains in relation to their respective vehicle control value show an increase at any dose level and the mean net nuclear value is 5.0 or higher.
2. The percentage of cells in repair show an obvious shift to higher values at any dose as compared to their respective vehicle control value.

Criteria for a negative response
The test substance is generally considered to be inactive in the DNA repair test if the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains as well as the percentage of cells in repair, do not significantly differ from the respective vehicle control value at any dose level.

Exceptions may be dependent on applicant assessment - see OECD TG 486 for further details.

Statistics:

Mean values and the standard deviations calculated. Comparison with historical laboratory mean control data from in vivo/in vitro DNA repair data

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no mortalities

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

mean net grain for this group was >5 (5.29 2 0.53) and >20% of cells (55%) were in repair. This could therefore be judged as a positive response [Butterworth et al., 1987]

Additional information on results:

1. There was no increase above the control level for either mean net grains observed or for % of cells in repair by administration of the test item.

2. The test item did not induce DNA repair at 2 or 14 hour time points. (Repair = Percentage of cells with net grain > or = 5.0)

3. In the hepatic function assay (female PVG rats, 250 mg/kg): No significant increases were seen in levels of AST, ALB, TP, and BA at any of the sample times. Significant increases were seen in T-BiI (24 hr; 2.6 ± 0.3 µmol/L), ALP (48 hr; 366 ± 24 iU/L), and ALT (1 week; 70.5 ± 6.6 iU/L) compared to concurrent controls. The authors considered these increases small, not time-related, and all of the measured values were within historical control ranges. It was therefore assumed that the statistical significance achieved for these results is due to normal physiological variation and that they are of no toxicological significance. The results of liver function tests presented show no evidence of functional impairment or damage to the livers of female PVG rats 1, 2, or 7 days after receiving a single oral dose of 250 mg/kg.

4. In the S-phase assay (female and male PVG rats, 250 mg/kg): it was considered there was a time-related increase in the incidence of cells undergoing semiconservative DNA synthesis with maximum S-phase induction occurring 24 h after dosing. This was also confirmed in parallel micronucleus assay and testing in female F344 rats. Male PVG rats were dosed orally with the same dose of test item and an increase in S-phase synthesis was observable both 24 and 48 hr after dosing. In males, induction of DNA synthesis was greater 48 hr after dosing. The authors indicated the results suggest the test item induces S-phase synthesis in the livers of female rats 14, 24, and 48 hrs after a single oral dose of 250 mg/kg. However, is not a male/female hepatocarcinogen.

Table 1.0 - Table 1. Group mean net grain count values

Treatment	Dose / mg/kg	Sample time / hours	Net grain #1		% Repair #2
Vehicle control	0	2	-4.2	± 0.19	0
Vehicle control	0	14	-1.7	± 0.14	0
Test item	125	2	-2.8	± 0.28	2
Test item	250	2	-1.9	± 0.13	1
Test item	125	14	-2.0	± 0.17	0
Test item	250	14	-2.7	± 0.115	0
Positive control	75 mg/kg	14	+5.3	± 0.53	55

#1 : Net grain = grains over nucleus minus grains over nuclear size area of cytoplasm.

#2 : Percentage of cells with net grain > or = 5.

#3 : PC = 2-acetylaminofluourene, 75 mg/kg

 

Table 2.0 - Induction of S-Phase Synthesis in Rat Hepatocytes Following In Vivo Treatment

					S-phase cells / 1000 heptocytes
Strain	Sex	Treatment	Dose / mg/kg	Sample time / hours	x #1		SD	Mean %S #2
PVG	F	Vehicle control	0	2 – 48	1.9	±	1.2	0.19
	M	Vehicle control	0	24 – 48	2.0	±	1.5	0.20
	F	Test item	250	14	8.6	±	2.3	0.86
	F	Test item	250	24	11.4	±	2.4	1.14 #1
	F	Test item	250	48	6.8	±	3.5	0.68
	M	Test item	250	24	18.4	±	2.7	1.84 #1
	M	Test item	250	48	23.5	±	7.6	2.35 #1
F344	F	Vehicle control	0	24	3.0		-	0.30
	F	Test item	250	24	26.9	±	5.9	2.69 #1

Criteria for positive response according to Mirsalis et al. [ 1989]: <0.5% = negative; >1.0% = positive; 0.5-1.0% = equivocal.

Reference: Mirsalis J.C. et al., (1989) Measurement of unscheduled DNA synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds. Environ Mutagen 14: 155-164.

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study, no evidence of test item induction of DNA damage that could be interpreted as suggestive of genotoxic properties of the substance to heptocytes.

Executive summary:

The test item was investigated in a method equivalent or similar to OECD TG 486, for unscheduled DNA synthesis (UDS) in vivo within PVG strain rat hepatocytes. The assay is designed to measure potential test item related unscheduled DNA synthesis in freshly isolated rat liver cells (hepatocytes) after in vivo treatment with the test item. The assay included additional investigations including an investigation of hepatic function and a Liver S-phase assay (to determine S-phase induction 24 hours after dosing). Hepatocytes are isolated by perfusion techniques described in the literature, and unscheduled DNA-synthesis caused by the test item or its metabolites is detected by reference to the incorporation of tritium-labelled thymidine (3H-TdR) into DNA during in vitro culture. DNA-repair is determined by scoring autoradiographs (counts of silver grains over the nuclei and counts of silver grains over nuclear-sized cytoplasmic areas). For the UDS assay, the test item was suspended in 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium and administered orally at single doses of 125 and 250 mg/kg and a dose volume of 10 mL/kg. The vehicle was used as negative control and 2-acetylaminofluourene at 75 mg/kg in corn oil was used as a positive control at a dose volume of 10 mL/kg. Hepatocytes from the treatment groups and negative control groups were isolated 2 and 14 hours after administration of the test item and slides were prepared. Hepatocytes from the positive control group (2-acetylaminofluourene at 75 mg/kg) were isolated 14 hours after administration. For the liver S-phase assay, male/female PVG rats were given a single oral dose of 250 mg/kg then hepatocytes were isolated at 2, 14, 24 and 48 hours. In the hepatic function assay, female PVG rats were given a single oral dose of 250 mg/kg, to assess test item related hepatotoxicity.

In the UDS assay, there was no increase above the control level for either mean net grains observed or for % of cells in repair by administration of the test item. Additionally, the test item did not induce DNA repair at 2 or 14-hour time points. (Repair = Percentage of cells with net grain > or = 5.0). In the parallel in vivo micronucleus assay there was additionally no positive result. In the hepatic function assay, no significant increases were seen in levels of AST, ALB, TP, and BA at any of the sample times. Significant increases were seen in T-BiI (24 hr; 2.6 ± 0.3 µmol/L), ALP (48 hr; 366 ± 24 iU/L), and ALT (1 week; 70.5 ± 6.6 iU/L) compared to concurrent controls. The authors considered these increases small, not time-related, and all of the measured values were within historical control ranges. It was therefore assumed that the statistical significance achieved for these results is due to normal physiological variation and that they are of no toxicological significance. In the liver cell S-phase assay, it was considered there was a time-related increase in the incidence of cells, and induction of cells undergoing S-Phase synthesis 14 to 48 hour after treatment. The authors indicated the results suggest the test item induces S-phase synthesis in the livers of female rats 14, 24, and 48 hrs after a single oral dose of 250 mg/kg, but without detectable hepatocellular damage or functional impairment. Under the conditions of the study, the test item was not considered a male/female hepatocarcinogen. The authors did not exclude potential effects to the bladder.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable. Mode of action is inconclusive based on current dataset.

Additional information

Eq. or similar to OECD TG 471, 1983 - The study was performed using methods equivalent or similar to the requirements of OECD Guideline 471, to evaluate the potential mutagenicity of two hundred and fifty substances in an inter laboratory study which included the test item. The method used was the pre-incubation method bacterial reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, in both the presence and absence of S-9 mix. The test item was an 88% purity grade sample obtained from a commercial source. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower using distilled water or dimethyl sulfoxide (DMSO). If the chemical was insoluble or not sufficiently soluble in water. Alternatively, ethanol (95%) or acetone was typically used if the chemical was not soluble or stable in DMSO. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a positive, i.e. mutagenic response in Salmonella typhimurium strains TA98, TA100, TA1535 and/or TA1537 in either the presence or absence of S-9 mix.

 

Eq. or similar to OECD TG 471, 1985 - The study was performed using methods equivalent or similar to the requirements of OECD Guideline 471, to evaluate the potential mutagenicity of sixty substances in an inter laboratory study which included the test item. The method used was the plate-incubation method bacterial reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in both the presence and absence of S-9 mix. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower. The vehicle was not specified. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a positive, i.e. mutagenic response in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in either the presence or absence of S-9 mix.

 

Eq. or similar to OECD TG 486, 1992 - The test item was investigated in a method equivalent or similar to OECD TG 486, for unscheduled DNA synthesis (UDS) in vivo within PVG strain rat hepatocytes. The assay is designed to measure potential test item related unscheduled DNA synthesis in freshly isolated rat liver cells (hepatocytes) after in vivo treatment with the test item. The assay included additional investigations including an investigation of hepatic function and a Liver S-phase assay (to determine S-phase induction 24 hours after dosing). Hepatocytes are isolated by perfusion techniques described in the literature, and unscheduled DNA-synthesis caused by the test item or its metabolites is detected by reference to the incorporation of tritium-labelled thymidine (3H-TdR) into DNA during in vitro culture. DNA-repair is determined by scoring autoradiographs (counts of silver grains over the nuclei and counts of silver grains over nuclear-sized cytoplasmic areas). For the UDS assay, the test item was suspended in 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium and administered orally at single doses of 125 and 250 mg/kg and a dose volume of 10 mL/kg. The vehicle was used as negative control and 2-acetylaminofluourene at 75 mg/kg in corn oil was used as a positive control at a dose volume of 10 mL/kg. Hepatocytes from the treatment groups and negative control groups were isolated 2 and 14 hours after administration of the test item and slides were prepared. Hepatocytes from the positive control group (2-acetylaminofluourene at 75 mg/kg) were isolated 14 hours after administration. For the liver S-phase assay, male/female PVG rats were given a single oral dose of 250 mg/kg then hepatocytes were isolated at 2, 14, 24 and 48 hours. In the hepatic function assay, female PVG rats were given a single oral dose of 250 mg/kg, to assess test item related hepatotoxicity.

In the UDS assay, there was no increase above the control level for either mean net grains observed or for % of cells in repair by administration of the test item. Additionally, the test item did not induce DNA repair at 2 or 14-hour time points. (Repair = Percentage of cells with net grain > or = 5.0). In the parallel in vivo micronucleus assay there was additionally no positive result. In the hepatic function assay, no significant increases were seen in levels of AST, ALB, TP, and BA at any of the sample times. Significant increases were seen in T-BiI (24 hr; 2.6 ± 0.3 µmol/L), ALP (48 hr; 366 ± 24 iU/L), and ALT (1 week; 70.5 ± 6.6 iU/L) compared to concurrent controls. The authors considered these increases small, not time-related, and all of the measured values were within historical control ranges. It was therefore assumed that the statistical significance achieved for these results is due to normal physiological variation and that they are of no toxicological significance. In the liver cell S-phase assay, it was considered there was a time-related increase in the incidence of cells, and induction of cells undergoing S-Phase synthesis 14 to 48 hour after treatment. The authors indicated the results suggest the test item induces S-phase synthesis in the livers of female rats 14, 24, and 48 hrs after a single oral dose of 250 mg/kg, but without detectable hepatocellular damage or functional impairment. Under the conditions of the study, the test item was not considered a male/female hepatocarcinogen. The authors did not exclude potential effects to the bladder.

 

Eq. or similar to OECD TG 474, 1992 - The test item was investigated in a method equivalent or similar to OECD TG 474, for mammalian bone marrow erythrocyte micronucleus assessment for genotoxicity in F344 female rats. The test item was administered orally at single doses to 5 to 9 females within 0.3% [w/v] METHOCEL™ cellulose ether K15M Premium at doses of 125, 250 and 500 mg/kg bw and a dose volume of 10 mL/kg. The vehicle was used as the negative control and the positive control was cyclophosphamide at 10 mg/kg in sterile water at 10 mL/kg. At 24 and 48 hours exposure time, main study group and negative control and 24 hours for positive control the exposure was terminated, and bone marrow smears were made immediately. The slides were immediately stained with haematoxylin and eosin according to procedure of literature. 2,000 polychromatic erythrocytes (PE) were analysed for the presence of micronuclei. Five hundred erythrocytes per animal were scored to determine the percentage of PE among all erythrocytes. Statistical analysis was performed using likelihood ratio test. Due to lethality at 500 mg/kg bw (6 out of 8 mortalities) the 500 mg/kg bw dose was not utilised for further assessment. The test item did not induce micronucleus formation at either time point, nor was there any reduction in the percentage of PE in the bone marrow of treated animals. Under the conditions of this study, there was no evidence of test item induction of micronucleated erythrocytes and genotoxic properties.

 

Further notes:

In accordance with REACH Regulation (EC) No. 1907/2006 Annex VIII, column 2 section 8.4.2 and 8.4.3 further studies does not need to be conducted if there is adequate data from an in vivo cytogenicity and/or in vivo mammalian gene mutation available. There is (1) an available in vivo mammalian erythorocyte micronucleus test concerning in vivo cytogenicity (in somatic cells) and (2) an available unscheduled DNA synthesis (UDS) with mammalian liver cells test concerning in vivo mammalian gene mutation (in somatic cells) and (3) an available in vivo carcinogenicity study. According to ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7a: Endpoint Specific Guidance, R.7.7, July 2017) the study does not need to be conducted.

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for mutagenicity: germ cell mutagen category 2: H341: Suspected of causing genetic defects

 

This classification is applied by the applicant on a precautionary basis – using the following within the weight of evidence:

1. The substance includes the presence of impurity > 1%wt : 4-nitrosophenol (CAS 104-91-6) with harmonised classification: CLP Regulation (EC) 1272/2008: germ cell mutagen category 2: H341: Suspected of causing genetic defects.

2. Available in vitro bacterial reverse mutation assays (Haworth, 1983 and Dunkel, 1985) either utilised technical grades of test item (ca. 88%wt or the test item purity was not reported). This exceeds the typical substance lower purity range (> or = 90 up to 100%wt) as placed on the market, and this may have been responsible for observed positive responses in the in vitro assays. Which could only be identified by a posteriori deduction and by examining the weight of evidence.

3. There was no observed positive responses in available in vivo cytogenicity and/or in vivo gene mutation assays (Westmoreland, 1992) however, the utilised test item was > 97% purity. Given the range of composition placed on the market, mutagenic effects to germ cells of lower purities resulting from impurity: 4-nitrosophenol (CAS 104-91-6), cannot be excluded. This is in line with CLP Regulation (EC) 1272/2008 section 3.5.2.3.9.

4. The applicant notes that the test item will be additionally classified for CLP Regulation (EC) 1272/2008: carcinogenicity: category 2: H351: Suspected of causing cancer, on the basis limited information and observed bladder neoplasms in female rats. No statement of purity was given in the relevant study (NCI, 1979) although it can be presumed that the substance was within the atypical purity range (> 85 to 95%wt) which was the same conclusion during international review of the substance (WHO IARC, 1982: IARC Monograph 29). At the present time, there is limited evidence of carcinogenicity, confined to only one experiment and there are remaining unresolved questions associated with the study design and only a narrow range of tissues or organs indicated promoting activity, and additionally there is potential confounding properties to interpretation to the presence of impurities such as 4-nitrosophenol (CAS 104-91-6) at atypical levels in the substance, as placed on the market.