Proxan-sodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1.06.2021 to 14.09.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study
A weak positive (equivocal) result was obtained even after repeating the assay
This result is similar to that seen for the main metabolite carbon disulphide

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22/2020/DPL

Type of assay:

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

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In the main test, positive control (PC; 3-Methylcholanthrene at 100 μg/mL) and a negative control (NC; solvent) were used simultaneously
5 concentrations of the test item (360 μg/mL, 167 μg/mL, 78 μg/mL, 36 μg/mL, 17 μg/mL.
As the result for treatment without metabolic activation system (S9-) was equivocal and was repeated with the following concentrations: 360 μg/mL, 227 μg/mL, 143 μg/mL, 90 μg/mL, 57 μg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results for treatment with presence of the metabolic activation system (S9+, run 2) were clearly negative. However, the results for the treatment in the absence of the metabolic activation system (S9-, run 1) were equivocal (neither clearly negative nor clearly positive). Therefore the experiment was repeated with modified conditions (S9-, run 3). In this repeated experiment the clearly positive result was only observed at the highest concentration, where the RS was below 10% (very high cytotoxicity). Therefore, this positive result should be taken with care.

Conclusions:

A weak positive (equivocal) result was obtained without S-9 even after repeating the assay
Negative in the presence of S-9
This result is similar to that seen for the main metabolite carbon disulphide

Executive summary:

A preliminary test (solubility test and cytotoxicity test) was performed prior to the main test.
In the main test, positive control (PC; 3-Methylcholanthrene at 100 µg/mL) and a negative control
(NC; solvent) were used simultaneously with 5 concentrations of the test item (360 µg/mL, 167
µg/mL, 78 µg/mL, 36 µg/mL, 17 µg/mL. However, the result for treatment without metabolic
activation system (S9-) was equivocal and was repeated with the following concentrations: 360
µg/mL, 227 µg/mL, 143 µg/mL, 90 µg/mL, 57 µg/mL.

Results of the studies:
The results for treatment with presence of the metabolic activation system (S9+, run 2) were clearly
negative. However, the results for the treatment in the absence of the metabolic activation system
(S9-, run 1) were equivocal (neither clearly negative nor clearly positive). Therefore the experiment
was repeated with modified conditions (S9-, run 3). In this repeated experiment the clearly positive
result was only observed at the highest concentration, where the RS was below 10% (very high
cytotoxicity). Therefore, this positive result should be taken with care.

Interpretation of the study results:
All acceptance criteria were met but the results remained equivocal even after repeating the
experiment. Therefore, the test should be considered likely to be positive in this study and further
testing with another system is recommended to clarify the genotoxicity properties of the test item.

Note that this is consistent with the main metabolite, carbon disulphides and the result was not unexpected. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

from 1.06.2021 to 14.09.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

A weak positive (equivocal) result was obtained even after repeating the assay This result is similar to that seen for the main metabolite carbon disulphide

Justification for type of information:

Read-across from sodium salt

The sodium salt xanthates is considered suitable as a key source of data for the others sodium salt xanthates
Considered valid for read-across for purposes of classification.
No further vertebrate testing can be justified.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22/2020/DPL

Type of assay:

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

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In the main test, positive control (PC; 3-Methylcholanthrene at 100 μg/mL) and a negative control (NC; solvent) were used simultaneously
5 concentrations of the test item (360 μg/mL, 167 μg/mL, 78 μg/mL, 36 μg/mL, 17 μg/mL.
As the result for treatment without metabolic activation system (S9-) was equivocal and was repeated with the following concentrations: 360 μg/mL, 227 μg/mL, 143 μg/mL, 90 μg/mL, 57 μg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results for treatment with presence of the metabolic activation system (S9+, run 2) were clearly negative. However, the results for the treatment in the absence of the metabolic activation system (S9-, run 1) were equivocal (neither clearly negative nor clearly positive). Therefore the experiment was repeated with modified conditions (S9-, run 3). In this repeated experiment the clearly positive result was only observed at the highest concentration, where the RS was below 10% (very high cytotoxicity). Therefore, this positive result should be taken with care.

Conclusions:

A weak positive (equivocal) result was obtained without S-9 even after repeating the assay
Negative in the presence of S-9
This result is similar to that seen for the main metabolite carbon disulphide

Executive summary:

A preliminary test (solubility test and cytotoxicity test) was performed prior to the main test.
In the main test, positive control (PC; 3-Methylcholanthrene at 100 µg/mL) and a negative control
(NC; solvent) were used simultaneously with 5 concentrations of the test item (360 µg/mL, 167
µg/mL, 78 µg/mL, 36 µg/mL, 17 µg/mL. However, the result for treatment without metabolic
activation system (S9-) was equivocal and was repeated with the following concentrations: 360
µg/mL, 227 µg/mL, 143 µg/mL, 90 µg/mL, 57 µg/mL.

Results of the studies:
The results for treatment with presence of the metabolic activation system (S9+, run 2) were clearly
negative. However, the results for the treatment in the absence of the metabolic activation system
(S9-, run 1) were equivocal (neither clearly negative nor clearly positive). Therefore the experiment
was repeated with modified conditions (S9-, run 3). In this repeated experiment the clearly positive
result was only observed at the highest concentration, where the RS was below 10% (very high
cytotoxicity). Therefore, this positive result should be taken with care.

Interpretation of the study results:
All acceptance criteria were met but the results remained equivocal even after repeating the
experiment. Therefore, the test should be considered likely to be positive in this study and further
testing with another system is recommended to clarify the genotoxicity properties of the test item.

Note that this is consistent with the main metabolite, carbon disulphides and the result was not unexpected. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31.05.2021-15.07.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Performed to guidelines

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

the study was carried out in accordance with microplate format of the Ames fluctuation assay, which differs from the methods described in details in the OECD 471 Guideline and EU Method B.13/14.

GLP compliance:

yes (incl. QA statement)

Remarks:

22/2020/DPL

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

94±1%

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The following components from Xenometrix were used to prepare the metabolic activity system:
◆ phenobarbital/β-naphtoflavone induced S9 fraction (cat. no. PRS-PB02, lot. no. F1903114). The Certificate of Analysis of Phenobarbital/β-naphtoflavone induced S9 – Postmitochondiral Supernatant was presented in Appendix no. 5.
◆ S9-Buffer Salts (cat. no. PCO-0800, lot. no. ND09648P), containing 0.20 M NaH2PO4, and 0.25 M MgCl*6H2O and 1 M KCl.
◆ 0.04 M S9-NADP (cat. no. PCO-0830, lot. no. X1904SNAA),
The metabolic activity system was supplemented with 0.20 M glucose-6-phosphatase solution (G-6-P; Sigma-Aldrich, cat. no. G7879, lot. no. SLBZ7814).
The defrosted reagents were stored on ice. To prepare 30 % mixture of the S9 fraction, the following volumes of the reagents were mixed (for 5 strains): 3.106 mL S9 – buffer – salts, 0.136 mL G-6-P, 0.540 mL S9 – NADP and 1.620 mL phenobarbital/β-naphtoflavone induced S9 fraction. This mixture was prepared immediately before use and added to the bacteria in the exposure media. The final concentration of S9 in culture was 4.5%.
The S9 fraction can be toxic for Escherichia coli strains. So, the number of positive wells may be lower than in other strains

Test concentrations with justification for top dose:

5.000 mg/mL, 1.581 mg/mL, 0.500 mg/mL, 0.158 mg/mL, 0.050 mg/mL, 0.016 mg/mL.
There were three replicates of each concentration.

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

other: N4-aminocitidine, 2-aminoanthracene

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item was tested in Bacterial Reverse Mutation Test to indicate potential mutagenic risk. The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and for point mutation-inducing activity. There was demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests, but the correlation is not absolute [5]. The test was performed with Ames MPFTM Penta I Microplate Format Mutagenicity Assay kit (Xenometrix).
Statistically significant difference (p<0.05) was noticed in only one result. This result was also outside the historical data of Negative Control. However, the average number of positive wells were not bigger than twice the baseline value and no concentration-response correlation was observed (r≥0.75). Therefore, genotoxic properties of the test item were not detected.
Hence, it may be concluded that the test item under the test conditions is not a mutagenic in the tested species.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

31.05.2021-15.07.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across from sodium salt
The sodium salt xanthates is considered suitable as a key source of data for the others sodium salt xanthates
Considered valid for read-across for purposes of classification.
No further vertebrate testing can be justified.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

the study was carried out in accordance with microplate format of the Ames fluctuation assay, which differs from the methods described in details in the OECD 471 Guideline and EU Method B.13/14.

GLP compliance:

yes (incl. QA statement)

Remarks:

22/2020/DPL

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

94±1%

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The following components from Xenometrix were used to prepare the metabolic activity system:
◆ phenobarbital/β-naphtoflavone induced S9 fraction (cat. no. PRS-PB02, lot. no. F1903114). The Certificate of Analysis of Phenobarbital/β-naphtoflavone induced S9 – Postmitochondiral Supernatant was presented in Appendix no. 5.
◆ S9-Buffer Salts (cat. no. PCO-0800, lot. no. ND09648P), containing 0.20 M NaH2PO4, and 0.25 M MgCl*6H2O and 1 M KCl.
◆ 0.04 M S9-NADP (cat. no. PCO-0830, lot. no. X1904SNAA),
The metabolic activity system was supplemented with 0.20 M glucose-6-phosphatase solution (G-6-P; Sigma-Aldrich, cat. no. G7879, lot. no. SLBZ7814).
The defrosted reagents were stored on ice. To prepare 30 % mixture of the S9 fraction, the following volumes of the reagents were mixed (for 5 strains): 3.106 mL S9 – buffer – salts, 0.136 mL G-6-P, 0.540 mL S9 – NADP and 1.620 mL phenobarbital/β-naphtoflavone induced S9 fraction. This mixture was prepared immediately before use and added to the bacteria in the exposure media. The final concentration of S9 in culture was 4.5%.
The S9 fraction can be toxic for Escherichia coli strains. So, the number of positive wells may be lower than in other strains

Test concentrations with justification for top dose:

5.000 mg/mL, 1.581 mg/mL, 0.500 mg/mL, 0.158 mg/mL, 0.050 mg/mL, 0.016 mg/mL.
There were three replicates of each concentration.

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

other: N4-aminocitidine, 2-aminoanthracene

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item was tested in Bacterial Reverse Mutation Test to indicate potential mutagenic risk. The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and for point mutation-inducing activity. There was demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests, but the correlation is not absolute [5]. The test was performed with Ames MPFTM Penta I Microplate Format Mutagenicity Assay kit (Xenometrix).
Statistically significant difference (p<0.05) was noticed in only one result. This result was also outside the historical data of Negative Control. However, the average number of positive wells were not bigger than twice the baseline value and no concentration-response correlation was observed (r≥0.75). Therefore, genotoxic properties of the test item were not detected.
Hence, it may be concluded that the test item under the test conditions is not a mutagenic in the tested species.

Read-across from sodium salt

The sodium salt xanthates is considered suitable as a key source of data for the others sodium salt xanthates
Considered valid for read-across for purposes of classification.
No further vertebrate testing can be justified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Publication with limited detail
However, the results are widely cited by international reviews on safety of xanthates.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosomal Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

unspecified

Species:

rat

Sex:

not specified

Route of administration:

other: inhalation and oral

Duration of treatment / exposure:

For 1, 15, 45 or 75 days

Sex:

not specified

Genotoxicity:

ambiguous

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: Only headline results provided in this study

10mg/kg dose or 13.64 mg/m³concentration increase the number of chromosomal disorders in bone marrow cells.

1.74 mg/m³concentration had no mutagenic effect.

Conclusions:

Interpretation of results: ambiguous

Executive summary:

This was a non-standard study and although only limited details provided in the data summary, it shows that over prolonged periods of dosing, there was a dose-related effect.

It must be noted that oral dosing would have resulted in expoure to the hydrolysis products Carbon Disulphide and Alcohol.  Carbon disulphides has been shown to lead to chromosomal effects in exposed workers.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1000 polychromatic erythrocytes were evaluated per animal, rather than 2000

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 8 to 11 weeks old
- Assigned to test groups randomly: yes, under following basis: by a computer generated randomization program
- Housing: 5 mice/cage
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow #5002 ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for seven days before being placed on study

ENVIRONMENTAL CONDITIONS
- Temperature: Reported in the study to be 72 ± 6 ºF (approximately 22.2 ºC)
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9% sodium chloride

Details on exposure:

Thirty randomly assigned mice/group (15 males/15 females) were treated with isopropanol dissolved in 0.9% sodium chloride at dose levels of 0 (vehicle control), 350, 1173 and 3500 mg isopropanol/kg bw by intraperitoneal injection. Mice were observed for toxic symptoms and/or mortalities immediately after dosing and twice daily for the duration of the assay

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Single exposure

Post exposure period:

None

Remarks:

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

Remarks:

Doses / Concentrations:
1173 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 80 mg/kg bw

Tissues and cell types examined:

Bone marrow was extracted from 10 mice/group (5 males/5 females) at 24, 48, and 72 hours after dosing. An additional group of 10 mice (5 males/5 females) was treated with 3500 mg/kg bw and held to ensure that 10 mice were available for bone marrow extraction at each interval. These mice were scheduled for use only if mortalities occurred in the primary dose group. As needed, mice were randomly selected from this secondary group for bone marrow extraction and unused mice were euthanized at the completion of the trial. Bone marrow for the positive control group was harvested at 24 hours. Terminal body weights were collected on all animals prior to euthanization for bone marrow harvest.

Details of tissue and slide preparation:

At the appropriate harvest time, the animals were euthanized with CO2 and the adhering soft tissue and epiphyses of both tibias were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 mL fetal calf serum. Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol and stained in May-Gruenwald solution followed by Giemsa. After being air-dried, the slides were coverslipped using Depex mounting medium. The coded slides were then scored blind for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Statistics:

ANOVA followed by Tukey's

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mutagenic activity of Propan-2-ol (Isopropyl alcohol) detected.

All animals in the 3500 mg/kg bw dose group became prostrate after dosing. Within 22 hours after dosing, 35 of the 40 animals dosed at 3500 mg/kg had expired. Due to this toxicity, the 3500 mg/kg bw dose level was eliminated from the study. Immediately after dosing, the animals receiving 2500 mg/kg became prostrate. Approximately four hours after dosing, the animals were languid with squinted eyes. The following morning, approximately 23 hours after dosing, one male (#8371) was languid with squinted eyes. This animal also exhibited dyspnea approximately 30 hours after dosing. All other animals appeared normal; however, within 46 hours of dosing three animals (male #8371; female #’s 8319 and 8354) were found dead. Another female (#8369) expired approximately 53 hours after dosing and several other animals were languid. On the third morning, approximately 70 hours after dosing, two males (#’s 8326 and 8405) were found dead. All remaining test article dosed animals had rough haircoats and this condition remained at the 72 hour harvest time. A gross necropsy was performed on all animals which expired during the observation period. Male #8371 had a moderate amount of clear yellow fluid in the trachea and thoracic cavity and an irregular black stomach mucosa. Female #8369 also had a moderate amount of a clear orange fluid in the thoracic cavity. The other necropsied animals had moderate to significant distension of the stomachs or colons, two with an abnormally thin fluid content. No other abnormalities were noted in the animals examined. The terminal body weight range of the animals harvested in this trial of the micronucleus assay was 28.1 - 37.4 grams for males and 19.0 - 26.9 grams for females. Terminal body weight gains were significantly lower at the 48 and 72 hour sacrifice intervals in mice treated with 2,500 mg/kg bw compared to corresponding vehicle control body weight gains indicating that there was a test article related reduction in body weights. The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 3.24% ± 0.84% and 1.22% ± 0.19% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 1). The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.56% ± 0.42% and 1.66% ± 0.39% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 2).

Conclusions:

Interpretation of results : negative
No mutagenic activity of Propan-2-ol (Isopropyl alcohol) detected.
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1000 polychromatic erythrocytes were evaluated per animal, rather than 2000

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 8 to 11 weeks old
- Assigned to test groups randomly: yes, under following basis: by a computer generated randomization program
- Housing: 5 mice/cage
- Diet (e.g. ad libitum): Purina Certified Laboratory Chow #5002 ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Quarantined for seven days before being placed on study

ENVIRONMENTAL CONDITIONS
- Temperature: Reported in the study to be 72 ± 6 ºF (approximately 22.2 ºC)
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9% sodium chloride

Details on exposure:

Thirty randomly assigned mice/group (15 males/15 females) were treated with isopropanol dissolved in 0.9% sodium chloride at dose levels of 0 (vehicle control), 350, 1173 and 3500 mg isopropanol/kg bw by intraperitoneal injection. Mice were observed for toxic symptoms and/or mortalities immediately after dosing and twice daily for the duration of the assay

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Single exposure

Post exposure period:

None

Remarks:

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

Remarks:

Doses / Concentrations:
1173 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
3500 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 80 mg/kg bw

Tissues and cell types examined:

Bone marrow was extracted from 10 mice/group (5 males/5 females) at 24, 48, and 72 hours after dosing. An additional group of 10 mice (5 males/5 females) was treated with 3500 mg/kg bw and held to ensure that 10 mice were available for bone marrow extraction at each interval. These mice were scheduled for use only if mortalities occurred in the primary dose group. As needed, mice were randomly selected from this secondary group for bone marrow extraction and unused mice were euthanized at the completion of the trial. Bone marrow for the positive control group was harvested at 24 hours. Terminal body weights were collected on all animals prior to euthanization for bone marrow harvest.

Details of tissue and slide preparation:

At the appropriate harvest time, the animals were euthanized with CO2 and the adhering soft tissue and epiphyses of both tibias were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 mL fetal calf serum. Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol and stained in May-Gruenwald solution followed by Giemsa. After being air-dried, the slides were coverslipped using Depex mounting medium. The coded slides were then scored blind for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).

Statistics:

ANOVA followed by Tukey's

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No mutagenic activity of Propan-2-ol (Isopropyl alcohol) detected.

All animals in the 3500 mg/kg bw dose group became prostrate after dosing. Within 22 hours after dosing, 35 of the 40 animals dosed at 3500 mg/kg had expired. Due to this toxicity, the 3500 mg/kg bw dose level was eliminated from the study. Immediately after dosing, the animals receiving 2500 mg/kg became prostrate. Approximately four hours after dosing, the animals were languid with squinted eyes. The following morning, approximately 23 hours after dosing, one male (#8371) was languid with squinted eyes. This animal also exhibited dyspnea approximately 30 hours after dosing. All other animals appeared normal; however, within 46 hours of dosing three animals (male #8371; female #’s 8319 and 8354) were found dead. Another female (#8369) expired approximately 53 hours after dosing and several other animals were languid. On the third morning, approximately 70 hours after dosing, two males (#’s 8326 and 8405) were found dead. All remaining test article dosed animals had rough haircoats and this condition remained at the 72 hour harvest time. A gross necropsy was performed on all animals which expired during the observation period. Male #8371 had a moderate amount of clear yellow fluid in the trachea and thoracic cavity and an irregular black stomach mucosa. Female #8369 also had a moderate amount of a clear orange fluid in the thoracic cavity. The other necropsied animals had moderate to significant distension of the stomachs or colons, two with an abnormally thin fluid content. No other abnormalities were noted in the animals examined. The terminal body weight range of the animals harvested in this trial of the micronucleus assay was 28.1 - 37.4 grams for males and 19.0 - 26.9 grams for females. Terminal body weight gains were significantly lower at the 48 and 72 hour sacrifice intervals in mice treated with 2,500 mg/kg bw compared to corresponding vehicle control body weight gains indicating that there was a test article related reduction in body weights. The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 3.24% ± 0.84% and 1.22% ± 0.19% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 1). The test article, isopropanol, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.56% ± 0.42% and 1.66% ± 0.39% for the males and females, respectively. The vehicle and positive control rates of micronucleated PCEs were within the historical control values in this laboratory (TRIAL 2).

Conclusions:

Interpretation of results : negative
No mutagenic activity of Propan-2-ol (Isopropyl alcohol) detected.
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Isopropyl Alcohol has been demonstrated to form during hydrolysis in gastric fluid.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read-across from key metabolite, carbon disulphide
Carbon disulphide has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Further animal testing on the xanthate cannot be justified

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK)
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 16.1-26 g
- Assigned to test groups randomly: yes
- Housing: high density polypropylene cages with stainless steel taps
- Diet: ad libitum
- Water: supplied via a polythene bottle and sipper tube
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12

Route of administration:

inhalation: vapour

Vehicle:

no vehicle used

Details on exposure:

TYPE OF INHALATION EXPOSURE: snout only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
-Animals were exposed to the test material by snout-only inhalation. Prior to exposure of animals, the test material atmospheres were generated for each exposure chamber and samples analysed. Each mouse was placed in an individual polymethyl methacrylate restraining tube so that only the snout protruded. Each restraining tube was marked with the animal and group numbers. The restraining tubes were attached to the appropriate chamber so that the snout of each mouse projected into the lumen of the chamber. When the pre-exposure observations were complete, the syringe pump was switched on and the exposure timed for six hours following a 4.5 minute equilibration period, the theoretical time required for the concentration of vapour to reach 90% of its final value under the conditions of exposure employed (Silver and Arsenal, 1946). After six hours, the test atmosphere
supply was switched off and the mice removed from the restraining tubes for examination.

Duration of treatment / exposure:

6 h

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
0, 467, 1558, 4675 mg/m3 (150, 500, 1500 ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

10 (5 for positive control)

Control animals:

yes

Positive control(s):

chlorambucil
- Route of administration: oral
- Doses / concentrations: 30 mg/kg

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on the preliminary toxicity testing

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): samples were taken 24 and 48 h after treatment

DETAILS OF SLIDE PREPARATION: Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually in 5% Giemsa stain (in Sorensen's buffer: pH 6.8), washed in buffer, air-dried, cleared for five minutes in xylene and made permanent using DPX mountant.


METHOD OF ANALYSIS: The slides were examined under the light microscope. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature. Each erythrocyte scored was also examined for the presence or absence of micronuclei. Thereafter, the frequencies of micronucleated cells per 1000 erythrocytes were calculated. The ratio of polychromatic to mature cells was also determined (indicating the rythm of cell division). The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Evaluation criteria:

Positive for clastogenicity was a statistically and biologically significant increase in micronucleated polychromatic cells, compared to vehicle control, in at least one treatment group; particularly if supported by evidence of a dose-related response.

Statistics:

Mann-Whitney U procedure (Mann and Whitney, 1942), two-tailed test, one-tailed test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 45, 150, 450, 1500, 2000 ppm
- Clinical signs of toxicity in test animals: unconscious and death at 2000 ppm, unconscious, slow and laboured respiration, all extremities red coloured. No adverse reactions to treatment were observed in animals exposed to 450, 150, 45 ppm test substance.
- Evidence of cytotoxicity in tissue analyzed: no bone marrow toxicity observed
- Harvest times: 48 hours

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): see table
- Ratio of PCE/NCE (for Micronucleus assay): see table

The micronucleus test - group means and standard deviations (sd) by sex

test group	dose (ppm)	sampling time (h)	Sex	total polychromatic cells scored	total micronucleated polychromatic cells	mean micronucleated polychromatic cells per 1000 and sd	total mature cells scored	total micronucleated mature cells per 1000 and sd	polychromatic cells/mature cells
Air	0	24	M	5183	7	1.3±0.9	5652	4	0.7±0.4
			F	5709	3	0.5±0.5	5607	3	0.6±0.9
Carbon disulphide	150		M	5208	2	0.4±0.5	5871	4	0.6±0.9
			F	6030	6	1.0±0.7	5469	5	0.9±0.8
	500		M	6194	14	2.0±1.6	5505	2	0.4±0.5
			F	5788	10	1.6±1.1	5148	3	0.6±0.5
	1500		M	6713	8	1.0±1.2	5089	2	0.4±0.5
			F	7640	9	1.2±0.9	5129	2	0.4±0.5
Chlorambucil	30 mg/kg		M	5473	322	59.0±27.6	5176	3	0.6±0.9
			F	5807	403	69.3±27.5	5201	7	1.3±0.8
Air	0	48	M	5090	4	0.8±0.8	6101	7	1.1±0.8
			F	5588	4	0.7±0.8	4561	1	0.2±0.4
Carbon disulphide	150		M	5353	2	0.4±0.5	5699	4	0.7±0.6
			F	5976	9	1.5±1.1	5079	0	0.0±0.0
	500		M	6153	7	1.1±1.2	5300	3	0.6±0.9
			F	6518	6	0.9±0.6	5105	2	0.4±0.5
	1500		M	7324	8	1.0±0.9	5072	0	0.0±0.0
			F	6558	15	2.1±1.4	5022	0	0.0±0.0

 

Conclusions:

Interpretation of results : negative
No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected after exposure of the animals to CS2 via inhalation.
Read-across from key metabolite, carbon disulphide
Carbon disulphide has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Further animal testing on the xanthate cannot be justified

Executive summary:

The effect of carbon disulphide on chromosome structure in the bone marrow erythrocytes of mice was examined. The animals (males and females) were exposed via inhalation snout-only, for 6 h to the following concentrations: 0, 467, 1558, 4675 mg/m3 (0, 150, 500, 1500 ppm). The exposure concentrations were based on a preliminary toxicity test. Chlorambucil (30 mg/kg bw) was used as a positive control, adminstered via the oral route. Animals were sacrifised and examined 24 and 48 h after exposure. No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected, uder the present test condition, after exposure of the animals to CS2 via inhalation. Mice exposed at 1500 ppm, however, showed a small increase in the ratio of polychromatic/mature cells, which may indicate disturbance of erythropoiesis. Carbon disulphide was tested for induction of micronuclei in the bone marrow ertythrocytes of mice according to the OECD Guidelines 474 (1983).

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read-across from key metabolite, carbon disulphide
Carbon disulphide has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Further animal testing on the xanthate cannot be justified

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK)
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 16.1-26 g
- Assigned to test groups randomly: yes
- Housing: high density polypropylene cages with stainless steel taps
- Diet: ad libitum
- Water: supplied via a polythene bottle and sipper tube
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55 ±15 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light):12

Route of administration:

inhalation: vapour

Vehicle:

no vehicle used

Details on exposure:

TYPE OF INHALATION EXPOSURE: snout only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
-Animals were exposed to the test material by snout-only inhalation. Prior to exposure of animals, the test material atmospheres were generated for each exposure chamber and samples analysed. Each mouse was placed in an individual polymethyl methacrylate restraining tube so that only the snout protruded. Each restraining tube was marked with the animal and group numbers. The restraining tubes were attached to the appropriate chamber so that the snout of each mouse projected into the lumen of the chamber. When the pre-exposure observations were complete, the syringe pump was switched on and the exposure timed for six hours following a 4.5 minute equilibration period, the theoretical time required for the concentration of vapour to reach 90% of its final value under the conditions of exposure employed (Silver and Arsenal, 1946). After six hours, the test atmosphere
supply was switched off and the mice removed from the restraining tubes for examination.

Duration of treatment / exposure:

6 h

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
0, 467, 1558, 4675 mg/m3 (150, 500, 1500 ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

10 (5 for positive control)

Control animals:

yes

Positive control(s):

chlorambucil
- Route of administration: oral
- Doses / concentrations: 30 mg/kg

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on the preliminary toxicity testing

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): samples were taken 24 and 48 h after treatment

DETAILS OF SLIDE PREPARATION: Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually in 5% Giemsa stain (in Sorensen's buffer: pH 6.8), washed in buffer, air-dried, cleared for five minutes in xylene and made permanent using DPX mountant.


METHOD OF ANALYSIS: The slides were examined under the light microscope. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature. Each erythrocyte scored was also examined for the presence or absence of micronuclei. Thereafter, the frequencies of micronucleated cells per 1000 erythrocytes were calculated. The ratio of polychromatic to mature cells was also determined (indicating the rythm of cell division). The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Evaluation criteria:

Positive for clastogenicity was a statistically and biologically significant increase in micronucleated polychromatic cells, compared to vehicle control, in at least one treatment group; particularly if supported by evidence of a dose-related response.

Statistics:

Mann-Whitney U procedure (Mann and Whitney, 1942), two-tailed test, one-tailed test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 45, 150, 450, 1500, 2000 ppm
- Clinical signs of toxicity in test animals: unconscious and death at 2000 ppm, unconscious, slow and laboured respiration, all extremities red coloured. No adverse reactions to treatment were observed in animals exposed to 450, 150, 45 ppm test substance.
- Evidence of cytotoxicity in tissue analyzed: no bone marrow toxicity observed
- Harvest times: 48 hours

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): see table
- Ratio of PCE/NCE (for Micronucleus assay): see table

The micronucleus test - group means and standard deviations (sd) by sex

test group	dose (ppm)	sampling time (h)	Sex	total polychromatic cells scored	total micronucleated polychromatic cells	mean micronucleated polychromatic cells per 1000 and sd	total mature cells scored	total micronucleated mature cells per 1000 and sd	polychromatic cells/mature cells
Air	0	24	M	5183	7	1.3±0.9	5652	4	0.7±0.4
			F	5709	3	0.5±0.5	5607	3	0.6±0.9
Carbon disulphide	150		M	5208	2	0.4±0.5	5871	4	0.6±0.9
			F	6030	6	1.0±0.7	5469	5	0.9±0.8
	500		M	6194	14	2.0±1.6	5505	2	0.4±0.5
			F	5788	10	1.6±1.1	5148	3	0.6±0.5
	1500		M	6713	8	1.0±1.2	5089	2	0.4±0.5
			F	7640	9	1.2±0.9	5129	2	0.4±0.5
Chlorambucil	30 mg/kg		M	5473	322	59.0±27.6	5176	3	0.6±0.9
			F	5807	403	69.3±27.5	5201	7	1.3±0.8
Air	0	48	M	5090	4	0.8±0.8	6101	7	1.1±0.8
			F	5588	4	0.7±0.8	4561	1	0.2±0.4
Carbon disulphide	150		M	5353	2	0.4±0.5	5699	4	0.7±0.6
			F	5976	9	1.5±1.1	5079	0	0.0±0.0
	500		M	6153	7	1.1±1.2	5300	3	0.6±0.9
			F	6518	6	0.9±0.6	5105	2	0.4±0.5
	1500		M	7324	8	1.0±0.9	5072	0	0.0±0.0
			F	6558	15	2.1±1.4	5022	0	0.0±0.0

 

Conclusions:

Interpretation of results : negative
No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected after exposure of the animals to CS2 via inhalation.
Read-across from key metabolite, carbon disulphide
Carbon disulphide has been demonstrated to form during hydrolysis in gastric fluid.
Any oral toxicity on the xanthate needs to consider oral effects of carbon disulphide and the corresponding alcohol
Further animal testing on the xanthate cannot be justified

Executive summary:

The effect of carbon disulphide on chromosome structure in the bone marrow erythrocytes of mice was examined. The animals (males and females) were exposed via inhalation snout-only, for 6 h to the following concentrations: 0, 467, 1558, 4675 mg/m3 (0, 150, 500, 1500 ppm). The exposure concentrations were based on a preliminary toxicity test. Chlorambucil (30 mg/kg bw) was used as a positive control, adminstered via the oral route. Animals were sacrifised and examined 24 and 48 h after exposure. No evidence of induced chromosomal or other damage leading to the formation of micronuclei in erythrocytes of the bone marrow was detected, uder the present test condition, after exposure of the animals to CS2 via inhalation. Mice exposed at 1500 ppm, however, showed a small increase in the ratio of polychromatic/mature cells, which may indicate disturbance of erythropoiesis. Carbon disulphide was tested for induction of micronuclei in the bone marrow ertythrocytes of mice according to the OECD Guidelines 474 (1983).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No studies were available for assessing the genotoxicity of Sodium isobutyl xanthate or other xanthates.

The results should be considered together with the results of test with the 3 assays from FDA genetic toxicity set, designed from the FDA archives and openly published results of genetic toxicity tests date for:

Bacterial gene mutation in vitro

• The Microbial composite (Sal_Ecoli_Bac) (A7A,3644 records)
• TheSalmonella typhimurium5 strains (A7B, 3535 records)
• The E.coli composite (A7C, 527 records)
• The E.coli WP strains (A7D, 280 records)

Mammalian gene mutationin vitro

• The Mammalianin vitroCHO V79 hgprt loci (A7O, 617 records)
• The Mammalianin vitroML5178y tk loci Ct (A7N, 806 records)
• The Mammalianin vitroML5178y tk loci A (AN7, 565 records)
• The Mammalianin vitroML5178y tk loci B (AN8, 563 records)

The Chromosomal aberrationsin vitro

• The Chromosomal Absin vitrocomposite (A7U, 1385 records)
• The Chromosomal Absin vitroCHO cells (A7V, 686 records)
• The Chromosomal Absin vitrocells (A7W, 744 records)
• The Chromosomal Absin vitroHuLym (A7X, 187 records)
• The Chromosome Absin vitroUndefined cells (A8H, 280 records)

 

The results of tests with the FDA GeneTOX sets are summarized in the following tables.

 

Important notice:The RCA method expert call is a computer-generated output that is considered, along with other available information, in formulating the final conclusion.The Final Conclusion is a conclusion made by the reviewer, taking into account all the available evidence, including thein-silicoand available experimental results.

Table 1. Ames test

Compound	A7A	A7B	A7C	A7D	RCA Method Expert Call (Overall)	Review expert
O-ethyl dithiocarbonic acid	-	-	-	-	-*	-

 

Table 2. Mammalian gene mutationin vitro

Compound	A7O	A7N	AN7	AN8	RCA Method Expert Call (Overall)	Review expert
O-ethyl dithiocarbonic acid	-	-	-	-	-*	-

 

Table 3. The Chromosomal aberrationsin vitro

Compound	A7U	A7V	A7W	A7X	A8H	RCA Method Expert Call (Overall)	Review expert
O-ethyl dithiocarbonic acid	-	-	-	-	-	-*	-

 

 

Table4.  Summary of results and overall conclusions for the genotoxicity tests

Compound	Ames		MAin vitro		CAin vitro		FINAL CONCLUSION
	RCAMethod Expert Call	Review Expert	RCAMethod Expert Call	Review Expert	RCAMethod Expert Call	Review Expert
O-ethyl dithiocarbonic acid	-*	-	-*	-	-*	-	-

AMES= bacterial mutation assay; MA = mammalian; CA = chromosomal aberration;

+ positive; (+) potentially positive; - negative; (-) potentially negative. ? –inconclusive * possible structural coverage problems

 

The RCA paradigm, implemented in the RCA decision support system, requires the following criteria for a chemical to be designated as POSITIVE:

1.     To be active in more than one test assay within a test battery

2.     To be active in a test assay, there must be two or more structurally similar fragments across the modules of the test assay.

Tables 1 - 4 reveal these conditions were not met by test compound in FDA Genetic Toxicity Set. The unknown structural features, which were detected in all test batteries, are typical for xantate moieties.

The xantates were not reported a genetoxic in publicly available sources and no component of this product present at levels greater than or equal to 0.1% is identified as probable, possible or confirmed human carcinogen by. The tested substance was reported in NICNAS Priority existing chemical assessment report (1995, Vol.6, 66p) and it was not listed there as genetic toxicant for humans. The Final conclusion is that the tested chemical is considered to be nonthreatening to humans from this evaluation.

 

The tested molecule does not contain any confirmed alerts and is assumed to be inactive.

The probability that the prediction is accurate is 80%.

 

Other (Q)SAR predictions of:

Lazar Toxicity Predictions: Mutagenicity-Salmonella typhimurium (CPDB) and

 

Tox Predictfor:(http://apps.ideaconsult.net:8080/ToxPredict)

The above QSAR software models indicate that sodium isobutyl xanthate (CAS# 140-90-9) and other xanthates are inactive as a mutagenic substance and does not contain alert structures according to Benigni/Bossa rules.

Justification for selection of genetic toxicity endpoint
The Final conclusion is that the tested chemical is considered to be nonthreatening to humans from this evaluation.

Endpoint Conclusion: No adverse effect observed (negative)