2-isopropyl-N-hydroxyethyl 1,3-oxazolidine;methyl carbonato-N-ethyl-2-isopropyl-1,3-oxazolidine;reaction mass of: carbonato-bis-N-ethyl-2-isopropyl-1,3-oxazolidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male ICR mice. In addition, the test item was not mutagenic in the Ames test in the absence and presence of metabolic activation. Positive results were observed in a chromosome aberration assay with and without metabolic activation. The test item is nevertheless not considered to be gentoxic gentoxic notwithstanding the positive results obtained in the chromosome aberration test since negative results were obtained in the in vivo micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1998-03-09 to 1998-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

cited as: Directive 92/69/EEC method B14 of 1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 8, 40, 200, 1000, 5000 µg/plate
Concentration range in the main test (with and without metabolic activation): 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

- Solvent: Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: without S9: TA102 with Glutaraldyhyde; with S9: TA102 with 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

DURATION for plate incorporation (Experiment 1)
- Exposure duration: 72h

DURATION for pre-incubation method (Experiment 2)
- Pre-incubation period: incubated for 2 hour at 37 °C,
- Exposure duration: 72h

NUMBER OF REPLICATIONS: 3
Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn

Evaluation criteria:

The test item was considered to be mutagenic if :
1. the assay was valid
2. Dunnetts test gave a significant response, and the data set showed a significant dose-correlation
3. the positive responses described in 2) were reproducible.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

>5000µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

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

Key result

Species / strain:

S. typhimurium TA 98

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:

not examined

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:

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

Key result

Species / strain:

S. typhimurium TA 102

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed reversion rates were not different from the historical control range.
Other: Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Incozol LV did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested at concentrations up to 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

A study was conducted according to OECD TG 471 and Directive 92/69/EEC method B.14 to assess the mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol LV at 8-5000 µg/plate, plus negative (solvent) and positive controls. Following these range-finder treatments, no evidence of toxicity was observed. In order to assess the reproducibility of an increase in revertant numbers observed in the range-finder experiment, repeat treatments of strain TA100 in the absence of S-9 were performed alongside the remaining strains in Experiment 1. All Experiment 1 treatments were performed using the same dose range as employed in the range-finder experiment. Following these Experiment 1 treatments, once again no evidence of toxicity was observed. Experiment 2 retained 5000 µg/plate as the maximum test dose and employed a narrowed dose range, in order to more closely investigate those doses considered most likely to induce any mutation. Treatments in the presence of S-9 included a pre-incubation step, in order to increase the range of mutagenic chemicals that could be detected using this assay system. Additional strain TA102 treatments in the presence of S-9 were included using the plate-incorporation methodology, in order to more thoroughly investigate a small increase in revertant numbers seen in Experiment 1. Evidence of toxicity was observed following preincubation treatments of all strains performed at the higher test doses, in the presence of S-9. No toxic signs were observed following any treatments of the test strains performed in the absence of S-9, or following treatments of strain TA102 in the presence of S-9 using plate incorporation methodology. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No Incozol LV treatment of any of the test strains produced an increase in revertant numbers sufficient to be considered as clearly indicative of mutagenic activity. It was concluded that Incozol LV did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions employed for this study, which included treatments at concentrations up to 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-05-19 to 1998-12-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

cited as: Directive 92/69/EEC (Annex V (Cytogenetics) (1996)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

revised draft document, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Blood from three healthy, non-smoking female volunteers were used in this study. No donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals.
- Properly maintained: yess

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 600.3, 1225, 2500 µg/mL
Concentration range in the main test (without metabolic activation): 600.3, 1225, 2500 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Culture period before exposure: approximately 24h
- Exposure duration: 3 hours
- Fixation time : 20 hours

SPINDLE INHIBITOR: colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase.
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate cultures for each dose level, negative, vehicle and positive controls

NUMBER OF CELLS EVALUATED: at least 1000 cells counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

The test article is considered as positive in this assay if:
1) a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more concentrations, and
2) the proportion of cells with structural aberrations at such doses exceeds the normal range.

Increases in numbers of cells with gaps or increases in the proportions of cells with structural aberrations, not exceeding the normal range or occurring only at very high or very toxic concentrations, are concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Cells with exchange aberrations or cells with greater than one aberration are considered of particular biological significance.

Statistics:

Cochran-Armitage test for linear trend and Fisher's Exact Test to compare the percentage of cells with aberrations.

Key result

Species / strain:

mammalian cell line, other: Human lymphocytes

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: Human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no

Cultures treated for 3 hours (with 17 hours recovery) with Incozol LV in the absence and presence of S-9 exhibited frequencies of cells with structural aberrations which exceeded the historical negative control (normal) range in both replicate cultures at the highest concentration analysed (2500 mg/mL) . Statistical analysis using Fisher's exact test (10) was therefore performed and indicated that the aberrant cell frequencies seen at this dose were significantly increased above levels seen in concurrent solvent control cultures. Additionally, cells containing exchange-type aberrations were observed which are rarely seen in negative control cultures and are therefore considered to be of particular biological relevance. Cultures treated with the intermediate and low dose of Incozol LV (1225 and 600.3 mg/mL respectively) exhibited normal frequencies of aberrant cells.

Conclusions:

It is concluded that Incozol LV induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

Executive summary:

A study was conducted according to OECD TG 473 and Directive 92/69/EEC method B.10 to assess the induction of Chromosome Aberrations in Cultured Human Peripheral Blood Lymphocytes of Incozol LV. Therefore, the test article was dissolved in dimethyl sulphoxide (DMSO) and the highest dose level used, 2500 µg/mL, was in excess of the solubility limit in culture medium. In this study, treatment in the absence and presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest (3 + 17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of Incozol LV on mitotic index. Chromosome aberrations were analysed at three dose levels (see below). The highest concentration chosen for analysis, 2500 µg/mL, induced approximately 43 % and 39 % mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively. Appropriate negative (solvent) control cultures were included in the test system under each treatment condition. Cultures treated for 3 hours (with 17 hours recovery) with Incozol LV in the absence and presence of S-9 exhibited frequencies of cells with structural aberrations which exceeded the historical negative control (normal) range in both replicate cultures at the highest concentration analysed (2500 µg/mL). Statistical analysis using Fisher's exact test was therefore performed and indicated that the aberrant cell frequencies seen at this dose were significantly increased above levels seen in concurrent solvent control cultures. Additionally, cells containing exchange-type aberrations were observed which are rarely seen in negative control cultures and are therefore considered to be of particular biological relevance. Cultures treated with the intermediate and low dose of Incozol LV (1225 and 600.3 µg/mL respectively) exhibited normal frequencies of aberrant cells. In conclusion, Incozol LV induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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:

from 1998-07-31 to 1999-03-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted: 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 4 - 8 weeks old
- Weight at study initiation: 25 - 32 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: yes
- Housing: appropriate caging
- Diet:ad libitum ,Special Diets Services Ltd, RM1.[E].SQC.
- Water: ad libitum, tab water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes: 15 air changes per hour
- Photoperiod : light for 12 hours, from 6:00 to 18:00

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing preparations were made by suspending Incozol LV (with homogenisation) in corn oil to give the top concentrations specified below and dilutions were made using corn oil. The test article preparations in the main study were protected from light, maintained as an even suspension (by multiple inversion) and used within 2.5 hours of initial formulation.

Duration of treatment / exposure:

2 days

Frequency of treatment:

daily o two consecutive days

Post exposure period:

Micronucleus assay: 24 or 48 hours

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

8 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) was freshly dissolved in saline at 2 mg/mL to serve as the positive control.

Tissues and cell types examined:

Bone marrow erythrocytes (polychromatic erythrocytes, PCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Observed toxicity in Dose Range Finding assay
An initial range finding study was performed using groups of three male and three female mice. Animals were dosed once daily for two consecutive days with the test article. Observations were made over a two day period following the first administration. Clinical signs of toxicity and body weight over this period were recorded and a maximum acceptable dose determined based on these data. This was used as the highest dose level in the main study.

SAMPLING TIME: Bone marrow was sampled 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow was removed from the femurs of the animals in a dose group. Following centrifugation to pellet the tissue, the supematant was removed by aspiration and portions of the pellet were spread on slides, air dried and stained in May-Grünwald solution followed by Giemsa.

METHOD OF ANALYSIS:
Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue/purple enucleate cells, and normochromatic erythrocytes (NCE), seen as yellow/grey stained enucleate cells, were detennined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.

Evaluation criteria:

The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No toxicity was seen at the maximum level of 2000 mg/kg in male or female mice.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
There was no sign of ill effect to the animals treated.
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 and 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: No clinical signs of toxicity observed.
- Evidence of cytotoxicity in tissue analyzed: no

RESULTS OF DEFINITIVE STUDY
Groups of male mice treated with Incozol LV exhibited PCE/NCE ratios which were similar to vehicle controls. Group mean frequencies of micronucleated PCE were also similar to those seen in the vehicle control group and were not significantly different by 2 % analysis, except at 1000 mg/kg bw/day, where a small but significant increase was observed (p<0.05). Additionally, the statistical test for linear trend indicated a small, but significant dose-response relationship (p<0.05) ( Appendix 5). Insofar as the frequency of micronucleated PCE both at 1000 mg/kg bw/day and the other doses analysed fell within the historical negative control range , these effects were considered to be of no biological relevance.

Conclusions:

It is concluded that Incozol LV did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw/day.

Executive summary:

 A study was conducted according to OECD TG 474 and Directve 92/69/EEC method B.12 to assess the induction of micronuclei in the bone marrow of treated mice of Incozol LV. Incozol LV was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which Incozol LV, made up in corn oil, was administered to mice orally by gavage. The test article was administered once daily on two consecutive days to groups of three male and three female mice at 500, 1000 and 2000 mg/kg bw/day. Observations were made over a 2 day period following the first administration and signs of toxicity and body weight recorded. For the micronucleus test, Incozol LV was made up as described and administered at 500, 1000 and 2000 mg/kg bw/day. Since no difference in toxicity was observed between the two sexes, male mice in groups of eight, killed 24 hours after the second administration were treated in the main study. The highest dose used (2000 mg/kg bw/day) was considered an acceptable top dose for the rodent micronucleus assay. The negative (vehicle) control in the study was corn oil also administered orally by gavage once daily on two consecutive days. A groups of eight male mice treated with this was killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered orally as a single dose at 40 mg/kg to a group of eight male mice which was killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2x2 contingency x2 test).

Seven mice from each group were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control (normal) ranges. Male mice treated with Incozol LV at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and also fell within normal ranges. It is concluded that Incozol LV did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

A study was conducted according to OECD TG 471 and Directive 92/69/EEC method B.14 to assess the mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments. An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of Incozol LV at 8 - 5000 µg/plate, plus negative (solvent) and positive controls. Following these range-finder treatments, no evidence of toxicity was observed. In order to assess the reproducibility of an increase in revertant numbers observed in the range-finder experiment, repeat treatments of strain TA100 in the absence of S-9 were performed alongside the remaining strains in Experiment 1. All Experiment 1 treatments were performed using the same dose range as employed in the range-finder experiment. Following these Experiment 1 treatments, once again no evidence of toxicity was observed. Experiment 2 retained 5000 µg/plate as the maximum test dose and employed a narrowed dose range, in order to more closely investigate those doses considered most likely to induce any mutation. Treatments in the presence of S-9 included a pre-incubation step, in order to increase the range of mutagenic chemicals that could be detected using this assay system. Additional strain TA102 treatments in the presence of S-9 were included using the plate-incorporation methodology, in order to more thoroughly investigate a small increase in revertant numbers seen in Experiment 1. Evidence of toxicity was observed following preincubation treatments of all strains performed at the higher test doses, in the presence of S-9. No toxic signs were observed following any treatments of the test strains performed in the absence of S-9, or following treatments of strain TA102 in the presence of S-9 using plate incorporation methodology. Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. No Incozol LV treatment of any of the test strains produced an increase in revertant numbers sufficient to be considered as clearly indicative of mutagenic activity. It was concluded that Incozol LV did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) when tested under the conditions employed for this study, which included treatments at concentrations up to 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system (S-9).

Chromosome aberration test

A study was conducted according to OECD TG 473 and Directive 92/69/EEC method B.10 to assess the induction of Chromosome Aberrations in Cultured Human Peripheral Blood Lymphocytes of Incozol LV. Therefore, the test article was dissolved in dimethyl sulphoxide (DMSO) and the highest dose level used, 2500 µg/mL, was in excess of the solubility limit in culture medium. In this study, treatment in the absence and presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest (3 + 17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of Incozol LV on mitotic index. Chromosome aberrations were analysed at three dose levels (see below). The highest concentration chosen for analysis, 2500 µg/mL, induced approximately 43 % and 39 % mitotic inhibition (reduction in mitotic index) in the absence and presence of S-9 respectively. Appropriate negative (solvent) control cultures were included in the test system under each treatment condition. Cultures treated for 3 hours (with 17 hours recovery) with Incozol LV in the absence and presence of S-9 exhibited frequencies of cells with structural aberrations which exceeded the historical negative control (normal) range in both replicate cultures at the highest concentration analysed (2500 µg/mL). Statistical analysis using Fisher's exact test was therefore performed and indicated that the aberrant cell frequencies seen at this dose were significantly increased above levels seen in concurrent solvent control cultures. Additionally, cells containing exchange-type aberrations were observed which are rarely seen in negative control cultures and are therefore considered to be of particular biological relevance. Cultures treated with the intermediate and low dose of Incozol LV (1225 and 600.3 µg/mL respectively) exhibited normal frequencies of aberrant cells. In conclusion, Incozol LV induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.

Micronuclei in the Bone Marrow

A study was conducted according to OECD TG 474 and Directve 92/69/EEC method B.12. Incozol LV was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which Incozol LV, made up in corn oil, was administered to mice orally by gavage. The test article was administered once daily on two consecutive days to groups of three male and three female mice at 500, 1000 and 2000 mg/kg bw/day. Observations were made over a 2 day period following the first administration and signs of toxicity and body weight recorded. For the micronucleus test, Incozol LV was made up as described and administered at 500, 1000 and 2000 mg/kg bw/day. Since no difference in toxicity was observed between the two sexes, male mice in groups of eight, killed 24 hours after the second administration were treated in the main study. The highest dose used (2000 mg/kg bw/day) was considered an acceptable top dose for the rodent micronucleus assay. The negative (vehicle) control in the study was corn oil also administered orally by gavage once daily on two consecutive days. A groups of eight male mice treated with this was killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered orally as a single dose at 40 mg/kg bw to a group of eight male mice which was killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2x2 contingency x2 test).

Seven mice from each group were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control (normal) ranges. Male mice treated with Incozol LV at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and also fell within normal ranges. It is concluded that Incozol LV did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw/day.

Justification for classification or non-classification

Based on the results of the two in vitro studies and the in vivo study and according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) 2017/776, the test item is not classified for genetic toxicity.