Fenamiphos

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Endpoints for in vitro mutagenicity are covered by available in vitro studies in bacteria, mammalian cells and human lymphocytes.

 

The test substance is not mutagen in bacteria or mammalian cells.

Additionaly, clastogenicity was only shown at clearly cytotoxic concentrations in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June to August 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Lymphocytes from the blood of healthy test subjects, one male and one female, were used for the tests. Four cultures per test group were prepared, two per donor.

For lymphocytes:
- Sex, age and number of blood donors: not specified
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: yes, phytohemagglutinin additive

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix was used as the metabolic activation system; it was prepared from the livers of at least 6 adult male Sprague-Dawley rats weighing approximately 200-300 g. For the induction of elevated microsomal enzyme activity, each rat was given a single intraperitoneal injection of Aroclor 1254 dissolved in corn germ oil at a dosage of 500 mg/kg body weight 5 days prior to sacrifice. The animals were prepared in accordance
with the procedure of Ames, et al. (1975), with no fasting. For this purpose, the rats were sacrificed by cervical dislocation and their livers were removed immediately thereafter under sterile conditions.

Test concentrations with justification for top dose:

The concentrations used were selected on the basis of a range-finding
test. +/-S9: 0, 25, 100, 400 μg/mL

Vehicle / solvent:

DMSO

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Four cultures per test group were prepared, two per donor (male and female donor)
- Number of independent experiments: 1

METHOD OF EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
Forty-eight hours after starting the cultures, the test compound, dissolved in DMSO, was added to the culture at a volume of 0.1 mL. The solvent alone was used as the negative control. The positive controls cyclophosphamide and Mitomycin C (dissolved in Hanks' saline solution) were added, also at a volume of 0.1 mL.
- Exposure duration: 24 h
Twenty-one hours after the test compound was added, Colcemid was added to the cultures, to give a final concentration of 0.4 µg/mL, in order to arrest mitosis in the metaphase stage. Three hours later, the cultures were processed.


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colcemid (demecolcine), 0.4 µg/mL
- Methods of slide preparation and staining technique used including the stain used: To prepare the slides, the cultures were carefully shaken and each transferred into a graduated tube. The slides were stained in 5% Giemsa stain for approximately 5 minutes and then rinsed briefly twice in water.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Two to three microscopic slides were prepared per culture. Both with and without S-9 mix, approximately 200 metaphases in each case were examined for structural changes of the chromosomes at each concentration. This means that approximately 100 metaphases were evaluated per culture.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): see "Evaluation criteria"
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): see "Evaluation criteria"

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: In addition to aberrations, metaphases were also found that exhibited chromosome disintegration as an indication of a cytotoxic effect.

Evaluation criteria:

Both with and without S-9 mix, approximately 200 metaphases in each case were examined for structural changes of the chromosomes at each concentration. This means that approximately 100 metaphases were evaluated per culture. Only when there were fewer than 100 metaphases in a culture were fewer metaphases evaluated. The evaluation of this structural chromosome damage was performed essentially in accordance with the following definition of terms:
1. Gap: A gap is an achromatic gap within one chromatid arm, with no dislocation of the end segment.
2. Break: This is an end segment of a chromatid that is noticeably dislocated, but must still be contained in the affected metaphase. Breaks are the precondition for all further chromosome damage.
3. Fragment: This is a chromosome fragment without a centromere resulting from a break; it is not located in the same metaphase with the affected chromosome.
4. Deletion: This is the loss of a chromatid segment resulting from a break, where the broken off segment is no longer present in the metaphase.
5. Exchange: This is an exchange mutation between two or more chromosomes
(interchange) or within one chromosome (intrachange).
6. Multiple aberrations: If more than 4 cases of structural damage (not including gaps) were found in a metaphase, this was evaluated as a multiple aberration.

In addition to these aberrations, metaphases were also found that exhibited chromosome disintegration as an indication of a cytotoxic effect. Chromosome disintegration was identified when no chromosomal structures could be recognized in the metaphase.

Statistics:

The one-tailed, corrected Chi-Square Test was used for the statistical analysis. For the mitotic index, a difference was regarded as significant when the probability of error was less than 1% and there was a decrease in the value of the treated group in comparison to the negative control. This high threshold was selected since the mitotic index is subject to large fluctuations, which are caused by the biological variability of the system. For the aberration rates, a difference was regarded as significant when the probability of error was less than 5% and there was an increase in the value for the treated group in comparison to the negative control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Positive findings were observed only in the clearly cytotoxic range.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-dependent at and above 400 µg/mL (with S9) dose-dependent at and above 100 µg/mL (without S9) Haemolysis: 400 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results of the determination of the mitotic index showed a concentration-related cytotoxic effect of the test substance both with and without S-9 mix. Haemolysis was found at 400 µg/mL.
With respect to the parameters relevant for the evaluation of the clastogenic effect (metaphases with aberrations including and excluding gaps, metaphases with exchanges) compound-related effects were found only in the clearly cytotoxic dose range with and without S-9 mix.
The results of the positive controls Mitomycin C and cyclophosphamide showed a clearly chromosome-damaging effect and demonstrated the sensitivity of the system.

Conclusions:

In conclusion, it must be stated that the test item, under the reported conditiosn and at concentrations of up to and including 400 µg/mL both without and with S-9 mix, exhibited a chromosome-damaging effect in the cytogenetic in vitro test on human lymphocyte cultures. Positive findings, however, were observed only in the clearly cytotoxic range. The relevance of this finding for the in vivo situation must therefore be regarded as questionable.

Executive summary:

Tthe test item was dissolved in DMSO and tested for chromosome-damaging effect in human lymphocytes cultures at concentrations up to and including 400 µg/mL both with and without S-9 mix. Mitomycin C and Cyclophosphamide were tested as positive controls.

The results of the determination of the mitotic index showed a concentration-related cytotoxic effect of the test substance both with and without S-9 mix. Haemolysis was found at 400 µg/mL.

With respect to the parameters relevant for the evaluation of the clastogenic effect (metaphases with aberrations including and excluding gaps, metaphases with exchanges) compound-related effects were found only in the clearly cytotoxic dose range with and without S9 mix.

A higher incidence of chromosome damage was found at cytotoxic concentrations of the test substance in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984-09-14 to 1985-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 cells

Metabolic activation:

with and without

Metabolic activation system:

The S9 was prepared according to established procedures. Adult male Fischer rats, 200 – 250 g, were induced by a single intraperitoneal injection of Aroclor-1254 at a dosage of 500 mg/kg bw two days prior to sacrifice. The animals were sacrificed and the livers removed. The excised tissue was rinsed and homogenised. The supernatant fraction (S9) was collected following centrifugation, portioned into aliquots.

Test concentrations with justification for top dose:

100, 110, 120 and 130 µg/mL (-S9), 0, 170, 190,
210, 230 μg/mL (+S9) based on preliminary study

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10E5 cells per flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 5 h

FOR GENE MUTATION:
- Expression time: 7 - 9 days
- Selection time: 7 - 9 days
- If a selective agent is used: 6-Thioquanine (TG, 2-amino-6-mercaptopurine), 20 mM in 0.1 M NaOH

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Replicates from each treatment condition were pooled and subcultured in triplicate, at a densitiy of 100 cells/ 60 mm dish. After 7 - days incubation the colonies were fixed with methanol, stained with 10 % aqueous Giemsa and counted.

METHODS FOR MEASUREMENTS OF GENOTOXICITY: The mutation frequency (MF) or each treatment condition has been calculated dividing the total number of mutant colonies by the number of cells selected and corrected for cloning efficiency of cells prior to mutant selection.

Evaluation criteria:

The cytotoxicity effects of each treatment condition are expressed relative to the solvent treated-control (relative cloning efficiency). The mutation frequency (MF) or each treatment condition has been calculated dividing the total number of mutant colonies by the number of cells selected and corrected for cloning efficiency of cells prior to mutant selection and is expressed TG-resistant mutants per 10E6 clonable cells. The assay is considered positive in the event a dose-dependent increase in mutation -frequency is observed with one or more of the four concentrations tested inducing a MF which is at least twice that of the solvent control. The assay is considered suspect if there is no dose response but one or more doses induce a mutation frequency which is at least twice that of the solvent control. The assay is considered negative if none of the doses tested induce a MF which is at least twice that of the solvent control.

The cloning efficiency must be no less than 50 % for solvent and untreated controls. The spontaneous MF in the solvent and untreated controls must fall within the range of 0-20 per 10E6 clonable cells.
The positive control must induce a MF at least three times that of the solvent control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test substance did not induce gene mutations in Chinese hamster ovary cells CHO-K1-BH4.

Executive summary:

The test item was dissolved in DMSO and tested for its potential to induce forward mutations at the HGPRT locus of CHO cells. Concentrations up to 120 µg/mL were tested without the addition of S-9 mix and up to 230 µg/mL with S-9 mix. Benzopyrene and ethylmethanesulfonate were used as positive control agents.

The mutant frequency of the test item treated samples was not increased in comparison to the solvent control samples both in the absence and in the presence of S9-mix. The positive controls demonstrated a good sensitivity of this assay.

Therefore, the test item was considered to be negative in the CHO/HGPRT mutation assay in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October to November 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

The study was conducted in accordance with the Salmonella/microsome test as described by Ames et al. (1973a, 1975). There were no deviations from the regulatory guideline considered to compromise the scientific validity of the study. Although no E. Coli strain has been tested the study has been considered adequate to address the endpoint of point mutations also during the 1st peer review of the test substance. Although the study was conducted in 1985, it is considered to comply in general with the OECD Guideline of 1997; any deviations from that guideline are considered not to have had a siginificant impact on the overall conclusion of the study.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Partly deficiency in lipopolysaccharide side chains, ampicillin resistance, crystal violet sensitivity, UV sensitivity

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix was used to metabolise. It was made from the livers of at least six adult male Sprague Dawley rats, approx. 200 - 300 g in weight. For enzyme induction the animals received a single intraperitoneal injection of aroclor 1254 at a dose of 500 mg/kg body weight, dissolved in peanut oil, five days before preparation. The animals were prepared as specified by Ames et al. (1975), and the S-9 fraction stored at -80° C in 10 ml portions. These portions were thawed slowly for use. The S-9 mix was prepared fresh (Ames et al., 1973a), the S-9 fractions being taken from the preparation dated 28.8.1984 and forming 30 % of the finished S-9 mix.

Test concentrations with justification for top dose:

The dose ranges were 0, 20, 100, 500, 2500, 2500 μg/plate in the preliminary assay and 0, 125, 250, 500, 1000, 2000 μg/plate in the confirmatory assay. 2000 µg per plate was used in the confirmatory test due to the substance's toxicity.

Vehicle / solvent:

DMSO, water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and demineralized water for endoxan

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA), trypaflavine, endoxan

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Four plates per strain per dose were used for each substance
- Number of independent experiments: 2

METHOD OF TREATMENT:
- Test substance added in medium (nutrient broth)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 48 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


METHODS FOR MEASUREMENTS OF GENOTOXICITY: mutant count

Rationale for test conditions:

The test followed the directions of Ames et al. (1973a, 1975).

Evaluation criteria:

A reproducible dose-related increase in the mutant counts of at least one strain is considered positive, and about double the negative control count should be reached.

Key result

Species / strain:

E. coli WP2

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 12500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2000 µg/plate and 12500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test substance did not induce point mutations in S. typhimurium under the examined conditions.

Executive summary:

The test substance was evaluated in the Salmonella/microsome test, in doses up to 12500 µg per plate, on four Salmonella typhimurium LT2 mutants for mutagenic effect with and without S-9. The strains concerned were the histidine auxotrophs TA 1535, TA 100, TA 1537 and TA 98. Endoxan, trypaflavine, MMS, 4-NQO and 2-amino-anthracene were used as positive controls. The dose ranges were 0, 20, 100, 500, 2500, 2500 μg/plate in the preliminary assay and 0, 125, 250, 500, 1000, 2000 μg/plate in the confirmatory assay, both using DMSO as solvent. Test item concentrations up to 12500 µg/plate did not produce an increase in the mutant count with and without S-9 mix. Bacteriotoxic effects after 500 µg/plate was observed, both with and without S-9 mix. The positive controls demonstrated a clear mutagenic effect in comparison to the negative controls. The test substance was non-mutagenic in the reverse mutation assay with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-05-13 to 2004-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. PMSB/ELD Notification No. 554.

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (5% v/v), isocitric acid (8.7 mM), NADP (1.6 mM) in R0. The co-factors were neutralised with 1N NaOH and filter sterilised before adding to S9 fraction and R0. S9 fraction was prepared from a group of ca. 10 rats. Mixed function oxidase systems in the rat livers were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in corn oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed.

Test concentrations with justification for top dose:

- 0, 10, 40, 80, 100, 120, 140, 160, 180 μg/mL with 3 h exposure (+/- S9), for repeat study (3 h exposure) the dose levels were 0, 5, 120, 150, 160, 170, 175 µg/mL without S9 and 0, 5, 120, 180, 185, 190, 195, 200 µg/mL with S9;
- 0, 5, 25, 40, 50, 60, 70, 80, 90 µg/mL (no independent repeat) with 24 h exposure (-S9)
All concentrations were based on a preliminary toxicity study.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2.0 x 10E6 cells/mL (3 h), 0.3 x 10E6 cells/mL (24 h)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 3 and 24 h
- Harvest time after the end of treatment: after 24 and 48 hours (3 and 24 h exposure)

FOR GENE MUTATION:
- Expression time: at least 7 days
- Selection time: approximately 10-14 days for mutant plates
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent: 4 µg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 10E3 cells/well
- Criteria for small and large colonies: The colony size distribution in the solvent and positive control was examined to ensure that there was an adequate recovery of small colony mutants.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Method: cloning efficiency (CE)

ACCEPTANCE CRITERIA
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in solvent and positive control cultures.

The criteria used to assess whether an assay was valid are based on those published by Sofuni et al, 1997 and Moore et al, 2000 and Moore et al, 2003. Sufficient cultures should be available for analysis of MF; normally scorable plates from at least 3 solvent control cultures, 1 positive control culture and from both cultures from at least 4 test substance concentrations.
Ideally, an acceptable range of suspension growth and CE for the solvent controls should be 8-32 and 65-120% respectively.
The concurrent solvent control should have a MF preferably of 0.00005-0.0002. The solvent and positive control MF should lie within the historical solvent and positive control range respectively.
There should be approximately 50-70% small colony mutants in the solvent control and more small colony mutants in the positive control than in the solvent control. For test substances showing a steep toxicity curve it may not always be possible to achieve an RTG of 10-20% even with 8 concentrations. In such cases a test will be considered to be acceptable if there are closely-spaced concentrations (less than 2-fold dose spacing) and the highest concentration tested shows RTG from approximately 20% to total death.

Evaluation criteria:

Valid tests were assessed, using data for MF where the RTG exceeds 10 %. The Study Director used her judgement to classify the results as negative, positive or equivocal using the following guidelines in sequence.

1. If all test substance concentrations are associated with increases in MF <= 1.5 times the concurrent solvent control value, the result is considered negative.
2. If any test substance concentration is associated with increases in MF >= 3.0 times the concurrent solvent control value, the result is considered positive.
3. If there is a statistically significant (P<=0.01) increase in MF following exposure to the test substance (Dunnett’s test) with a corresponding RTG of 10% or greater, the maximum observed response is compared with the 95% confidence limits for the laboratory historical solvent control range. If the observed increase lies within the 95% confidence limits, the test substance is considered to have given statistically significant but biologically unimportant responses in the assay and the result is considered negative.
4. If the increase in MF exceeds the 95% confidence limits for the historical solvent control range, the nature of the concentration-effect relationship will be assessed. For example, if there is evidence of a concentration relationship over at least two, preferably high, concentrations and a significant positive linear trend (P<=0.01) the result is considered positive.
5. The reproducibility in any increases in MF between cultures and between tests was assessed. Increases in the number of mutant colonies (as opposed to mutant frequency) were considered. Results that are considered inconclusive may need further testing and/or test modification. In rare cases the data set will preclude making a definite judgement about the activity of the test substance when results will be characterised as equivocal.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
A 3-hour exposure to 5.9-3034 μg/mL in the absence and presence of S9 mix resulted in the Relative Suspension Growth (RSG) values of 114-0% and 99-0% respectively. A continuous exposure to the test item, at concentrations of 5.9-3034 μg/mL for 24-hours in the absence of S9 mix resulted in RSG values of 103-0%. Concentrations used in the main test were based upon these data.

Three-hour treatment - first main test:
In the absence of S9 mix a 3-hour exposure to the test substance at concentrations of 10-200 μg/mL resulted in RSG values of 90-1 %. Cultures exposed to the test substance at concentrations of 10 up to 180 μg/mL were assessed for determination of mutant frequency. RTG values of 83-5 %, and cloning efficiencies of 97-86 %, were obtained, relative to the solvent control. There were no statistically significant increases in mutant frequency after exposure to the test item, at concentrations within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced a large increase in mutant frequency and an increase in the number of small colony mutants.
In the presence of S9 mix a 3 hour exposure to the test substance at concentrations of 5-200 µg/mL resulted in RSG values of 99-17 %. Cultures exposed to the test substance at concentrations of 5-200 µg/mL were assessed for determination of mutant frequency. RTG values of 104-15 %, and cloning efficiencies of 117-90 %, were obtained, relative to the solvent control. Data are presented for concentrations tested which span the toxicity range of approximately 10-100% RTG. There were no statistically significant increases in mutant frequency after exposure to the test substance, at concentrations within acceptable levels of toxicity. The positive control, 3-methylcholanthrene, induced a large increase in mutant frequency and an increase in the number of small colony mutants.

24-hour treatment - second main test:
In the absence of S9 mix a 24 hour exposure to the test substance at concentrations of 5-100 µg/mL resulted in RSG values of 107-9 %. Cultures exposed to the test substance at concentrations of 5-90 µg/mL were assessed for determination of mutant frequency. RTG values of 112-12 %, and cloning efficiencies of 110-85 %, were obtained, relative to the solvent control. Data are presented for concentrations tested which span the toxicity range of approximately 10-100 % RTG. There were no statistically significant increases in mutant frequency after exposure to the test substance, at concentrations within acceptable levels of toxicity. The positive control, methyl methanesulphonate, induced a large increase in mutant frequency and an increase in the number of small colony mutants.

Validity criteria:
All reported mutagenicity tests were assessed for validity, and were observed to have met acceptance criteria.

Conclusions:

The test item did not demonstrate a mutagenic or clastogenic potential in the mouse lymphoma assay under the examined conditions.

Executive summary:

The test item (purity 95.6%, lot no. 2380NCL40) was analysed for its genotoxic potential in mouse lymphoma cells L5178Y TK +/- and a test item dose range of 10-200 μg/mL with 3 h exposure (+/- S9) and 5-100 μg/mL with 24 h exposure (-S9). DMSO was used as solvent and the positive control items were without metabolic activation (-S9 mix): Methyl methanesulphonate and with metabolic activation (+S9 mix): 3-methylcholanthrene.

Relative Total Growth [RTG] (–S9) for the 3 h exposure was: 5% at 180 μg/mL and 15% at 175 μg/mL; RTG (+ S9): 32-38% at 180 μg/mL and 15% at 200 μg/mL. RTG (–S9) for the 24 h exposure was 12% at 90 μg/mL.
Precipitation was not observed in the main test (in preliminary toxicity test: above 1616 μg/mL).

The positive controls induced a large increase in mutant frequency and an increase in the number of small colony mutants for both samling times and with and wthout S9. All reported mutagenicity tests were assessed for validity, and were observed to have met acceptance criteria.

The test item did not demonstrate a mutagenic or clastogenic potential in the mouse lymphoma assay under the examined conditions.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: lymphocytes, human

For lymphocytes:
- Sex, age and number of blood donors: one male and one female
healthy donor
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix was used for the simulation of mammalian metabolism of SRA 3886. It was made from the livers of at least six adult male Sprague Dawley rats approximately 200 to 300 g in weight. For enzyme induction, the animals received a single Intraperitoneal injection of Aroclor 1254 at a dose of 500 mg/kg body weight dissolved in corn oil five days before sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975).

Test concentrations with justification for top dose:

0, 25, 50, 75, 100 μg/mL (-S9); 0, 100, 150, 225, 350 μg/mL (+S9);
The concentrations used were based on a prior study (T 3022874, Report No. 15406) in which concentrations were used: 25 µg/ml, 100 µg/mL and 400 µg/mL.

Vehicle / solvent:

DMSO

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
There were four cultures in each group, two per donor. Per donor and treatment group only one culture was evaluated. The second culture was maintained as a spare and was only evaluated if it was found during culture or evaluation that the first culture could not be used.
- Number of independent experiments

METHOD OF EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
- Exposure duration: 24 h
Colcemid was added at a final concentration of 0.4 µg/mL, to the cultures twenty-one hours after the substance, in order to arrest the mitoses in the metaphase stage. Three hours later, the cultures were terminated, and the slides were prepared.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colcemid, final concentration: 0.4 µg/mL
- Methods of slide preparation and staining technique used including the stain used: To prepare the slides, the cultures were carefully shaken and each transferred into a graduated tube. The slides were stained in 5% Giemsa stain for approximately 5 minutes and then rinsed briefly twice in water.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Two to three microscopic slides were prepared per culture. A light microscope at approximately 1000x magnification with planapochromatic lenses was used for the evaluation.
The mitotic index was determined by counting 1000 cells per culture including the spare cultures. The numbers of mitotic and non-mitotic cells were noted. The spare cultures were also used to enlarge the data base for that parameter, which varies over a wide range. Approximately 200 metaphases per concentration, both with and without S-9 mix, were examined for structural changes in the chromosomes, i.e. approximately 100 metaphases were evaluated per sex in each test group. The spare cultures were not used for this evaluation. Only if less than 100 metaphases could be evaluated in a culture, then this number was brought up to 100 by reading the slides of the respective spare culture. Less than 100 metaphases were read only if less than 100 metaphases could be found in normal and in the spare cultures together.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: In addition to aberrations, metaphases were also found that exhibited chromosome disintegration as an indication of a cytotoxic effect. Chromosome disintegration was assumed if virtually no chromosomal structures could be recognized in the metaphase.

Evaluation criteria:

The evaluation of this structural chromosome damage was performed essentially in accordance with the following definition of terms:
1. Gap: A gap is an achromatic gap within one chromatid arm, with no dislocation of the end segment.
2. Break: This is an end segment of a chromatid that is noticeably dislocated, but must still be contained in the affected metaphase. Breaks are the precondition for all further chromosome damage.
3. Fragment: This is a chromosome fragment without a centromere resulting from a break; it is not located in the same metaphase with the affected chromosome.
4. Deletion: This is the loss of a chromatid segment resulting from a break, where the broken off segment is no longer present in the metaphase.
5. Exchange: This is an exchange mutation between two or more chromosomes (interchange) or within one chromosome (intrachange).
6. Multiple aberrations: If more than 4 cases of structural damage (not including gaps) were found in a metaphase, this was evaluated as a multiple aberration.


ACCEPTANCE CRITERIA
An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the negative control rate was in the expected range according to this laboratory's experience.

1. A test was considered positive if there was a dose - dependent and statistically significant test was considered negative if there was no such
increase in any of the concentrations tested.

2. A test was considered equivocal if there was an increase which was statistically significant but not concentration - related, or if a concentration -
related increase occurred which was not statistically significant increase in the aberration rate.

Statistics:

The one-sided corrected chi2 test was used for the statistical evaluation. A difference was considered as significant if the probability of error was below 5%. With reference to the mitotic index the test was only performed if the treatment group value was lower than the negative control value. In reference to the aberration rates, the test was only performed if this rate was higher in the treated group compared to the negative control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 350 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 225 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

1. with S-9 mix:
The test item did not increase the number of aberrations at concentrations up to 225 µg/mL. With respect to the parameters relevant for evaluation (metaphases with aberrations including or excluding gaps, and metaphases with exchanges), no statistically significant variations were noted. However, at 350 µg/mL, a significant increase was found. Aberrant metaphases including and excluding gaps were significantly increased. But no exchanges
were found. But, these differences were found only in the strongly cytotoxic concentration range.

2. without S-9 mix:
The test ite, did not lead to a relevant increase in the incidence of aberrations.
With respect to the parameters relevant for evaluation (metaphases with aberrations including or excluding gaps, and metaphases with exchanges) no statistically significant variations were noted.

The positive controls mitomycin C and cyclophosphamide had a clear clastogenic effect, thereby demonstrating the system's sensitivity to clastogenic agents.

Conclusions:

The test substance did induce chromosome aberrations in human lymphocytes (with metabolic activation only) under the examined conditions.

Executive summary:

The test item was disolved in DMSO and tested for chromosome-damaging effects in human lymphocytes cultures at concentrations up to 100 µg/mL without S-9 mix and up to 350 µg/mL with S-9 mix. Mitomycin C and Cyclophosphamide served as positive control agent.

Without the addition of S-9 mix no differences were found between the test substance test concentrations and the control sample.

In the presence of S-9 mix a positive test result was obtained only for the highest concentration (350 µg/mL) which, however, also demonstrated a clear cytotoxic effect. Therefore, the variations observed at this dose level were not regarded as an indication for a clastogenic effect of the test substance. The positive controls showed a good sensitivity of the test system.

The test substance was found to induce no clastogenic effects to human lymphocytes at non-cytotoxic in vitro concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Endpoints for in vivo mutagenicity are covered by available animal studies.

 

The test substance is not clastogenic in an in vivo micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-08-13 to 1995-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

1000 PCE counted per animal; only one dose level tested

Principles of method if other than guideline:

Deviations from OECD 474:
- Only one dose level was used. This level was clearly toxic to the animals. The choice of dose level was based on a pilot test in which animals were exposed to 25 or 100 mg/kg bw of 10 GR. Animals of both dose levels showed toxic symptoms and all animals in the high dose died.
- Only 1000 PCE were scored instead of 2000.
In this case the results for males (5) and females (5) could be taken together (according to the guidelines it is sufficient to test a single sex when there are no substantial differences in toxicity between the sexes).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Hsd/Win: NMR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: Males: 36.0 - 43.0 g (mean: 37.3 g); females 27.0 - 34.0 g (mean: 29.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were kept singly in type I cages with bedding of soft wood granules
- Diet (ad libitum): Altromin 1324 standard diet (Altromin GmbH, Lage, Germany)
- Water (ad libitum): Tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 23.5
- Humidity (%): 40.0 - 70.0
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in deionized water using sonication for 5 minutes, and formed a light-brown turbid suspension. The suspension was stirred with a magnetic mixer during administration and injected intraperitoneally. Cyclophosphamide was dissolved in deionized water and administered in the same way. The negative control received deionized water by the same method. In all of the groups, the administered volume was 10 mL/kg body weight.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single application

Post exposure period:

16, 24 or 48 h

Dose / conc.:

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

No. of animals per sex per dose:

5 animals per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES: 16, 24 and 48 h
Sacrifice of the negative and positive controls was after 24 hours

DETAILS OF SLIDE PREPARATION:
From each sacrificed animal, one intact femur was prepared. Bone marrow cells were washed out by injecting fetal bovine serum into one end of the dissected epiphyses of each femur. Following centrifugation, excess supernatant was removed and the cells re-suspended in a small volume of serum remaining in the tube. A drop of the suspension was used to make a smear. The smears were dried overnight dried with heat for a short period. The smears were stained automatically. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry before covering.

METHOD OF ANALYSIS:
One slide per animal was examined at 1000-fold magnification by light microscopy. Normally, 1000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A relevant, treatment related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated polychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones. Before an effect can be observed in normochromatic erythrocytes, there must be a much greater increase in micronucleated polychromatic erythrocytes, due to the "dilution effect" of the "old" cells, i.e. normochromatic erythrocytes already present at the start of the test, and this effect would have been observed previously.

VALIDITY CRITERIA
(1) The frequency of micronucleated polychromatic erythrocytes in the negative control group was within an acceptable range based on the historical control data in the testing facility;
(2) The frequency of micronucleated polychromatic erythrocytes in the positive control group was statistically higher than that of the concurrent vehicle control group.

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which was within the range of negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

Statistics:

The test item groups with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The dose selectionof the test item was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 25 mg/kg and 100 mg/kg test substance. The following symptoms were recorded for up to 48 hours, starting at 25 mg/kg: apathy, roughened fur, spasm, extension spasm and leaping spasm. In addition, 5 of 5 animals died in the 100 mg/kg group. Based on these results, 25 mg/kg test subtance was chosen for this test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: 1.7/1000 (1s=1.4, negative contro)l, 1.8/1000 (1s=1.7), 1.1/1000 (ls=1.0) and 1.5/1000 (1s=1.0) in the test item 16, 24 and 48 h groups
- Ratio of PCE/NCE: 1000:1047 (1s=506, negative control), 1000:1027 (1s=268, the 16 hours group), 1000: 865 (1s=208, 24 hours group) and 1000:939 (1s=471, 48 hours group)

After single intraperitoneal administration of 25 mg/kg of the test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

Concerning the assessment of the clastogenic potential of the test item, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test item, being 1000:1047 (1s=506) in the negative control, 1000:1027 (1s=268) in the 16 hours group, 1000: 865 (1s=208) in the 24 hours group and 1000:939 (1s=471) in the 48 hours group. No relevant variations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 25 mg/kg the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.7/1000 (1s=1.4) in the negative control, and 1.8/1000 (1s=1.7), 1.1/1000 (ls=1.0) and 1.5/1000 (1s=1.0) in the the test item groups.
Similarly, there could be no biologically significant variation between the negative control and the test item groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 18.7/1000 (ls=6.7), which represents a biologically relevant increase in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones. No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [1000:862 (ls=221), as against 1000:1047 in the
negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.
All validity criteria were met.

Conclusions:

There was no indication of a clastogenic effect of an intraperitoneal dose of 25 mg/kg bw the test subtance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Executive summary:

The test item was analysed for its clastogenic potential in the in vivo micronucleus test.

Mice (5/sex/dose) were dosed by intraperitonial injection of the test substance at a dose of 0 or 25 mg/kg bw (equivalent to 2.5 mg test item/kg bw). Deionized water was used as vehicle and cyclophosphamid served as positive control test item. The bone marrow was sampled 16, 24 and 48 h after dosing. 1000 polychromatic erythrocytes (PCE) were counted per animal. The number of normochromatic erythrocytes (NCE) per 1000 polychromatic ones was noted. In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established.

After single intraperitoneal administration of 25 mg/kg of the test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

The following PCE/NCE ratios were established: 97%, 115%, 106% for 16, 24 and 48 h after dosing, respectively (the ratio for positive control was 121% and for the negative control 96%). The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test item. No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 25 mg/kg of the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. Similarly, there could be no biologically significant variation between the negative control and test item groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. All validity criteria were met in this micronucleus test.

It was concluded that there was no indication of a clastogenic effect of an intraperitoneal dose of 25 mg/kg bw of the test item in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The information provided below was taken from the original plant protection dossier on the active substance submitted in 2017 for inclusion of the test substance to Annex I of Directive 91/414/EEC and has been previously evaluated in the Draft Assessment Report (DAR) according to the Commission Regulation (EU) No 1107/2009 (2003; Renewal Assessment Report (RAR) 3rd Revision: 2018) and subject to peer review by EFSA and Member States (2006).

 

In vitro genetic toxicity

Several in vitro mutagenicity assays are performed with the test substance. In an Ames test (Herbold, 1985) with doses up to 12.5 mg/plate, the test item did not induce point mutations in S. typhimurium, neither with nor without metabolic activation. In addition, a gene mutation assay (HGPRT) in CHO-K1-B4H4 cells (Yang, 1985) was negative.  Two in vitro cytogenic assays (Herbold, 1987; 1988) were performed in human lymphocytes. In the first, 100 μg/mL test item induced 9.5 % metaphases with aberrations (excluding gaps) without metabolic activation, compared to 1.0 % in the control group. With metabolic activation, an increase in metaphases with aberrations was only observed at highly cytotoxic levels (mitotic index <0.1 %). However, the dose levels in combination with metabolic activation were not appropriate, since at 100 μg/mL toxicity was not observed and at 400 μg/mL haemolysis was observed. In the second assay, no increase in cells with chromosome aberrations was found without metabolic activation between the test substance and control values. In the presence of S9, a dose related increase in metaphases with aberrations excluding gaps was observed at the highest dose (10% metaphases with aberrations excluding gaps vs. 3.5% in the control group). At lower concentrations no increase in cells with chromosome aberrations were observed. In addition, a mouse lymphoma test (Soanes, 2004) was performed, with doses up to 200 μg/mL. The positive result in the chromosome aberration study in human lymphocytes without metabolic activation at a dose level of 100 μg/ml coulL not be confirmed in this assay. The test item did not demonstrate a mutagenic or clastogenic potential in this assay.

 

Herbold (1985): The test substance was evaluated in the Salmonella/microsome test equivalent to OECD 471, in doses up to 12500 µg per plate, on four Salmonella typhimurium LT2 mutants for mutagenic effect with and without S-9. The strains concerned were the histidine auxotrophs TA 1535, TA 100, TA 1537 and TA 98. Endoxan, trypaflavine, MMS, 4-NQO and 2-amino-anthracene were used as positive controls. The dose ranges were 0, 20, 100, 500, 2500, 2500 μg/plate in the preliminary assay and 0, 125, 250, 500, 1000, 2000 μg/plate in the confirmatory assay, both using DMSO as solvent. Test item concentrations up to 12500 µg/plate did not produce an increase in the mutant count with and without S-9 mix. Bacteriotoxic effects after 500 µg/plate was observed, both with and without S-9 mix. The positive controls demonstrated a clear mutagenic effect in comparison to the negative controls. The test substance was non-mutagenic in the reverse mutation assay with and without metabolic activation.

 

The following statement as presented in the RAR (2018), prepared according to Regulation (EC) No 1107/2009, is given below to adress test acceptability.

Although no E. Coli strain has been tested, the study has been considered adequate to address the endpoint of point mutations of the test item.

This deviation had not been considered significant enough so that not to consider the study valid for drawing a final conclusion regarding point mutations in bacteria. The study has been considered acceptable and conclusive for the strains tested. More specifically, the following are noted:
The study was conducted in 1985 in accordance with the Salmonella/microsome test as described by Ames et al. (1973a, 1975). It has been considered that the study complies in general with the OECD Guideline of 1997. Although no E. Coli strain has been tested, the study has been considered adequate to address the endpoint of point mutations; any deviations from the OECD guideline are considered not to have had a significant impact on the overall conclusion of the study. Overall, the study is considered acceptable in order to address the issue of point mutations in bacteria.

 

 

Yang (1985): The test item was dissolved in DMSO and tested for its potential to induce forward mutations at the HGPRT locus of CHO cells equivalent to OECD 476. Concentrations up to 120 µg/mL were tested without the addition of S-9 mix and up to 230 µg/mL with S-9 mix. Benzopyrene and ethylmethanesulfonate were used as positive control agents. The mutant frequency of the test item treated samples was not increased in comparison to the solvent control samples both in the absence and in the presence of S9-mix. The positive controls demonstrated a good sensitivity of this assay. Therefore, the test item was considered to be negative in the CHO/HGPRT mutation assay in vitro.

 

Soanes (2004): The test item was analysed for its genotoxic potential in mouse lymphoma cells L5178Y TK +/- according to OECD 476 and a test item dose range of 10-200 μg/mL with 3 h exposure (+/- S9) and 5-100 μg/mL with 24 h exposure (-S9). DMSO was used as solvent and the positive control items were without metabolic activation (-S9 mix): Methyl methanesulphonate and with metabolic activation (+S9 mix): 3-methylcholanthrene. Relative Total Growth [RTG] (–S9) for the 3 h exposure was: 5% at 180 μg/mL and 15% at 175 μg/mL; RTG (+ S9): 32-38% at 180 μg/mL and 15% at 200 μg/mL. RTG (–S9) for the 24 h exposure was 12% at 90 μg/mL. Precipitation was not observed in the main test (in preliminary toxicity test: above 1616 μg/mL). The positive controls induced a large increase in mutant frequency and an increase in the number of small colony mutants for both samling times and with and wthout S9. All reported mutagenicity tests were assessed for validity, and were observed to have met acceptance criteria. The test item did not demonstrate a mutagenic or clastogenic potential in the mouse lymphoma assay under the examined conditions.

 

Both studies (Yang, 1985; Soanes, 2004) were assessed in a weight-of-evidence approach. Based on their experimental outcomes, it is concluded that the test item is not mutagenic in mammalian cells.

 

 

Herbold (1988): Test item was disolved in DMSO and tested for chromosome-damaging effects in human lymphocytes cultures similar to OECD 473 at concentrations up to 100 µg/mL without S-9 mix and up to 350 µg/mL with S-9 mix. Mitomycin C and Cyclophosphamide served as positive control agent. Without the addition of S-9 mix no differences were found between the test substance test concentrations and the control sample. In the presence of S-9 mix a positive test result was obtained only for the highest concentration (350 µg/mL) which, however, also demonstrated a clear cytotoxic effect. Therefore, the variations observed at this dose level were not regarded as an indication for a clastogenic effect of the test substance. The positive controls showed a good sensitivity of the test system. The test substance was found to induce no clastogenic effects to human lymphocytes at non-cytotoxic in vitro concentrations.

 

Herbold (1987): Test item was dissolved in DMSO and tested for chromosome-damaging effect in human lymphocytes cultures similar to OECD 473 at concentrations up to and including 400 µg/mL both with and without S-9 mix. Mitomycin C and Cyclophosphamide were tested as positive controls. The results of the determination of the mitotic index showed a concentration-related cytotoxic effect of the test substance both with and without S-9 mix. Haemolysis was found at 400 µg/mL. With respect to the parameters relevant for the evaluation of the clastogenic effect (metaphases with aberrations including and excluding gaps, metaphases with exchanges) compound-related effects were found only in the clearly cytotoxic dose range with and without S9 mix. A higher incidence of chromosome damage was found at cytotoxic concentrations of the test substance in vitro.

 

Based on the experimental outcomes of both in vitro micronucleus tests (Herbold 1988, 1987), it was concluded that the test substance is not clastogenic in vitro .

 

In vitro genotoxicity studies and according results which are considered acceptable for the overall evaluation of the test substance are summarized in Table 1.

 

Table 1: Summary of in vitro genetic toxicity studies

Indicator cells	Endpoint	Result without S9 activation	Result with S9 activation	Reference
S. typh. TA 98 TA 100 TA 1535 TA 1537	frame shifts base pair substitutions base pair substitutions frame shifts	- - - -	- - - -	Herbold, 1985
Chinese hamster ovary cells CHO-K1-BH4	gene mutations (HGPRT)	-	-	Yang, 1985
Human lymphocytes	chromosome aberration	+	n.a.	Herbold, 1987
Human lymphocytes	chromosome aberration	-	+	Herbold, 1988
mouse lymphoma cells	gene mutation / clastogenicity	-	-	Soanes, 2004

 

In vivo genetic toxicity:

Herbold (1997): The test item was analysed for its clastogenic potential in the in vivo micronucleus test according to OECD 474. Mice (5/sex/dose) were dosed by intraperitonial injection of the test substance at a dose of 0 or 25 mg/kg bw (equivalent to 2.5 mg test item/kg bw). Deionized water was used as vehicle and cyclophosphamid served as positive control. The bone marrow was sampled 16, 24 and 48 h after dosing. 1000 polychromatic erythrocytes (PCE) were counted per animal. The number of normochromatic erythrocytes (NCE) per 1000 polychromatic ones was noted. In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. After single intraperitoneal administration of 25 mg/kg of the test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups. The following PCE/NCE ratios were established: 97%, 115%, 106% for 16, 24 and 48 h after dosing, respectively (the ratio for positive control was 121% and for the negative control 96%). The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test item. No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 25 mg/kg of the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. Similarly, there could be no biologically significant variation between the negative control and test item groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. All validity criteria were met in this micronucleus test. It was concluded that there was no indication of a clastogenic effect of an intraperitoneal dose of 25 mg/kg bw of the test item in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

 

Herbold (1980): A non-GLP micronucleus test was conducted with oral administration in mice. The mice (5/sex/dose) were orally dosed twice 0, 0.625, 1.25 or 2.5 mg/kg bw of the test substance, 24 h apart, and the bone marrow was sampled once, 6 h after the second dose. Because of severe toxicity at the highest dose (mortality in 4 tested animals after first dose) it was not possible to evaluate these animals for micronuclei formation. Systemic toxicity was observed at dose level: 2 x 2.5 mg/kg bw. The PCE/NCE ratio was 108 %, 151 %, 96 % for 0, 2 x 0.625, 2 x 1.25 mg/kg bw, respectively. Considering that the substance is not able to reach the target organ (please refer to section 7.1), no conclusions on chromosome aberrations in vivo can be drawn from this study.

 

In vivo genotoxicity studies and according results are summarized in Table 2.

 

Table 2: Summary of in vivo genetic toxicity studies

Species	Endpoint	Result	Reference Applicant
micronucleus test (i.p.)	micronuclei/ chromosomal damage	-	Herbold, 1997
micronucleus test (oral)	micronuclei/ chromosomal damage	-	Herbold, 1980

 

The following statement as presented in the RAR (2018), prepared according to Regulation (EC) No 1107/2009, is given below to adress test item's bone marrow exposure following i.p. administration in Herbold (1997):

During the commenting period on the RAR (October 2017), reservations were expressed regarding the acceptability of the in vivo micronucleous test following i.p. route of administration and whether bone marrow exposure is sufficient to conclude. It is noted that during the 1st peer review of the test item similar concerns had been raised regarding the bone marrow exposure in the study of Herbold (1980a) where the test item had been administered orally. However, the test item was concluded not to be genotoxic based on the available negative in vivo MN assay following i.p. administration of the test item (EFSA Scientific Report, 2006) considering that since the test substance was given i.p., it has reached the bone marrow. This conclusion has been considered still valid during the reevaluation of the study, i.e. it has been considered that i.p administration can be considered representative of the oral administration or even a worst case. It is further noted that, based on the available TK data (Beaudoin, 2015), radioactivity in plasma was detected following oral administration of radiolabelled test substance and, thus, in general there should be no concerns regarding the bone marrow exposure following oral administration. Regarding the bone marrow/plasma exposure following the i.p. administration of the test item, the systemic toxicity observed in the study of Herbold (1997), i.e. apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing, further supports the conclusion drawn that there is enough evidence of systemic exposure to fenamiphos, meaning also plasma and, consequently, bone marrow exposure.

Overall, it is considered that there is adequate weight of evidence to support that bone marrow has been exposed in the available MN assay following i.p. administration. The applicant has presented the following additional arguments/information when responding to the Reporting Table – Comment 2(29):

“The distribution comment quoted is taken from a whole body autoradiography study at a single sampling time. The level of discrimination in identifying the absolute presence or concentration of a material in such a study is not great, and the study cannot be used to determine that the bone marrow was not exposed to the test item or metabolites. Similarly, the observation of rapid excretion does not indicate that the bone marrow was not exposed. The intraperitoneal route of administration is generally considered a route that allows for rapid absorption of a chemical into the blood, and consequent rapid distribution throughout the animal. It would therefore be expected that the intraperitoneal route would have resulted in exposure of the bone marrow (a well-perfused tissue), albeit not in the same concentratrions as other tissues (as reported in the whole body autoradiography study). However, this will be the case for many other chemicals. Such findings should not be used to indicate that the bone marrow micronucleus assay has limited value in genotoxicity evaluations. 1000 PCE per animal were evaluated, however, this was from 5 males and 5 females, giving a total number of PCEs evaluated of 10,000. This is acceptable under OECD 474. A single dose level was used, however, this was a dose causing toxicity and is therefore an acceptable maximum tolerated dose, so giving a robust evaluation of the test material.

In response to the specific request for the applicant set by EFSA, i.e. EFSA Request No 14. Applicant is given the opportunity to provide further information/justification to support the non-genotoxic potential of the substance and notably on the reliability of the in vivo test. The applicant has argued that it is widely accepted that an i.p. dose will give bone marrow exposure, not surprising since along with other clinical observations the animals showed the ultimate systemic toxicity i.e. death, similar to observations reported in the oral study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.