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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
8 May 1992 to 1 June 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
There is no indication of the duration of the incubation period in Experiment 2. This is not expected to effect the validity of the results. This read-across is based on the hypothesis that source and target substances have similar toxicological properties because of their structural similarities and they are assumed to have similar toxicokinetic profiles i.e. they are expected to be metabolised in a similar fashion. The target substance Epoxidized Palm Oil (EPO) and source substance Epoxidized Soybean Oil (ESBO) are derived respectively from their raw materials, palm oil and soybean oil, and are of variable composition consisting of fatty acid triglycerides. Both EPO and ESBO are organic UVCB sub-type 1: substances of biological nature that have been modified in chemical processing. Both are manufactured by the reaction of the respective oils with an epoxidizing agent (50-60% hydrogen peroxide at 60-75°C). The olefinic bonds of the oils are converted to epoxy oxirane groups. As Palm oil has lower unsaturated bonds than Soybean Oil, less of the epoxidizing agent hydrogen peroxide is required and thus EPO has a lower epoxidized adduct content than ESBO and is therefore expected to be less chemically reactive. The % epoxidation in ESBO is 6-8% while the % epoxidation in EPO is 2.5-3.5%. The target substance (EPO) and source substance (ESBO) have a structurally similar backbone which is an epoxidized triglyceride structure derived from one glycerol molecule and three fatty acid molecules. Therefore, the source and the target substances share structural similarities with common functional groups and side chains varying in their length and the amount of epoxide groups. The target substance contains 8 fatty acids with the largest components being C16:0 palmitic acid (44%), C18:1 oleic acid (39.2%) and C18:2 linoleic acid (10.1%). The source substance contains 5 fatty acids with the largest components being C16:0 palmitic acid (11.3%), C18:1 oleic acid (39.2%) and C18:2 linoleic acid (55.8%). ESBO does not contain lauric (C12), myristic (C14) and arachidic (C20) acids while they are present in very low amounts in EPO (0.2, 1.1 and 0.3% respectively). Stearic acid (C18:0) is present in both substances at similar levels (4.5% in EPO and 3.4% in ESBO) while α-linolenic acid (C18:3) is present at 0.4% in EPO and 6.4% in ESBO. So, the main component of the triglyceride structure of both EPO and ESBO is C16 (44%; 11.3%) and C18 (54.2%; 88.7%). The data gap for the target substance EPO is an in vitro bacterial reverse mutation (Ames test) study (Annex VII, 8.4.1). No reliable data from an in vitro bacterial reverse mutation (Ames test) of EPO is available. Therefore, read-across from an existing in vitro bacterial reverse mutation (Ames test) study of the source substance, ESBO, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.4.1 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: EEC Annex V Test B14
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Epoxidised Soybean Oil
IUPAC Name:
Epoxidised Soybean Oil
Constituent 2
Reference substance name:
ESBO
IUPAC Name:
ESBO
Constituent 3
Reference substance name:
Soybean oil, epoxidized
EC Number:
232-391-0
EC Name:
Soybean oil, epoxidized
Cas Number:
8013-07-8
IUPAC Name:
8013-07-8
Details on test material:
- Name of test material (as cited in study report): Epoxidised Soybean Oil
- Abbreviation: ESBO
- Alias name: Reoplast 39
- Physical state: Clear yellow Liquid
- Lot/batch No.: 08380306
- Expiration date of the lot/batch: July 1994
- Stability under test conditions: Stable
- Storage condition of test material: Dark at room temperature
- Other:
- Colour value (Gardner): 1 - 2
- Refractive Index at 20 °C: 1.4731
- Iodine Value: 4.3 g J2/100g
- Acid Value: 0.27 mg KOH/g
- Oxirane Value: 6.4 %

Method

Target gene:
histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98; TA100; TA 1535; TA1537 and TA102
Additional strain / cell type characteristics:
other: biotin and histidine required for growth
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Please see Table 1 and 2 below.
An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1. For experiment 2 treatments, the dose range was narrowed to 312.5 - 5000 µg/plate in order to investigate those concentrations most likely to exhibit a mutagenic response.
Vehicle / solvent:
Test chemical solutions were prepared by dissolving Epoxidised Soybean Oil in analytical grade acetone, immediately prior to assay to give the required maximum concentration treatment solution. Further dilutions were then made using acetone. The test chemical solutions were protected from light and used within approximately 4 hours of the initial formulation of the test agent.
Controls
Untreated negative controls:
yes
Remarks:
solvent acetone
Positive controls:
yes
Remarks:
Please Table 2 below
Details on test system and experimental conditions:
An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1.

Five strains of bacteria were used in this study. For all assays, bacteria were cultured for about 10 hours at 37 °C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102). Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character and resistance to ampicillan (TA98 and TA100) or ampicillin plus tetracycline (TA102). Checks were carried out according to Maron and Ames and De Serres and Shelby. For all treatments, cultures were used within 2 hours of the end of the incubation period.

Epoxidised Soybean Oil was tested for mutation in 5 strains of Salmonella typhurium at the concentrations detailed in Table 1. Triplicate plates with and without S-9 mix were used. Negative (solvent) and positive controls were included in both assays, in quintuplicate without and with S-9 mix. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S-9.

Because the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step, where the quantities of test chemical or control solution, bacteria and S-9 mix detailed, were mixed together and incubated for 1 hour at 37 °C, before the addition of 2.5 mL molten agar at 46 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Colony Counting:
Colonies were counted electronically using a Seescan Colony Counter or manually where minor agar damage might have interfered with automatic counting, and the background lawn inspected for signs of toxicity.
Evaluation criteria:
Acceptance Criteria:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range as defined in Appendix 4
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9.
iii) no more than 5 % of the plates were lost through contamination or some other unforeseen event

Evaluation criteria:
A test compound was considered to be mutagenic if
i) the assay was valid
ii) Dunnett's test gave a significant response (p <= 0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible
Statistics:
The m-statistic was first calculated to check that the data were Poisson-distributed and then Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was then checked by linear regression analysis.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity:
Only treatments of strain TA102 in Experiment 2 (+ S-9 only) showed signs of toxicity (as indicated by thinning of the background bacterial lawn) in this study. In this case, toxic effects were seen mostly at the 3 highest doses. It would appear that the use of a pre-incubation step particularly enhanced the toxicity of the test agent to this test strain.

Range-finder and Experiment 1 treatments were carried out using final concentrations of Epoxidised SOybean Oil at 8, 40, 200, 1000 and 5000 µg/plate, Precipitation, in the form of oil droplets, was observed at concentrations of 1000 and 5000 µg/plate. For experiment 2, testing was again carried out up to maximum concentration of 5000 µg/plate (despite the observation of precipitation), as it was possible that the compund formed an emulsion within the test system and it was felt important to maximise the exposure of the cells to this. A narrowed dose range was also used in this experiment (312.5 - 5000 µg/plate) in order to examine those doses most likely to exhibit a mutagenic response. Oil droplets were observed on test plates in this experiment, following treatments of 1250 µg/plate and above.

Mutation:
The individual plate counts were averaged to give mean values. From the data it can be seen that mean solvent control counts fell within the normal historical range, that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that < 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

The mutation data were evaluated as follows:
No treatment with Epoxidised Soybean Oil of any of the tester strains, earlier in the absence or presence of S-9, resulted in a significant increase in revertant numbers. The data obtained therefore gave no indication of an ability of the test agent to induce mutation.
Remarks on result:
other: strain/cell type: S. typhimurium TA 102
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Read-Across Justification: Full report is attached in study summary

3 Analogue approach justification

3.1. Physicochemical properties

Physicochemical data shows that the physicochemical properties of the target and source substances are similar as outlined in the data matrix (Table 3). Both are liquids and the structural differences in the side chains do not significantly influence the physicochemical properties of both substances, i.e. vapour pressure, water solubility and partition co-efficient (log Pow). Both substances are highly insoluble in water; <0.01 mg/L at 30°C for EPO and <0.02 µg/L at 20°C (calculated) for ESBO. Neither of the substances is volatile, with a vapour pressure of 0.5 kPa at 25°C for EPO and 8.4 x 10-8 Pa at 25°C for ESBO. Both substances are highly lipophilic; mean Log Pow >6.2 at 25°C for ESBO and log Pow >10 (calculated) for EPO.

3.2. Toxicokinetics

No specific experimental data on absorption, distribution, metabolism or excretion is available for the source or target substance. Read-across was performed for all human health toxicity endpoints to Epoxidised Soybean Oil (ESBO, CAS No. 8013-07-8). An OECD SIDS report is available that concluded on a proposed metabolic pathway for epoxidised fatty acid esters, including ESBO (OECD, 2006). The toxicokinetic analysis is based on physicochemical data from EPO, read-across ESBO data from in vivo animal models and the OECD SIDS report in the literature (OECD, 2006).

Physicochemical data

The molecular weight of EPO and ESBO is > 500 g/mol and is not in the range for favourable oral absorption (<500 g/mol). The calculated log Pow of EPO (>10) and mean Log Pow >6.2 at 25°C for ESBO indicate they are highly lipophilic and water solubility (<0.01 mg/L at 30°C) for EPO and <0.02 µg/L at 20°C (calculated) for ESBO indicates they are both insoluble in water. These characteristics will not facilitate transport of EPO or ESBO via passive diffusion. Based on its high lipophilicity, absorption of EPO and ESBO via the lymphatic system through micellular solubilisation by bile salts is likely, similar to other vegetable oils. Insolubility in water of both EPO and ESBO indicates low dermal uptake while the high log Pow values for both are an indication for a high uptake into the stratum corneum but little or no penetration into the lower layers of the epidermis and dermis. Overall, the physical state, molecular weight, calculated log Pow and water insolubility indicate that dermal absorption of EPO and ESBO is unlikely. Due to the low vapour pressure of EPO (0.5 kPa at 25°C) and ESBO (8.4 x 10-8 Pa at 25°C) and physical state (liquid), exposure via the inhalation route of both is expected to be negligible. Based on the information available for the analogue ESBO in the OECD SIDS report (OECD, 2006; see ‘Other data in the literature’), during metabolism, breakdown products are produced that are more water soluble than the parent substance i.e. free fatty acids, so it is expected that any EPO metabolites will be excreted in the urine.

Other data in the literature

The OECD produced a report on Epoxidised Oils and Derivatives in 2006 (OECD, 2006), which included ESBO. The OECD SIDS concluded that epoxidised fatty acid esters, such as ESBO and therefore we assume EPO, produce metabolic products with similar primary constituents as other vegetable oils and are assumed to have similar metabolic pathways e.g. breakdown in the gastrointestinal tract by esterases (pancreatic lipase) to epoxidised fatty acids and glycerol which enter the normal nutritional pools (JECFA, 1974). Pancreatic lipase works at the oil/water interface since triglycerides are insoluble. During metabolism in the GI tract, pancreatic lipase preferentially hydrolyses triglycerides to release the free fatty acids from the SN-1 and SN-3 (terminal) positions of the glycerol backbone. The other products of metabolism are mono- and di-glycerides (OECD, 2006). The EFSA Panel on Contaminants in the Food Chain agreed with this assessment for ESBO in 2011 (EFSA, 2011). Overall, the proposed metabolic pathway for ESBO is enzymatic breakdown to epoxidised fatty acids and glycerol; a similar pathway is predicted for EPO. Based on the information available for the analogue ESBO in the OECD SIDS report, during metabolism, breakdown products are produced that are more water soluble than the parent substance i.e. free fatty acids, so it is expected that any EPO metabolites will be excreted in the urine.

Available in vivo toxicological data

The in vivo read-across data from ESBO indicate no adverse effects if oral absorption occurs (acute oral LD50 of >5,000 mg/kg (3), 2 year combined chronic/carcinogenicity toxicity study

NOEL (male) of 1000 mg/kg bw/day and NOEL (female) of 1400 mg/kg bw/day (13), pre-natal developmental toxicity maternal/developmental NOAEL of 1000 mg/kg bw/day (14). The in vivo read-across data from ESBO indicates is poorly absorbed via the dermal route (slightly irritating in the in vivo skin irritation study in rabbits (6) and non-sensitising in Guinea pig maximization test (8)). Any significant dermal absorption is unlikely.

3.3. Comparison of data from human health endpoints

3.3.1 Toxicity data of the target and source substances

There is no existing human health toxicity data for the target substance, EPO. As is presented in the data matrix (Table 3), the acute oral (LD 50 (male/female) >5,000 mg/kg bw) and acute dermal toxicity data (LD50 >20mL/kg bw) shows very low toxicity for the source chemical, ESBO, in rats and rabbits. The source chemical is slightly irritating to skin and eye in rabbits. The source substance is not a skin sensitizer in the guinea pig maximization test. In the in vitro chromosomal aberration study and in vitro gene mutation study in mammalian cells, the source substance ESBO was negative in the presence and absence of metabolic activation. The source substance ESBO is not genotoxic. In a combined chronic toxicity/carcinogenicity study in rats for 104 weeks, a NOEL value of 1000 mg/kg bw/day (male) and 1400 mg/kg bw/day (female) was derived. In a pre-natal developmental toxicity study in rats, the NOAEL (maternotoxic, embryofetal) was 1000 mg/kg bw/day with no adverse effects noted. In accordance with Column 2 of ANNEX IX of the REACH Regulation, a two generation reproductive toxicity study does not need not to be conducted as the existing read across combined chronic toxicity/carcinogenicity study from ESBO is available and does not indicate clear adverse effects on reproductive organs or tissues.

The data gap for the target substance EPO is an in vitro bacterial reverse mutation (Ames test) study (Annex VII, 8.4.1). No reliable data from an in vitro bacterial reverse mutation (Ames test) of EPO is available. Therefore, read-across from an existing in vitro bacterial reverse mutation (Ames test) study of the source substance, ESBO, is considered as an appropriate adaptation to the standard information requirements of Annex VII, 8.4.1 of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Two read-across in vitro bacterial reverse mutation studies (Ames test) are available. The key read-across study (RL2) was conducted according to OECD 471 and GLP. In this study, strains of S. typhimurium TA98, TA100, TA 1535, TA1537 and TA102 were exposed to ESBO in acetone in experiment 1 (plate incorporation) at concentrations 8, 40, 200, 1000 and 5000 µg/plate (derived from initial toxicity range-finder with TA100) and in experiment 2 (pre-incubation) at concentrations of 312.5 - 5000 µg/plate. Both experiments were carried out in the presence and absence of S9 mammalian metabolic activation. Appropriate positive controls were used for each strain and solvent negative controls were used. ESBO was tested up to limit concentration (5000 µg/plate).

Only treatments of S. typhimurium TA102 in Experiment 2 (+S9 only) showed signs of toxicity (as indicated by thinning of the background bacterial lawn) in this study. In this case, toxic effects were seen mostly at the 3 highest doses. It would appear that the use of a pre-incubation step particularly enhanced the toxicity of the test agent to this test strain. Range-finder and Experiment 1 treatments were carried out using final concentrations of ESBO at 8, 40, 200, 1000 and 5000 µg/plate, Precipitation, in the form of oil droplets, was observed at concentrations of 1000 and 5000 µg/plate. For experiment 2, testing was again carried out up to maximum concentration of 5000 µg/plate (despite the observation of precipitation), as it was possible that the compound formed an emulsion within the test system and it was felt important to maximise the exposure of the cells to this. A narrowed dose range was also used in this experiment (312.5 - 5000 µg/plate) in order to examine those doses most likely to exhibit a mutagenic response. Oil droplets were observed on test plates in this experiment, following treatments of 1250 µg/plate and above. The mean solvent control counts fell within the normal historical range, and the positive control chemicals all induced large increases in revertant numbers in the appropriate strains. None of the treatments with ESBO on the tester strains, either in the absence or presence of S9, resulted in a significant increase in revertant numbers. Therefore ESBO was negative in the presence and absence of metabolic activation in the in vitro bacterial reverse mutation (Ames test) key study. EPO is also predicted to be negative in this study.

The supporting read-across study (RL2) was conducted according to the Ames method. In this study, strains of S. typhimurium TA98, TA100, TA 1535, TA1537 were exposed to ESBO in acetone at concentrations of 25, 75, 225, 675 and 2025 µg/0.1 mL. At the concentrations of 675 and 2025 µg/0.1 mL the substance precipitated in soft agar. The experiment was carried out in the presence and absence of rat liver microsome and co-factors metabolic activation. Appropriate positive controls were used for each strain and solvent negative controls were used. ESBO was tested up to precipitating concentrations. There was no evidence of induced mutant colonies over background. Therefore ESBO was negative in the presence and absence of metabolic activation in the in vitro bacterial reverse mutation (Ames test) supporting study. EPO is also predicted to be negative in this study.

3.3.2 Effect of structural differences between target and source chemical

The target substance consists of 8 fatty acids with the largest components being C16:0 palmitic acid (44%), C18:1 oleic acid (39.2%) and C18:2 linoleic acid (10.1%). The source substance consists of 5 fatty acids with the largest components being C16:0 palmitic acid (11.3%), C18:1 oleic acid (39.2%) and C18:2 linoleic acid (55.8%). ESBO does not contain lauric, myristic and arachidic acids while they are present in very low amounts in EPO (0.2, 1.1 and 0.3% respectively). Stearic acid is present in both substances at similar levels (4.5% in EPO and 3.4% in ESBO) while α-linolenic acid is present at 0.4% in EPO and 6.4% in ESBO. So, the main component of the triglyceride structure of both EPO and ESBO is C16 (44%; 11.3%) and C18 (54.2%; 88.7%). When these epoxidized fatty acid products are released during metabolism (see Section 3.2) they are not expected to have any adverse effects in the body as all are naturally occurring fatty acids and they are all ‘Not Classified’ in the ECHA Classification and Labelling (C&L) inventory (checked 24-08-15). The only exception is α-linolenic acid which is indicated as a skin sensitizer but this is not relevant to ESBO and EPO as the former is negative in the guinea pig maximization test and EPO is also predicted to be negative also. The main structural difference is that palm oil has lower unsaturated bonds than soybean oil therefore less of the epoxidizing agent hydrogen peroxide is required to produce the epoxidized derivatives. EPO has a lower epoxidized adduct content than ESBO and is therefore expected to be less chemically reactive. The % epoxidation in ESBO is 6-8% while the % epoxidation in EPO is 2.5-3.5%.

3.3.3 Classification and labelling

According to the ECHA Classification and Labelling (C&L) inventory, the source substance, ESBO, is ‘Not Classified’ (647 notifiers, joint entry; checked 24-08-15). The target substance, EPO, is not listed in the C&L inventory (checked 24-08-15). EPO is predicted to be negative based on the results from the ESBO read-across in vitro cytogenicity study in mammalian cells and the ESBO read-across in vitro gene mutation study in mammalian cells. Together with the predicted result from this read-across in vitro bacterial reverse mutation (Ames test), EPO does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.

4. Conclusion

The structural similarities between the source and the target substances and estimated similar toxicokinetics presented above support the read-across hypothesis. The structural differences between the target and source substance are not expected to have an impact on the prediction. Adequate, reliable and available scientific information indicates that using the source substance for read across to the target substance is acceptable.

Therefore, based on the considerations above, it can be concluded that the in vitro bacterial reverse mutation study conducted with ESBO is likely to predict the in vitro bacterial reverse mutation effects of EPO and is considered as adequate to fulfill the information requirement of Annex VII, 8.4.1.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system.
According to Directive 67/548/EEC, no classification is warranted.
According to Regulation (EC) No. 1272/2008, no classification is warranted.
Executive summary:

Epoxidised Soyban Oil was assayed for mutation in 5 -histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

It was concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system. According to Directive 67/548/EEC, no classification is warranted. According to Regulation (EC) No. 1272/2008, no classification is warranted.