N-(2-hydroxypropyl)oleamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity tests were performed (OECD 471, 476, 487)

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:

2013-01-14 to 2013-03-14

Reliability:

1 (reliable without restriction)

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:

Histidine

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:

Rat Liver Microsomal fraction S9-mix

Test concentrations with justification for top dose:

In the bacteriostatic assay : 5000 - 1500 - 500 - 150 - 50 µg/plate
In the 1st mutagenic assay :
- without metabolic activation :
5000 - 1500 - 500 - 150 - 50 µg/plate in strains TA1535, TA1537 and TA98
5000 - 1500 - 500 - 150 - 50 - 15 µg/plate in strain TA100
5000 - 1500 - 500 - 150 - 50 - 15 - 5 µg/plate in strain TA102
- with metabolic activation :
500 - 150 - 50 - 15 - 5 - 1.5 µg/plate in strain TA1535
5000 - 1500 - 500 - 150 - 50 - 15 µg/plate in strain TA100
5000 - 1500 - 500 - 150 - 50 - 15 - 5 µg/plate in strain TA102

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: As indicated by the Sponsor and specified in the Final Study Plan, the test item N-(2-hydroxypropyl)Oleamide was dissolved in ethanol.

As indicated by the Sponsor and specified in the Final Study Plan, the test item N-(2-hydroxypropyl)Oleamide was dissolved in ethanol (Merck, batches K43508483225 for the bacteriostatic and the 1st mutagenic assays and K42909883148 for the 2nd, 3rd and 4th assays).
The test item was soluble at a maximum concentration of 679.12 mg/mL in ethanol.
For the bacteriostatic assay, it was dissolved at the maximal initial concentration of 50 mg/mL in order to obtain the top dose of 5000 µg/plate when added at 100 µL/plate. Nevertheless, an unexpected toxicity with no dose-effect relationship, in particular in the negative control was observed at the microscopic examination of the background growth. It was thus decided in accordance with Amendment No. 1 to FSP-IPL 121202 to perform another toxicity assay with a volume of treatment decreased down to 50 µL/plate.
For the 2nd bacteriostatic assay and the 1st and the 2nd mutagenic assays, the test item was thus dissolved at a higher initial concentration of 100 mg/mL in order to obtain the top dose of 5000 µg/plate when added at 50 µL/plate.
Finally for the 3rd and the 4th mutagenic assays, the test item was dissolved at the maximal initial concentration of 30 mg/mL in order to obtain the top dose of 1500 µg/plate when added at 50 µL/plate (without S9-mix) or at a maximal initial concentration of 200 mg/mL to obtain the top dose of 5000 µg/plate when added at 25 µL/plate (with S9-mix, with pre-incubation).

The limiting factor for maximum dose in the toxicity assay was thus maximum dose according to OECD Guideline.

Successive dilutions were also prepared with ethanol and used at either 100 or 50 or 25 µL/plate.

The stability of the test item in the solvent was unknown but preparations for treatment were performed just before use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Remarks:

- Without S9-mix: sodium azide (TA1535, TA100) 9-amino-acridine (TA1537) 2-nitro fluorene (TA98) mitomycin C (TA102) - With S9-mix: 2-anthramine (TA1535, TA1537, TA98, TA100) benzo[a]pyrene (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (only for the 3rd and 4th assays with metabolic activation)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine withdrawal
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: triplicate for each dose and positive control, 6 replicates for negative control

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF TOXICITY
- Method: microscopic observation of the background growth

Evaluation criteria:

Criteria based on biological significance
A test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times (for strains TA1535, TA1537) or 2 times ( for strains TA98, TA100, TA102) the value for the solvent control, is considered positive in the assay.

Criteria based on statistical significance
see below

Reproducibility
If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant.

Comparison to historical control data
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen.

Statistics:

In parallel, data will be analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not applicable
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: unknown
- Precipitation:
In the first toxicity assay (Table 1), slight and moderate precipitates were observed in all strains both with and without metabolic activation at the 2 highest doses tested of 1500 and 5000 µg/plate, respectively. However, it is noteworthy that due to unexpected toxicity, a 2nd assay was performed.
In the 2nd toxicity assay (Table 1-continued), precipitates were observed as described below :
-strong precipitate in strain TA1537 without metabolic activation at the highest dose tested of 5000 µg/plate.
-moderate precipitates at the 2 highest doses tested of 1500 and 5000 µg/plate in strains TA1535, TA98 and TA102 both with and without metabolic activation and in strain TA100 without metabolic activation; at the highest dose of 5000 µg/plate in strains TA1537 and TA100 with metabolic activation; at the 2 doses of 1500 and 500 µg/plate in strain TA1537 without metabolic activation.
-slight precipitates at the 2 doses of 1500 and 500 µg/plate in strain TA1537 with metabolic activation; at the dose of 1500 µg/plate in strain TA100 with metabolic activation; at the 2 doses of 500 and 150 µg/plate in strain TA98 both with and without metabolic activation and in strain TA102 with metabolic activation; at the dose of 500 µg/plate in strain TA1535 both with and without metabolic activation and in strains TA100 and TA102 without metabolic activation; at the dose of 150 µg/plate in strain TA1537 without metabolic activation.
In the 4 mutagenicity assays, some slight to moderate precipitates were found again at the 1 to 3 highest doses tested.

- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the first toxicity assay (Table 1), slight to potent toxicity was observed in all strains both with and without metabolic activation but especially without metabolic activation and up to the 5 doses tested. Moreover, an unexpected toxicity was observed in the negative control with a decrease in the frequencies of spontaneous revertants. This toxicity could thus be due to the intrinsic toxicity of ethanol at high dose volume (100µL/plate). It was thus decided in accordance with the Amendment No. 1 to FSP-IPL 121202 to perform another toxicity assay with a volume of treatment decreased down to 50 µL/plate.

In the 2nd toxicity assay, toxicity was observed as described below :
-strong toxicity at the 2 highest doses tested of 1500 and 5000 µg/plate in strains TA1535, TA1537, TA100 and TA102 without metabolic activation.
-moderate toxicity at the highest dose tested of 5000 µg/plate in strain TA98 without metabolic activation and in strains TA1537 and TA98 with metabolic activation; at the 3 highest doses tested from 500 to 5000 µg/plate in strain TA100 and TA102 with metabolic activation; at the 2 doses of 150 and 500 µg/plate in strain TA1537, TA100 and TA102 without metabolic activation; at 500 µg/plate in strain TA1535 without metabolic activation.
-slight toxicity at the 2 doses of 1500 and 5000 µg/plate in strain TA1535 with metabolic activation; at the 3 doses from 150 to 1500 µg/plate in strain TA98 without metabolic activation; at the 4 doses from 50 to 1500 µg/plate in strains TA1537 and TA98 with metabolic activation but this toxicity was also observed in negative control; at the 2 doses of 50 and 150 µg/plate in strains TA100 and TA102 with metabolic activation; at 150 µg/plate in TA1535 without metabolic activation and at 50 µg/plate in strains TA1537, TA100 and TA102 without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
Concurrently to the main assays, tests were carried out on reference mutagenic test compounds in order to show the sensitivity of the strains tested and the efficiency of the metabolic activation system. Statistically and biologically significant increases in the numbers of revertants were observed in the presence of positive reference test substances. The values observed were within the limits of historical controls with some minor exceptions (See Below). The frequencies of spontaneous revertants (solvent controls) were within the limits generally observed under our experimental conditions with a minor exception. The frequencies of spontaneous revertants (solvent controls) in the 3rd assay in strain TA102 with metabolic activation with preincubation was below the lower limit generally observed under our experimental conditions (i.e. 159.0 vs. 200 – 568). Nevertheless, this deviation was considered as minor. It did not affect the quality or the integrity of the study data and did not impair the conclusion of the study.
The validity criteria for the test were fulfilled.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The search for any mutagenic activity ofN-(2-hydroxypropyl)Oleamide(Batch T22221 without solvent) provided by SEPPIC, was studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471 using the highest dose compatible with the toxic activity. Four to seven lower dilutions were also tested.

The summaries of the test results are given in Appendix No. 1 (Tables 2, 3, 4 and 5). The individual results for mutagenicity assays are shown in Appendix No. 2 (Tables 7 to 22).

In the third assay in strain TA98 in presence of metabolic activation(Table 19), a statistically significant increase was observed at the intermediary dose tested of 50 µg/plate with an induction ratio of 1.9,i.e.below the threshold ratio for a biologically significant response (set at 2 in this strain). No dose-response relationship was observed and the value for the mean number of revertants was within the range of values for negative control in historical data (i.e.11 – 57).

The N-(2-hydroxypropyl)Oleamide is not mutagenic in this condition.

In strain TA102 in absence of metabolic activation(Table 11), statistically significant increases in the number of revertants were noted at the 2 intermediary doses of 5 and 15 µg/plate with induction ratios of 1.9. The threshold ratio for a biologically significant response (set at 2 in this strain) was thus not reached, no dose-response relationship was observed and the value for the mean number of revertants was within the values for negative control in historical data (i.e.48 – 354) Furthermore, the second and the third assay were clearly negative, with neither statistically nor biologically significant increase in the number of revertants (Tables 16 and 21).

The N-(2-hydroxypropyl)Oleamide is not mutagenic in this condition.

In the third assay in strain TA102 in presence of metabolic activation(Table 21), statistically significant increases in the number of revertants were noted at the 4 intermediary doses from 50 to 1500 µg/plate with induction ratios from 1.5 to 1.9. The threshold ratio for a biologically significant response (set at 2 in this strain) was thus not reached and the values for the mean number of revertants was within the values for negative control in historical data (i.e.200 – 568). The statistically significant assay was certainly due to the low level ofspontaneous revertants but a dose-response relationship was observed up to the dose of 500 µg/plate. It was thus decided to reiterate the assay in this specific condition.

In the fourth assay in strain TA102 in presence of metabolic activation(Table 22), neither statistically nor biologically significant increase in the number of revertants was noted at all the doses tested.

The N-(2 -hydroxypropyl)Oleamide is not mutagenic in this condition.

Except the meaningless increases mentioned above, in three independent assays performed either with or without metabolic activation (the third assay with S9-mix was performed according to the pre-incubation protocol), no biologically significant increase in the mean number of revertants was noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the presence of N-(2-hydroxypropyl)Oleamide.

Furthermore, some statistically significant decreases in the mean number of revertants were observed. Nevertheless, these effects were probably due to the toxic activity of the test item and had no meaning in terms of mutagenic hazard.

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenic activity of the test item N-(2-hydroxypropyl)Oleamide (Batch T22221 without solvent) provided by SEPPIC was assessed by means of the Ames’s test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in three or four independent assays.

The validity criteria for the assay were fulfilled. The study was thus considered as valid.

Under these experimental conditions, no mutagenic activity was revealed.

Executive summary:

Bacterial Reverse Mutation Test

On five strains of Salmonella typhimurium His^-using B.N. AMES' Technique

SUMMARY

TEST ITEM                                 : N-(2-hydroxypropyl)Oleamide

BATCH NUMBER                        : T22221 without solvent

STUDY LOCATION                     : INSTITUT PASTEUR DE LILLE

                                                                       Genetic Toxicology Laboratory

                                                                       1, rue du Professeur Calmette - B.P. 245

                                                                       59019 LILLE CEDEX FRANCE

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

Study initiation date (date Study Director signed Study Plan):                                                   14/01/2013

Experimental start date:                                                                                                        14/01/2013

Experimental completion date:                                                                                               14/03/2013

AIM

The search for any mutagenic activity ofN-(2-hydroxypropyl)Oleamide(BatchT22221 without solvent) studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471.

METHODS

Strains used                                : TA1535, TA1537, TA98, TA100, TA102

Solvent                                        : ethanol (Merck, Batches K43508483225 and K42909883148)

Stability in the solvent                  : unknown (dilutions were prepared extemporaneously)

TOXICITY ASSAYS

Preliminary test of toxic activity     : carried out on 5 strains – Incubation period: 48 h

Sterility test                                 : absence of contamination

Limiting factor for maximum dose  : maximum dose according to OECD Guideline

Doses used in toxicity assay        :expressed asµg/plateN-(2-hydroxypropyl)Oleamide

Volumes of treatment                   : 1^stassay: 100 µL/plate as specified in the Final Study Plan

                                                     2^ndassay 50 µL/plate as specified in the Amendment No. 1

                                                     

1^stassay(under 100 µL/plate):

The examination of the background growth demonstrated unexpected toxicity in the negative control. This could be due to the intrinsic toxicity of ethanol at high dose volume of 100 µL/plate. It was this decided in accordance with Amendment No. 1 to FSP-IPL 121202 to perform another toxicity assay with a volume of treatment decreased down to 50 µL/plate.

2^ndassay(under 50 µL/plate):

The examination of the background growth demonstrated thatN-(2-hydroxypropyl)Oleamideinduced slight to strong toxicity in all strains depending on the condition or the dose tested. Moreover, slight to important precipitates in all strains were observed.

Therefore, the maximum doses retained for the first mutagenicity assay were:

-         Without metabolic activation:

o       In strains TA1535, TA1537, TA98 and TA102: 500 µg/plate

o       In strain TA100: 250 µg/plate

-         With metabolic activation: 5000 µg/plate in all strains.

The dose volume was set at 50 µL/plate.

MUTAGENICITY ASSAYS

Mutagenicity test                         : carried out on 5 strains both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254 – Incubation period: 48h

Number of assays                        : 2 or 3 (the last assay with metabolic activation was performed according to the pre-incubation method) or 4 (only in strain TA102 with metabolic activation, the 2 last assays were performed according to the pre-incubation method)

Limiting factor for maximum dose  : maximum dose according to OECD Guideline or toxic activity (depending on the strains and/or the experimental conditions)

Doses used in main test               :expressed asµg/plate pureN-(2-hydroxypropyl)Oleamide

Volume of treatment                     : 50 µL/plate as specified in the Amendment No. 1 to FSP-IPL 121202

Results:

In the 1st assay, no biologically significant increase in the mean number of revertants was observed. However, a too strong toxicity (that could be due to the batch of solvent) and invalidated positive controls were noted. When gathering these observations, in order to bring confident conclusions, it was decided to reiterate the assay in all the conditions even in the ones that were not invalidated.

In the 2nd assay, no biologically significant increase in the mean number of revertants was observed.

In the 3rd assay, no biologically significant increase in the mean number of revertants was observed.

In the 4th assay (performed only in strain TA102 in presence of metabolic activation), no biologically significant increase in the mean number of revertants was observed.

CONCLUSION

The mutagenic activity of the test item N-(2-hydroxypropyl)Oleamide (Batch T22221 without solvent) was assessed by means of the Ames’s test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in three or four independent assays.

The validity criteria for the assay were fulfilled. The study was thus considered as valid.

Under these experimental conditions, no mutagenic activity was revealed.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of the study 14/01/2013 - End of the study (draft study report) 03/04/2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 (Genetic toxicology :In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

- Type and identity of media:The basic culture medium used was the RPMI 0 medium containing sodium bicarbonate, non-essential aminoacids, penicillin, streptomycin and HCl. The RPMI 0 medium was supplemented with 10% inactivated horse serum, pluronic acid, L-Glutamine, sodium pyruvate and Amphotericine B.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix : The S9 fraction was prepared at Institut Pasteur de Lille using the method described by Ames et al. (1975) in male rat OFA Sprague Dawley induced by Aroclor 1254 (origin - Monsanto, Saint Louis, U.S.A) .

Test concentrations with justification for top dose:

Toxicity assay :
1.25 - 0.63 - 0.31 - 0.16 - 0.08 - 0.04 - 0.02 - 0.01 - 0.005 - 0.002 mM
Genotoxicity assays
Assay with 3 h treatment and 27 h recovery period:
-Without S9-mix:0.20 – 0.15 – 0.10 – 0.075 – 0.05 mM but only the following concentration were assessed : 0.20 - 0.15 - 0.10 mM
-With S9-mix (5% S9-mix):0.40 – 0.30 – 0.20 – 0.15 – 0.10 – 0.075 mM but only the following concentration were assessed : 0.30 – 0.20 – 0.15 mM
Assay with 27 h treatment and no recovery period:
- Without S9-mix:0.075 – 0.05 – 0.025 – 0.013 – 0.0063 – 0.0031 mM but only the following concentration were assessed : 0.075 – 0.05 – 0.025 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: As indicated by the Sponsor and specified in the Final Study Plan, the test item N-(2-hydroxypropyl)Oleamide was dissolved in ethanol.

The N-(2-hydroxypropyl)Oleamide was dissolved in ethanol (Merck, batch K429 09 883 148) at a maximum initial concentration of 2000 mM. Nevertheless, this concentration as well as the 2 concentrations below induced a strong precipitate when used at 1% in culture medium. The test item was thus dissolved at a maximum initial concentration of 125 mM and used at 1% in culture medium, giving a final concentration of 1.25 mM.
This concentration induced no variation in osmolality higher than 50 mOsmol when compared to the solvent control and was thus retained as top concentration to be tested in cytotoxicity assays. Successive dilutions were also performed in distilled water and used at 1%.

Furthermore, the pH was in the acceptable range of 6-8 at the highest concentrations tested from 1.25 to 0.31 mM.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Short treatment without S9-mix: mitomycin C 0.5 µg/mL and With S9-mix: cyclophosphamide 10 µg/mL Continuous treatment: mitomycin C 0.1 µg/mL and griseofulvin 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 hours with 24-hour recovery period both with and without metabolic activation and 27-hour without recovery period
- Expression time (cells in growth medium): 27-hours after the beginning of the treatment
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 27-hours after the beginning of the treatment

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): Giemsa reagent (at 2% in water)

NUMBER OF REPLICATIONS: 2 cultures per assay

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other:

OTHER:

Evaluation criteria:

The results obtained in the different treatment cultures are presented giving the mean number of micronuclei for 2000 mononucleated cells, and the RPD per concentration.
A test item is found to demonstrate clastogenic or aneugenic properties against TK6 cells:
-if it results in a statistically significant increase in the number of micronuclei compared with the control,
-if this increase amounts to at least a doubling of the control value and
-if the genotoxicity detected shows a concentration-effect relationship.

A test item is found to have no genotoxic effect on TK6 cells, if it does not comply with any of the 3 criteria listed above.
If neither situation occurs, the results are discussed case by case.

Statistics:

Statistical analysis of the results obtained in the cells treated at each concentration level is performed using the c2 test in comparison with those in control groups.

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: unknown
- Water solubility: poorly
- Precipitation: The N-(2-hydroxypropyl)Oleamide was dissolved in ethanol at a maximum initial concentration of 1000 mM. Nevertheless, this concentration as well as the 2 concentrations below induced a strong precipitate when used at 1% in culture medium. The test item was thus dissolved at a maximum initial concentration of 125 mM and used at 1% in culture medium, giving a final concentration of 1.25 mM.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay using a 3-hour treatment without metabolic activation, a potent toxicity was observed at the 3 highest concentrations from 1.25 to 0.31 mM. At the immediately lower concentration of 0.16 mM, a strong but acceptable toxicity was observed with a RPD of 49.1%, respectively corresponding to a percentage of cytostasis of 50.9%, when compared to the respective solvent control (Table 3). Under these conditions, the concentration of 0.20 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

In the preliminary toxicity assay using a 27-hour treatment without metabolic activation, a potent toxicity was observed at the 5 highest concentrations from 1.25 to 0.08 mM. At the immediately lower concentration of 0.04 mM, a moderate toxicity was observed with a RPD of 66.2%, respectively corresponding to a percentage of cytostasis of 33.8%, when compared to the respective solvent control (Table 5). Under these conditions, the concentration of 0.075 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

In the preliminary toxicity assay using a 3-hour treatment with metabolic activation, a potent toxicity was observed at the 2 highest concentrations of 1.25 and 0.63 mM. At the immediately lower concentration of 0.31 mM, a strong but acceptable toxicity was observed with a RPD of 45.2%, respectively corresponding to a percentage of cytostasis of 54.8%, when compared to the respective solvent control (Table 4).
Under these conditions, the concentration of 0.40 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay. A narrowed range of concentration was chosen in order to reach a concentration that induces a RPD of around 45 ± 5%.

COMPARISON WITH HISTORICAL CONTROL DATA: In the solvent control group, the number of micronucleated cells per mononucleated cells were within the limits of values generally observed under our experimental conditions.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity assay using a 3-hour treatment without metabolic activation (Table 6), the concentration of 0.20 mM induced a moderate toxicity with a RPD of 67.2%, corresponding to percentages of cytostasis of 32.8%, when compared to the respective solvent control. Under these conditions, the concentration of 0.20 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

In the mutagenicity assay using a 27-hour treatment without metabolic activation (Table 8), the concentration of 0.075 mM induced a moderate toxicity with a RPD of 67.8%, corresponding to percentages of cytostasis of 32.2%, when compared to the respective solvent control. Under these conditions, the concentration of 0.075 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

In the mutagenicity assay using a 3-hour treatment with metabolic activation (Table 7), the concentration of 0.40 mM induced a too strong toxicity. The immediately lower concentration of 0.30 mM induced a moderate toxicity with a RPD of 55.9%, corresponding to percentages of cytostasis of 44.1%, when compared to the respective solvent control. Under these conditions, the concentration of 0.30 mM was retained as the maximum concentration to be analyzed. Two lower concentrations were also assessed for genotoxicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The investigation of a genotoxic activity of the test item N-(2 -hydroxypropyl)Oleamide (batch T22221 without solvent) provided by SEPPIC has been carried outcompliance with the OECD Guideline 487, usingin vitromammalian cell micronucleus test onTK6 lymphoblastoid human cells.The test was performed at the highest dose compatible with the toxic activity of the test itemin the three assays with and without S9-mix, both with and without recovery period. Two concentrations below were also analysed.

All the concentrations tested in this study are expressed as mM of N-(2-hydroxypropyl)Oleamide.

In the short treatment without metabolic activation followed by a 24-hour recovery period(assay S9- 3h/+ 24h), the test item N-(2-hydroxypropyl)Oleamide induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.20 to 0.10 mM.

Indeed, 8 to 14 micronucleatedmononucleatedcells were observed per 2000 cells,vs.7 in the negative control without any dose-effect relationship.

The test item N-(2-hydroxypropyl)Oleamidewas thus considered as not genotoxic under this condition.

In the short treatment with metabolic activation followed by a 24-hour recovery period(assay S9+ 3h/24h), the test item N-(2-hydroxypropyl)Oleamide induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.30 to 0.15 mM.

Indeed, 7 and 8 micronucleatedmononucleatedcells were observed per 2000 cells,vs.8 in the negative control without any dose-effect relationship.

The test item N-(2-hydroxypropyl)Oleamide was thus considered as not genotoxic under this condition.

In the continuous treatment without metabolic activation without recovery period(assays S9- 27h/+0h), the test item N-(2-hydroxypropyl)Oleamide induced neither biologically nor statistically significant increase in the number of micronucleated cells at all the concentrations analyzed from 0.075 to 0.025 mM.

Indeed, 4 to 6 micronucleatedmononucleatedcells were observed per 2000 cells,vs.3 in the negative control.

The test item N-(2-hydroxypropyl)Oleamide was thus considered as not genotoxic under this condition.

Conclusions:

Interpretation of results (migrated information):
negative

The genotoxic activity of the test item N-(2-hydroxypropyl)Oleamide (batch T22221 without solvent) was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence and in absence of metabolic activation, either with a short or with a continuous treatment.

The acceptance criteria for the assay were fulfilled. The study is thus considered as valid.

Under these experimental conditions, no genotoxic activity was revealed.

Executive summary:

In Vitro MAMMALIAN CELL MICRONUCLEUS TEST

On TK6 lymphoblastoid human cells

SUMMARY

TEST ITEM                                 : N-(2-hydroxypropyl)Oleamide

BATCH NUMBER                        : T22221 without solvent

STUDY LOCATION                     : INSTITUT PASTEUR DE LILLE

                                                     Genetic Toxicology Laboratory

                                                     1, rue du Professeur Calmette - B.P. 245

                                                     59019 LILLE CEDEX FRANCE

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

Study initiation date (date Study Director signed Study Plan): 14/01/2013

Experimental start date: 14/01/2013

Experimental completion date: 13/03/2013

Study completion date 18/07/2013

AIM

The investigation of a genotoxic activity of the test itemN-(2-hydroxypropyl)Oleamide(batchT22221 without solvent) has been carried out in compliance with the OECD Guideline 487 (July 2010), usingin vitromammalian cell micronucleus test onTK6 lymphoblastoid human cells.

METHODS

Cell strain                                             :    TK6 lymphoblastoid human cells

Culture medium                                    :    RPMI 1640

Solvent used                                         :    ethanol (Merck, batch K429 09 883 148)

Stability in solvent                                 :    3 weeks at 4°C according to Sponsor’s information (dilutions were prepared extemporaneously)

Preliminary Cytotoxicity Assay

Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers.

Number of assays                                 :    3

Number of cultures/concentration           :    1

Factor limiting the maximum concentration analyzed                          :    solubility

Concentrations tested expressed as mM N-(2-hydroxypropyl)Oleamide

In the preliminary toxicity assay using a 3-hour treatment without metabolic activation, a potent toxicity was observed at the 3 highest concentrations from 1.25 to 0.31 mM. At the concentration of 0.16 mM, a strong but acceptable toxicity was observed with a percentage of cytostasis of 50.9%, when compared to the respective solvent control. The concentration of 0.20 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay and a narrowed range of concentration was tested.

In the preliminary toxicity assay using a 27-hour treatment without metabolic activation, a potent toxicity was observed at the 5 highest concentrations from 1.25 to 0.08 mM. At the concentration of 0.04 mM, a moderate toxicity was observed with a percentage of cytostasis of 33.8%, when compared to the respective solvent control. The concentration of 0.075 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay and a narrowed range of concentration was tested.

In the preliminary toxicity assay using a 3-hour treatment with metabolic activation, a potent toxicity was observed at the 2 highest concentrations of 1.25 and 0.63 mM. At the concentration of 0.31 mM, a strong but acceptable toxicity was observed with a percentage of cytostasis of 54.8%, when compared to the respective solvent control. The concentration of 0.40 mM was retained as the maximum concentration to be tested in the corresponding mutagenicity assay and a narrowed range of concentration was tested..

Genotoxicity assay

Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers.

Number of assays                                 :    3

Number of cultures/concentration           :    2

Number of analyzed cells                      :    2000 mononucleated cells / concentration

Factor limiting the maximum concentration analyzed                          :    cytotoxicity

Concentrations tested expressed as mM N-(2-hydroxypropyl)Oleamide

Assay with 3 h treatment and 27 h recovery period:

-    Without S9-mix                      :    0.20 – 0.15 – 0.10– 0.075 – 0.05 mM

-    With S9-mix (5% S9-mix)       :    0.40 –0.30 – 0.20 – 015– 0.10 – 0.075 mM

Assay with 27 h treatment and no recovery period:

-   Without S9-mix                      :    0.075 – 0.05 – 0.025– 0.013 – 0.0063 – 0.0031 mM

In bold, concentrations actually assessed

Positive controls

-Short treatment

Without S9-mix                   :    mitomycin C 0.5 µg/mL(Sigma, batch 098K1681)

 With S9-mix                       :    cyclophosphamide 10 µg/mL(Sigma, batch 079K1569)

-Continuous treatment                    :    mitomycin C 0.1 µg/mL(Sigma, batch 098K1681)

griseofulvin 10 µg/mL(Sigma, batch 031M1289V)

Results:

The test item N-(2 -hydroxypropyl)Oleamide induced no biologically or statistically significant increase in the number micronucleated cells either with orwithout metabolic activation.

CONCLUSION:

The genotoxic activity of the test item N-(2-hydroxypropyl)Oleamide (batch T22221 without solvent) was assessed by means of the in vitro micronucleus test in TK6 lymphoblastoid human cells treated in presence and in absence of metabolic activation, either with a short or with a continuous treatment.

The acceptance criteria for the assay were fulfilled. The study is thus considered as valid.

Under these experimental conditions,no genotoxic activitywas revealed.

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:

09/01/2013 – 15/04/2013

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

Remarks:

1 deviation: The control of concentration in treatment solutions was performed in a non GLP-compliant laboratory. However, the laboratory was compliant with ISO 9001, COFRAC 17025 and CIL 17043.

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

TK+/-
Type and identity of media: RMPI supplemented with 10 or 20% inactivated horse serum
Properly maintained: Yes
Periodically checked for: (1) Mycoplasma contamination: Yes; (2) Karyotype stability: No
Periodically cleansed against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation using S9 from rat liver induced by Aroclor 1254

Test concentrations with justification for top dose:

Concentrations tested expressed as mM N-(2-hydroxypropyl)Oleamide as supplied
• Without S9 mix : 0.150 – 0.130 – 113 – 0.099 – 0.086 – 0.075 – 0.065 – 0.056 (assay 1: 3-hour treatment)
0.080 – 0.070 – 0.060 – 0.050 – 0.040 – 0.030 – 0.020 (assay 2: 24-hour treatment)
• With S9 mix : 0.3 – 0.2 – 0.175 – 0.150 – 0.125 – 0.100 – 0.075 (assay 1)
0.175 – 0.163 – 0.150 – 0.125 – 0.100 – 0.075 (assay 2)

Vehicle / solvent:

ethanol (Merck, batch K429 09 883 148)
The N-(2-hydroxypropyl)Oleamide was dissolved in ethanol (Merck, batch K429 09 883 148) at a maximum initial concentration of 1000 mM (see solubility assays performed in study FSP-IPL 121204). Nevertheless, this concentration as well as the 2 concentrations below induced a strong precipitate when used at 1% in culture medium. The test item was thus dissolved at a maximum initial concentration of 125 mM and used at 1% in culture medium, giving a final concentration of 1.25 mM.
This concentration induced no variation in osmolality higher than 50 mOsmol when compared to the solvent control and was thus retained as top concentration to be tested in cytotoxicity assays. Successive dilutions were also performed in distilled water and used at 1%.

Furthermore, the pH was in the acceptable range of 6-8 at the highest concentrations tested from 1.25 to 0.31 mM.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

• Without S9-mix: methyl methanesulfonate 10 µg/mL (3-h treatment); 2 µg/mL (24-h treatment) (Aldrich, batch MKBD8856V) • With S9-mix: cyclophosphamide, 2 µg/mL (Sigma, batch 079K1569)

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Method of application: in medium
Duration:
- preincubation period: 3 days
- Exposure duration: 3h (or 24 h: 2nd assay in absence of S9-mix)
-Expression time: 2 days
-Selection time: 10-14 days
Selection agent: Trifluorothymidine
Number of replication: 2 assays bith with and W/O S9-mix
Dtermination of cytotoxicity: cloning efficiency and adjusted Relative Total Growth

Evaluation criteria:

Under our experimental conditions and when the criteria for validity are fulfilled, a test item is considered as mutagenic in this system if the following conditions are fulfilled:
1. The induced mutation frequency for at least one tested concentration is higher than the mutation frequency in the vehicle control cultures by at least the global evaluation factor of 126 x10-6 (Moore et al., 2006).
2. A statistical trend test demonstrates a positive concentration related increase in the mutation frequency (Moore et al., 2006).
3. The results have to be reproducible in an independent study, at least from a qualitative point of view.

Statistics:

Statistical evaluation of data for the total number of mutants and for small colony mutants is performed using the method proposed by Robinson et al. (1990). ROBINSON W.D., GREEN M.H., COLE J., HEALY M.J., GARTNER R.C., GATEHOUSE D.
Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical evaluation of mutagenicity test data. KIRKLAND D.J. (Ed). Cambridge University Press, Cambridge- New York, (1990) 102-140
Briefly, the statistical analysis procedure includes the following steps:

· Test for consistency of duplicate cultures at each dose level for a single experiment. The limit is 10.8 times the current heterogeneity factor (H) (10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. If the current heterogeneity factor is higher than 10.8 for survival heterogeneity factor (Hs) or mutation heterogeneity factor (Hm), this dose level is excluded from further consideration.
In case of heterogeneity in the negative control, the statistical evaluation of data is nevertheless performed.

· The new heterogeneity factor (Hexp) is calculated for doses not excluded. If it exceeds the value for that number of degrees of freedom in the experiment, the experiment may be discarded.

· For each dose level, mutant frequency (MF), log mutant frequency (LMF), the variance (V) of LMF and the weight (W) of MF are calculated for non-excluded dose levels (the value of MF could be slightly different in statistically evaluation compared to recapitulative tables because MF is calculated on pooled independent cultures, both series A and B).

· Each treatment is compared with the control by means of one-sided Dunnett's test for multiple comparisons with the same control.

· Test for linear trend of mutant frequency with dose is performed. The slope b and its variance var (b) are calculated to form the test statistic b2/var (b) that should be compared with tabulated critical values of chi2 with 1 degree of freedom.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Potent cytotoxicity with no cell growth at 0.15 mM. Very strong toxicity at 0.13 and 0.113 mM with adjusted RTG’s of 0.8 and 5.6%, respectively. Moderate level of cytotoxicity at 0.099 mM with an adjusted RTG of 29.7%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very strong toxicity at 0.08 and 0.07 mM with adjusted RTG’s of 1.8 and 6.3%, respectively. Moderate level of cytotoxicity at 0.06 mM with an adjusted RTG of 16.4%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Potent cytotoxicity with no cell growth at 0.3 mM. Very strong toxicity at 0.200 and 0.175 mM with adjusted RTG’s 1.1 and 7.9%, respectively. Moderate level of cytotoxicity at 0.150 mM with an adjusted RTG of 26.7%.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Very strong toxicity at 0.175 mM with adjusted an RTG’s 9%, respectively. Moderate level of cytotoxicity at 0.163 mM with an adjusted RTG of 13.4%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the second assay using a 3-hour treatment with metabolic activation, a statistically significant increase in the mutation frequency of total induced mutants (small and large colonies) were noted at the highest analysable concentration of 0.163 mM with a statistically linear trend. However, the induced mutation frequency (IMF) reached only 92.8x10-6 mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10-6 for a biologically significant effect (Moore et al., 2006).
Otherwise, no statistically significant increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentration tested. Moreover, no statistically linear trend was noted.

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Additional results for the determination of the cytotoxicity

In thecytotoxicity assay, the maximum dose was chosen in accordance with thesolubilityof the test item.

Without metabolic activation after a 3-hour treatment,N-(2-hydroxypropyl)Oleamiderevealed a potent cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 1.25 to 0.156 mM with no cell growth. The immediately inferior concentration of 0.078 mM induced no cytotoxicity with an adjusted RTG of 95.8%. The concentration of 0.150 mM was thus retained as the maximum concentration for the first mutagenicity test without S9-mix.

With metabolic activation,N-(2-hydroxypropyl)Oleamiderevealed a potent cytotoxicity in L5178Y cells at the 2 highest concentrations tested of 1.25 and 0.625 mM with no cell growth. The immediately inferior concentration of 0.313 mM induced a very strong cytotoxicity with 0.61% of adjusted RTG and the immediately lower concentration of 0.156 mM induced a moderate cytotoxicity with an adjusted RTG of 26.0%. The concentration of 0.3 mM was thus retained as the maximum concentration for the first mutagenicity test without S9-mix.

Without metabolic activation after a 24-hour treatment,N-(2-hydroxypropyl)Oleamiderevealed a potent cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 1.25 to 0.156 mM with no cell growth. The immediately inferior concentration of 0.078 mM induced a strong cytotoxicity with an adjusted RTG of 8.6%. The immediately lower concentration of 0.039 mM induced a slight cytotoxicity with an adjusted RTG of 68.6%. The concentration of 0.08 mM was thus retained as the maximum concentration for the second mutagenicity test without S9-mix.

Additional results for the determination of the genotoxicity

Acceptance criteria for the results

Concurrently to the main assays, tests were carried out with reference mutagenic compounds (methyl methanesulfonate in the absence of metabolic activation and cyclophosphamide in the presence of metabolic activation via S9-mix).

The plating efficiency of the negative control (mean of the 2 cultures) ranged from 65 to 120 % at T2.

The mutation frequency (MF) of the negative control is within the range of historical data of the laboratory (with one minor exception See above), and ranged from 50 to 170 x10^-6mutants

The suspension growth value of the negative control ranged from 8 to 32 in the 3-hour treatments, and were above 32 in the 24-hour treatment.

The induced mutation frequencies (IMF) for the positive controls were significantly increased when compared to the MF for the solvent control, and demonstrated an increase above the spontaneous background MF of at least 300 x10^-6mutants. The observed values were within or close to the limits of historical positive controls of the laboratory. The acceptance criteria for the results were thus fulfilled.

Additional results

In the maingenotoxicity assay, the maximum dose was chosen in accordance with thetoxicityof the test item.

In the second assay using a 3-hour treatment with metabolic activation, a statistically significant increase in the mutation frequency of total induced mutants(small and large colonies)werenoted at the highest analysable concentration of 0.163 mM with a statistically linear trend. However, the induced mutation frequency (IMF) reached only 92.8x10^-6mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10^-6for a biologically significant effect (Mooreet al., 2006).

Otherwise, no statistically significant increase in the mean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentration tested. Moreover, no statistically linear trend was noted.

Furthermore, in the first assay performed in the same experimental conditions, no significant increase in the mutation frequency of total induced mutants (small and large colonies) or in themean number of small colonies and in the mutation frequency of small colony mutants was noted at any concentrations tested in the presence of N-(2-hydroxypropyl)Oleamide.

In two independent assays without metabolic activation, no significant increase in the mutation frequency of total induced mutants (small and large colonies) or in themean number of small colonies and in the mutation frequency of small colony mutantswas noted at any concentrations tested in the presence of N-(2-hydroxypropyl)Oleamide.

Conclusions:

Interpretation of results (migrated information):
negative

The search for any mutagenic activity of N-(2-hydroxypropyl)Oleamide (batch T22221 without solvent), was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476, in 2 independent assays performed both without and with metabolic activation.

The acceptance criteria for the assay were fulfilled. The current study was considered as valid.

Under these experimental conditions, the test item induced no mutagenic activity.

Executive summary:

In VitroMAMMALIAN CELL GENE MUTATION TEST

At the TK Locus in L5178Y mouse lymphoma cells

using a Microtiter Cloning Technique

SUMMARY

TEST ITEM                :     N-(2-hydroxypropyl)Oleamide

BATCH NUMBER      :    T22221 without solvent

STUDY LOCATION   :    INSTITUT PASTEUR DE LILLE

                                       Genetic Toxicology Laboratory

                                       1, rue du Professeur Calmette - B.P. 245

                                       59019 LILLE CEDEX FRANCE

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

Study initiation date (date Study Director signed Study Plan): 14/01/2013

Experimental start date (solubility assay): 09/01/2013*

Experimental completion date: 15/04/2013

Study completion: 22/07/2013

*The solubility assay was performed before the finalisation of the Study Plan but the assays began on 14/01/2013 (i.e. after the signature of the study plan).

AIM

The search for any mutagenic activity of N-(2-hydroxypropyl)Oleamide (Batch T22221 without solvent) , was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476.

METHODS

     Cell strain: L5178Y mouse lymphoma cells

     Type of mutation study: TK Locus (Trifluorothymidine Resistance)

     Culture medium: RPMI 1640

     Solvent : ethanol

Stability in solvent: 111 days at ambient temperature according to INERIS’ information under the responsibility of the Sponsor (in all cases, dilutions were prepared extemporaneously)

CYTOTOXICITY ASSAY:

Carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor 1254.

-Treatment duration:

              • Without S9-mix: 3 hours (short treatment)

                                              24 hours (continuous treatment)

              • With S9-mix:3 hours

- Concentrations tested expressed as mM N-(2-hydroxypropyl)Oleamide as supplied: 1.25 – 0.625 – 0.313 – 0.156 – 0.078 – 0.039 – 0.020

- Factor limiting the maximum concentration tested: solubility

Without metabolic activation after a 3-hour treatment, N-(2-hydroxypropyl)Oleamide revealed a potent cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 1.25 to 0.156 mM with no cell growth. The immediately inferior concentration of 0.078 mM induced no cytotoxicity with an adjusted RTG of 95.8%. The concentration of 0.150 mM was thus retained as the maximum concentration for the first mutagenicity test without S9-mix.

With metabolic activation,N-(2-hydroxypropyl)Oleamiderevealed a potent cytotoxicity in L5178Y cells at the 2 highest concentrations tested of 1.25 and 0.625 mM with no cell growth. The immediately inferior concentration of 0.313 mM induced a very strong cytotoxicity with 0.61% of adjusted RTG and the immediately lower concentration of 0.156 mM induced a moderate cytotoxicity with an adjusted RTG of 26.0%. The concentration of 0.3 mM was thus retained as the maximum concentration for the first mutagenicity test without S9-mix.

Without metabolic activation after a 24-hour treatment,N-(2-hydroxypropyl)Oleamiderevealed a potent cytotoxicity in L5178Y cells at the 4 highest concentrations tested from 1.25 to 0.156 mM with no cell growth. The immediately inferior concentration of 0.078 mM induced a strong cytotoxicity with an adjusted RTG of 8.6%. The immediately lower concentration of 0.039 mM induced a slight cytotoxicity with an adjusted RTG of 68.6%. The concentration of 0.08 mM was thus retained as the maximum concentration for the second mutagenicity test without S9-mix.

MUTAGENICITY ASSAYS:

Carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor 1254.

     - Number of assays: 2

     - Number of replicate cultures:2 per concentration

     - Factor limiting the maximum concentration tested: toxicity

- Expression time: 2 days after treatment

     - Treatment duration:

• Without S9-mix: 3 hours (short treatment) and 24 hours (continuous treatment)

• With S9-mix: 3 hours

     - Concentrations tested expressed asmMN-(2-hydroxypropyl)Oleamideas supplied

• Without S9 mix                   :   0.150 – 0.130 – 113 – 0.099 – 0.086 – 0.075 – 0.065 – 0.056(assay 1)

                                                    0.080 – 0.070 – 0.060 – 0.050 – 0.040 – 0.030 – 0.020(assay 2)*

• With S9 mix                        :    0.3 – 0.2 – 0.175 – 0.150 – 0.125 – 0.100 – 0.075(assay 1)

                                                    0.175 – 0.163 – 0.150 – 0.125 – 0.100 – 0.075 (assay 2)

* As a negative response was obtained in the first assay using a 3-h treatment, the second assay was performed following a 24 hour-treatment

     - Positive controls:              

• Without S9-mix: methyl methanesulfonate 10 µg/mL (3-h treatment)

                                 methyl methanesulfonate 2 µg/mL (24-h treatment)

  • With S9-mix: cyclophosphamide, 2 µg/mL

In the second assay using a 3-hour treatment with metabolic activation, astatistically significant increase in the mutation frequency of total induced mutants(small and large colonies)werenoted at the highest analysable concentration of 0.163 mM with a statistically linear trend. However, the induced mutation frequency (IMF) reachedonly 92.8x10^-6mutants, that is to say clearly under the global evaluation factor (GEF) of +126 x10^-6for a biologically significant effect (Mooreet al., 2006).Furthermore, neither clear doseeffect relationship nor reproductibility, were noted (1st assay performed under the same experimental conditions was clearly negative). Otherwise, in the 1st assay without metabolic activation, an increase in the 3-hour treatment (IMF of +95.6 at 0.113 mM) was observed but at a too highly toxic concentration while clear negative results were observed at lower concentrations and after a long-term treatment even at high level of cytotoxicity. CONCLUSION: The search for any mutagenic activity of N-(2-hydroxypropyl)Oleamide (batch T22221 without solvent) , was studied by means of gene mutation test at the TK locus in L5178Y mouse lymphoma cell culture in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 476, in 2 independent assays performed both without and with metabolic activation. The acceptance criteria for the assay were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item induced no mutagenic activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo test available and no need to perform futher tests as the registered substance was negative in the three in vitro tests (OECD 471, 476, 487)

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance is not classified for mutagenicity/genotoxicity.