N-(2-hydroxypropyl)oleamide

Genetic toxicity: in vitro

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)oleamide

EC Number:

203-828-2

EC Name:

N-(2-hydroxypropyl)oleamide

Cas Number:

111-05-7

Molecular formula:

C21H41NO2

IUPAC Name:

N-(2-hydroxypropyl)oleamide

Details on test material:

- Name of test material (as cited in study report): N-(2-hydroxypropyl)Oleamide
- Substance type: chemical
- Physical state: beige waxy solid
- Analytical purity: 100 % dry matter
- Lot/batch No.: T22221 without solvent
- Expiration date of the lot/batch: 29/05/2014 (retest date)
- Stability under test conditions: unknown
- Storage condition of test material: at room temperature

Method

Target gene:

Histidine

Species / strain

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.

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.