Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames' assay, OECD TG 471): negative with and without metabolic activation in the 5 strains tested

Chromosomal aberration in mammalian cells (OECD TG 473): negative in human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD TG 476): negative in mouse lymphoma cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000/01/30 to 2001/03/09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OCDE guideline with no deviation, GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Mutation in Histidine biosynthesis for Salmonella typhimurium: his D 3052/strain TA98 (frameshift), his G 46/strains TA100 and TA1535 (base-pair substitution) and his C 3076/strain TA1537 (frameshift).
Mutation in Tryptophan biosynthesis for Escherichia coli : WP2uvrA- (deletion in an excision repair gene).

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

50 - 150 - 500 - 1500 - 5000 µg/plate

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for TA98 strain without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA100, TA1535 and WPEuvrA strains without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA1537 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 aminoanthracene

Remarks:

for TA100, TA1535, TA1537 and WP2uvrA with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

for TA98 strain with S9

Details on test system and experimental conditions:

(1) Preliminary evaluation of cytotoxicity of the substance
The cytotoxicity was performed of the Salmonella Typhimurium strain TA 100 and in the Escherichia Coli WP2uvrA- with and without metabolic activation (S9 mix). The substance was tested at 10 concentrations : 0.15 ; 0.5 ; 1.5 ; 5 ; 15; 50; 150 ; 500 ; 1500 and 5000 µg/dish. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was
plated onto a sterile plates of Vogel-Bonner Minimal agar. The plates were incubated at 37°C for 48 hours.
(2) Main study
4 strains of Salmonella Typhimurium were tested: TA98, TA100, TA1535 and TA1537. 1 strain of Escherichia Coli was tested : WP2uvrA-
As the concentration of 5000 µg/plate was not toxic in the strain TA 100 and UWP2uvrA- with and without metabolic activation during the
preliminary study, the following concentrations were tested during the main study: 50 ; 150 ; 500; 1500 and 5000 µg/dish.
Each concentration was tested 3 times under a constant volume of 0.1 ml with and without metabolic activation (S9 fraction obtained from rat livers pre-treated with ohenobarbitone/b-naphthoflavone at 80/100 mg/kg/day for 3 consecutive days) and used at on each strain. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was plated onto the surface of Vogel-Bonner Minimal agar plate and incubated at 37°C for 48 hours.
Acetone and positive controls, 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA-98; N-ethyl-N'-nitro-nitroguanidine at 3 µg/plate for
TA-100, at 5 µg/plate for TA-1535 and at 2 µg/plate for WP2uvrA-; 9-aminoacridine at 80 µg/plate for TA-1537. 2-aminoanthracene was used as
positive reference, in presence of S9 mix, at 1 µg/plate for TA100 ; at 2 µg/plate for TA1535 and TA1537 and at 10 µg/plate for WP2uvrA-.
Benzo(a)pyrene was used, in presence of S9 mix, at 5 µg/plate for TA98.
All the results were confirmed in a second study independent from the first.

Evaluation criteria:

(1) Preliminary cytotoxicity
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test material precipitation. The signs of
toxicity were noted.
(2) Reverse mutation
At the end of the incubation period, the plates were assessed for numbers of revertant colonies using a Domino colony counter. Manual counts were performed at and above 5000 µg/plate because of excessive test material precipitation.


The test material may considered to be positive in the test system if it should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments
then this is taken as evidence of a positive response.

Statistics:

Dunnett's method of linear regression (p<0.05)

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Under experimental conditions employed, no value obtained in the presence of the test article was greater than or equal to twice the value obtained
in the presence of the vehicle with and without metabolic activation on the bacterial strains used.
All the results obtained in presence of positive controls were significant with and without metabolic activation in the used bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test article LCE00051, is not mutagenic as it induces no significant increase in the number of revertants with and without metabolic
activation of Salmonella Typhimurium TA98, TA100, TA1535 and TA1537 and in Escherichia Coli WP2uvrA-.

Executive summary:

The substance LCE00051 was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation.

The 5 concentrations (50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first.

In each study was included a negative control (vehicle = acetone) and a positive control (specific standard mutagen).

Under the experimental conditions employed, the substance LCE00051 did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008/05/29 to 2008/06/28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OCDE with no deviation, GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

numerical and structural aberrations for chromosome or chromatid.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male donor.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

(1) solubility assay
Different solvents were tested: water, DMSO and ethanol with or without heating at 40°C. It was demonstrated that 1 µg/ml of test substance in
ethanol was the least obtainable soluble solution, considering that the suspension will deliver a reliable amount of the test substance into the test
system. Higher concentrations got saturated on cooling (37°C) and couldn't be testing on cultures.
(2) Cytotoxicity
According to the solubility and precipitation assay, 5 concentrations were tested for the cytotoxic assay: 0.41 - 0.512 - 0.64 - 0.8 and 1 µg/ml.
(3) Main study
According to the cytotoxicity assay, 5 concentrations were tested: 0.0625 - 0.125 - 0.25 - 0.5 and 1 µg/ml.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Details on test system and experimental conditions:

The assay was performed in cultured human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male donor.
A range finding study was first performed in a preliminary study to assess the cytotoxicity based on mitotic index and microscopic evaluation. This
preliminary study fixed the high dose for the main study.
According to the cytotoxic preliminary assay, the concentrations chosen for the main study were 0.0625 - 0.125 - 0.25 -0.5 and 1 µg/ml.
with (10% S9) and without S9. Vehicle (ethanol) and positive controls were tested in parallel. The solvent control received 1 % ethanol. Positive
controls employed for with and without S9 were cyclophosphamide and mitomycin C respectively, at concentrations 20 and 1 µg/ml respectively.
The cultures were tested in duplicates for all the treatment conditions.
The PHA-M stimulated cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ± 0.5 °C. After the test substance addition at the 48th hour with and without S9, cultures were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times corresponding to 36
hours. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical -colchicine at a concentration of 0.4 μg/ml.
Metaphase preparations were made from cells in fixative, after hypotonic shock with pre incubated 0.075M KCl at 37 °C and a series of washes with fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed preparations were stained with Giemsa. Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases with 46 +/6
centrometers were analyzed for aberrations. The observations were recorded and summarized as percentage numerical aberrations and mean
structural aberrations.

Evaluation criteria:

Evaluation of cytotoxicity of the substance according to the mitotic index and microscopic observation.
For each condition of the main study, the following parameters were studied :
- number of cells scored
- percentage mitotic indices
- frequencies of structural and numerical aberrations
- percentage of cells with aberration (excluding gaps): polyploidy, fragment, deletion, pulverized chromosome, endoreduplication, break, exchange, dicentric, ring

Statistics:

comparison between the various treatment groups according to the Student's T test (p<0.05)

Species / strain:

lymphocytes: human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male donor.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the preliminary study performed to evaluation the cytotoxicity of the substance, 5 concentrations were first employed 0.41 - 0.512 - 0.64 - 0.8
and 1 µg/ml, with (10% S9) and without metabolic activation system (S9). No cytotoxicity was observed at all concentrations with and without S9.
In the main study with 4 hours exposure time period and with continous exposure period, the % mitotic indices of the 5 concentrations tested (with
and without S9), 0.0625 - 0.125 - 0.25 - 0.5 and 1 µg/ml were comparable with the solvent control. Corresponding values were obtained in the
duplicate
At 0.0625 - 0.125 - 0.25 - 0.5 and 1 µg/ml in the 4h exposure time period and the continuous exposure time period, with and without S9, a lack of
induction of chromosome aberrations was observed. It was in consensus with the solvent control. The frequencies of polyploidy was insignificant
amongst the cultures treated with the substance and the vehicle in both the exposure periods and with and without S9.
The cultures treated with known mutagens in the 4h exposure time period and the continuous exposure time period, with and without S9, exhibited a statistically significant increase in the mean frequency of structural aberrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the test conditions employed, the substance LCE07105 did not induce any apparent numerical and structural aberrations both in presence
and absence of an exogenous mammalian metabolic activation component (S9) in the 4h exposure and continuous exposure time periods. Hence,
the substance LCE07105 may be considered non genotoxic in the in vitro chromosome aberrations assay on human lymphocytes.

Executive summary:

The ability of the substance LCE07105 to induce numerical and structural cytogenetic anomalies was evaluated by enumerating the incidence of chromosome aberrations in cultured human peripheral blood lymphocytes.

Because of the low solubility of the substance, different solvents were tested DMSO and ethanol with and without heating. The maximal miscible and not precipiting concentration was 1 µg/ml in heated (40°C) ethanol. Higher concentrations precipitated during cooling at 37°C.

A range finding study was performed to assess the cytotoxicity based on mitotic index, to fix the high dose for the main study. The following concentrations of the test substance were employed 0.41 - 0.512 - 0.64 - 0.8 and 1 µg/ml with (10% S9) and without metabolic activation system (S9). No cytotoxicity was observed at all concentrations. From the results of the range finding studies, 1 µg/ml was chosen as the high dose for the main study. The concentrations chosen for the main study were 0.0625 - 0.125 - 0.25 - 0.5 and 1 µg/ml with (10% S9) and without S9. Concurrent solvent (1% ethanol) and positive controls were tested. Positive controls employed for with and without S9 were cyclophosphamide and mitomycin C respectively, at concentrations 20 and 1 µg/ml respectively. The cultures were maintained in duplicates for all the treatment conditions.

The test substance, vehicle and positive controls were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical.

Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases were analyzed for aberrations. The observations were recorded and summarized as percentage numerical aberrations and mean structural aberrations.

Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested with and without metabolic activation in the 4 h exposure time period. Cultures treated with the test substance exhibited statistically comparable values of numerical and structural aberrations to that of the solvent control group with and without

S9. However, the positive control groups exhibited statistically increased frequencies of cytogenetic anomalies thus validating the sensitivity of the assay.

Based on the negative results from the 4 h exposure time period, an additional experiment was performed with continuous exposure in the presence of the test substance for 1.5 cell cycle time without S9. The concentrations employed were 0.0625 - 0.125 - 0.25 - 0.5 and 1 μg/ml and the experiment was performed in a similar pattern as detailed in the 4 h exposure time period. The

concentration 1 μg/ml exhibited a reduction in MI around 50% without metabolic activation (S9) compared to the solvent control.Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested without metabolic activation in the continuous exposure time period. Cultures treated with the test substance exhibited statistically comparable values of numerical and structural aberrations to that of the solvent control group. However, the

positive control group exhibited statistically increased frequencies of cytogenetic anomalies thus validating the sensitivity of the assay.

Thus, the substance LCE07105 was considered non genotoxic in the in vitro chromosome aberration assay on human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-10 to 2008-06-27

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (tk) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

According to the solubility of the product, the maximal non precipitant concentration tested was 0.5 µg/ml.
For the preliminary cytotoxicity assay, 9 concentrations were tested: 0.09 ; 0.11 ; 0.14 ; 0.17 ; 0.21 ; 0.26 ; 0.32 ; 0.4 and 0.5 µg/ml.
For the main study, 5 concentrations were tested: 0.0313 ; 0.0625 ; 0.125 ; 0.25 and 0.5 µg/ml.

Vehicle / solvent:

ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

(1) solubility of the substance
Different assay of solubility were performed in water, DMSO, ethanol with or without heating.
(2) Preliminary cytotoxicity assay
According to the solubility assay, 0.1 mg/ml in heated ethanol (80°C) was the maximal miscible concentration. The maximal concentration tested with the cells was 0.5 µg/ml.
L5178Y TK +/- cells treated for 3 hours with ethanol alone (solvent control) or with 9 concentrations of the test substance with or without S9 activation. After washing, cells were subsequently cultured for 24hours, 24 hours and 3 days after recounting and dilution. Viable clones were counted visually visually. The relative total growth (RTG) was calculated.
(2) evaluation of gene mutation potential (main study)
Cultures were treated for 3 or 24 hours with test substance, known mutagens (benzo-a-pyrene 2 and 3 µg/ml and methylmethanesulfonate 10 and 20 µg/ml) or vehicle (ethanol) with or without S9. The solubility of the test substance was assessed visually, at the begining and end of the treatment.
Each assay was performed in duplicate.
After the 3 or 24 hours treatment period, the cells were washed, pelleted and resuspended for 2 days. During this time, the tk- mutation was expressed. At the end of the expression period, cells were incubated for 2 weeks with or without trifluorothymidine (TFT) at 3 µg/ml (final concentration) was added to the cell to establish the TK mutation. The Mutant Frequency (MF) was calculated.

Evaluation criteria:

(1) Cytotoxicity
The highest concentration for the main study was chosen on the basis that it exhibited 10-20% RTG.
(2) Gene mutation
A concentration-related or a reproducible increase in the mutant frequency is amongst the several criteria's considered for determining a positive result.

Statistics:

The number of mutant colonies obtained at different concentrations of the test substance was statistically compared to the solvent control using Student's T test, for significance (p<0.05).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no concentration related and reproducible increase in the mutant frequencies of any of the tested concentrations and also no statistically significant dose-response relationship was observed. However, in the positive controls a statistically significant increase in the mutant frequencies was observed and a clear indication of clastogenic activity was indicated by a proportional increase in both the small and large colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Hence the test substance was considered as non mutagenic in the mouse lymphoma assay, under the given test conditions.

Executive summary:

A study in mouse lymphoma L5178Y TK+/- cell line was performed to measure mutation at thymidine kinase (TK) locus with and without exogenous mammalian (rat liver S9) microsomal metabolic activation in order to evaluate the ability of LCE07105 to induce gene mutation. A range finding study was performed with 3 h and 24 h treatments with and without S9. The concentrations chosen

were 0.09, 0.11, 0.14, 0.17, 0.21, 0.26, 0.32, 0.4 and 0.5 µg/ml, and the maximum possible dose being 0.5 µg/ml. The test substance was dissolved in ethanol. The %Relative Total Growth (RTG) for the above mentioned concentrations were 74.70, 76.74,

66.66, 58.51, 55.84, 55.96, 53.15, 50.27 and 51.02 respectively with S9 and 72.14, 82.54, 76.75, 67.70, 63.15, 62.62, 58.10, 51.43 and 51.24 respectively without S9 in the 3 h treatment. The % Relative Total Growth (RTG) for the above mentioned concentrations in the 24 h treatment without S9 were 70.42, 65.84, 56.12, 58.66, 48.63, 50.00, 56.55, 49.29 and 48.34 respectively. The high dose chosen for the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was 0.5 µg/ml, as the % RTG was found comparable to that of the solvent control.

Based on the results of the range finding study, the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was performed employing concentrations 0.0313, 0.0625, 0.125, 0.25 and 0.5 µg/ml.

Two positive controls, Methylmethanesulfonate (MMS) in DMSO at concentrations 10 and 20 µg/ml without S9 and Benzo [a] pyrene (BP) at concentrations 2 and 3 µg/ml with S9 were used. The solvent control received 0.1 ml of ethanol alone.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genetic toxicity was tested in-vitro in 3 studies. Each key study is quoted as reliability 1 according to Klimisch criteria (OECD studies and performed in accordance with GLP). No in-vivo data is available.

Gene mutation in bacteria (Ames):

The substance was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation. The 5 concentrations (50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first. In each study was included a negative control (vehicle = acetone) and a positive control (specific standard mutagen).

Under the experimental conditions employed, the substance did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.

Gene mutation in mammalian cells:

A study in mouse lymphoma L5178Y TK+/- cell line was performed to measure mutation at thymidine kinase (TK) locus with and without exogenous mammalian (rat liver S9) microsomal metabolic activation in order to evaluate the ability of the substance to induce gene mutation.

Based on the results of the range finding study, the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was performed employing concentrations 0.0313, 0.0625, 0.125, 0.25 and 0.5 µg/ml in 1% ethanol. Correspoding positive controls and solvent control were tested.

There was no concentration related and reproducible increase in the mutant frequencies of any of the tested concentrations and also no statistically significant dose-response relationship was observed.

Hence the test substance was considered as non mutagenic in the mouse lymphoma assay, under the given test conditions.

Chromosomal aberration in mammalian cells:

The ability of the substance to induce numerical and structural cytogenetic anomalies was evaluated by enumerating the incidence of chromosome aberrations in cultured human peripheral blood lymphocytes.

From the results of the range finding studies, the concentrations chosen for the main study were 0.0625 - 0.125 - 0.25 - 0.5 and 1 µg/ml with (10% S9) and without S9, in 1% ethanol. Concurrent solvent (1% ethanol) and positive controls were tested. The cultures were maintained in duplicates for all the treatment conditions.

The test substance, vehicle and positive controls were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical.

Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases were analyzed for aberrations.

Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested with and without metabolic activation in the 4 h exposure time period.

Based on the negative results from the 4 h exposure time period, an additional experiment was performed with continuous exposure in the presence of the test substance for 1.5 cell cycle time without S9. Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested without metabolic activation in the continuous exposure time period.

Thus, the substance LCE07105 was considered non genotoxic in the in vitro chromosome aberration assay on human lymphocytes.

Justification for classification or non-classification

Based on the classification criteria of UN/EU GHS, and considering the negative results in the three in vitro genetic toxicity tests using C20/22 alkyl phosphate, no classification for mutagenicity is required.