Initial in vitro screening approach to investigate the potential health and environmental hazards of Envirox™ – a nanoparticulate cerium oxide diesel fuel additive

1743-8977-4-12 1743-8977 Research Initial in vitro screening approach to investigate the potential health and environmental hazards of Envirox™ – a nanoparticulate cerium oxide diesel fuel additive Park Barry barry.park@oxonica.com Martin Patricia parc.pamartin@talktalk.net Harris Chris chris.harris@oxonica.com Guest Robert rguest@safepharm.co.uk Whittingham Andrew awhittingham@safepharm.co.uk Jenkinson Peter pjenkinson@safepharm.co.uk Handley John jhandley@safepharm.co.uk

Oxonica, 7 Begbroke Science Park, Sandy Lane, Yarnton, Oxfordshire, UK

Product Assessment & Regulatory Compliance, Sydney Road, Cradley Heath, West Midlands, UK

SafePharm Laboratories Ltd, London Road, Shardlow, Derby, UK

Particle and Fibre Toxicology 1743-8977 2007 4 1 12 http://www.particleandfibretoxicology.com/content/4/1/12 18053256 10.1186/1743-8977-4-12 20 3 2007 05 12 2007 05 12 2007 2007 Park et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Nanotechnology is the new industrial revolution of the 21st Century as the various processes lead to radical improvements in medicine, manufacturing, energy production, land remediation, information technology and many other everyday products and applications. With this revolution however, there are undoubted concerns for health, safety and the environment which arise from the unique nature of materials and processes at the nanometre scale.

The in vitro assays used in the screening strategy are all validated, internationally accepted protocols and provide a useful indication of potential toxicity of a chemical as a result of effects on various toxicological endpoints such as local site of contact (dermal) irritation, general cytotoxicity and mutagenicity.

The initial in vitro screening strategy described in this paper to investigate the potential health implications, if any, which may arise following exposure to one specific application of nanoparticulate cerium oxide used as a diesel fuel borne catalyst, reflects a precautionary approach and the results will inform judgement on how best to proceed to ensure safe use.

Background

Nanotechnology is the production and application of structures, devices and systems by controlling shape and size at the nanometre scale (1 nanometre = 1 × 10^-6mm). The range of structures covered by the term nanotechnology is also extensive and encompasses existing materials with at least one dimension of < 100 nm and includes nanoparticulates of various metals and their oxides, as well as more diverse engineered nanomaterials such as carbon nanotubes (CNT) and fullerenes.

The downstream applications of nanotechnology are equally diverse, from electronics and engineering, medicinal/medical diagnostic devices, remediation of environmental pollution, personal care products and also food/beverages as well as information technology. The general public is already exposed to a variety of consumer products containing nanoparticulates e.g. titanium dioxide in paints, and cosmetic products, e.g. sunscreens containing zinc oxide and/or titanium dioxide.

Envirox™ is a cerium oxide based fuel borne catalyst and supplied as a dispersion of 2% w/v cerium oxide in a hydrocarbon carrier (which is wholly compatible with diesel). This is diluted with diesel for delivery to the vehicle at a ratio of 1:4000, yielding a final cerium oxide concentration in the vehicle diesel fuel of 5 ppm. The efficacy of Envirox™ is primarily due to the vastly increased surface area of the nanoparticulate cerium oxide. Once in the combustion chamber nanoparticulate cerium oxide works to modify the combustion of diesel fuel while extending and improving fuel burn. In addition the inclusion of nanoparticulate cerium oxide reduces the temperature at which carbon combusts. This feature provides a secondary benefit to the performance of a diesel fuel engine, as it facilitates the removal of hard carbon deposits and soot, which further increases fuel efficiency and potentially reduces wear in the engine and, most importantly, results in reduced particulate emissions from the exhaust.

Despite the fact that humans have been exposed to nano-sized particles throughout their evolution from natural/anthropogenic sources, it is the proliferation of the new (nano)materials which has prompted concerns to be raised 1 over human health and environmental safety following their suspected inevitable release into the environment at some stage of the 'product application' lifecycle. These concerns are intrinsically inter-linked with the extremely small size of these particles. There is an inverse relationship between particle size and surface area, i.e. the smaller the particle the larger the surface area and the greater the proportion of atoms or molecules which are distributed on the surface rather than the interior of the material. These surface changes may lead to an increase in the number of 'reactive sites' on the particle which in turn may lead to an increase in (eco)-toxicological effects 23.

In 2003 the UK Government commissioned a review, conducted by the Royal Society and the Royal Academy of Engineering 4, into the likely developments in nanotechnology and an examination of whether this would raise new health and safety, environmental, social or ethical issues which might lead to a requirement for new controlling legislation. This report, together with others 56, identified a dearth of eco/toxicological hazard information on nanomaterials that needed to be supplemented prior to better understanding the potential risks, if any, posed by manufactured nanoparticulates.

Recent publications 789 have begun the process required to address the development of sound scientific strategies for the safety evaluation, or hazard identification and thence risk assessment of nanomaterials. The above publications agree that any robust approach to hazard identification for nanomaterials may be predicated upon the following three elements:

- physical-chemical characterisation

- in vitro assays, and

- in vivo studies.

Robust characterisation of each test material is an essential prerequisite of any investigation to ensure that comparison of results between laboratories is based on sound science. This characterisation may include measurement of some, or all, of the following where appropriate: particle size and distribution, shape, agglomeration state, crystal structure, chemical composition, porosity, surface area, surface charge and surface chemistry.

In vitro assays of the test material in any given matrix allow the examination of specific biological response(s) and/or mechanism of action under controlled conditions which are not easily studied in complex in vivo situations. The in vitro assays used in the current preliminary human safety assessment were employed to investigate local toxicological effects (skin irritation), any immediate effects on the structure, function or pathology of cells (cytotoxicological effects) and whether there was any initial adverse effect on the cell's genetic material (Ames test) using a variety of readily available and well-characterised cells in culture. Additional tests were conducted to address the potential for adverse ecotoxicological and environmental fate effects. The results of all these initial tests will be used to inform the stepwise development of a comprehensive testing and safety assessment strategy for nanoparticulate cerium oxide.

The approach outlined in this paper describes an initial in vitro preliminary screening strategy to examine the potential for human health hazards following exposure to nanoparticulate cerium oxide. All of the tests conducted have included non-nanoparticulate cerium oxide as a reference material for comparison with nano cerium oxide. The work was carried out within the departments for Alternative Testing and Genetic Toxicology at SafePharm Laboratories Ltd (SPL), Derby UK and is part of a broader investigation of potential health effects.

Results

Characterisation of test materials

Data generated from a series of tests to characterise the two forms of cerium oxide used as test materials in the subsequent assessments of toxicological potential are shown in Table 1.

Table 1

Physico-chemical characteristics of the test materials

Test

Property

Nano

Non-Nano

XRD

Crystal Form

Cerianite

EDX

Gross Elemental Analysis

Ce, O

BET

Surface Area

94.7 m²/g

2.64 m²/g

Mean Particle Size

9 nm

320 nm

XPS

Surface Chemistry

Ce, O

Transmission Electron Microscopy (TEM) photographs of each of the nanoparticulate and non-nanoparticulate cerium oxide samples are shown in Figures 1 and 2. There are significant differences in particle size in the two test material samples and this is clearly illustrated in the photographs.

Figure 1

Nanoparticulate cerium oxide

Nanoparticulate cerium oxide.

Figure 2

Non-nanoparticulate cerium oxide

Non-nanoparticulate cerium oxide.

Epiderm™ skin model

Table 2 illustrates the mean OD₅₄₀and mean viability for tissues exposed to the test materials. The nano-cerium oxide and the non-nano cerium oxide test materials did not directly reduce MTT.

Table 2

Results of the Epiderm™ skin model study

Test Materials

Exposure time (mins.)

Mean OD₅₄₀

% Viability

ET₅₀mins.

MIP

NEGATIVE CONTROL

960

1.685

100

1440

NANO-CeO₂

960

2.010

119.29

1517.18

<0.01

1200

1.138

67.54

1440

1.089

64.63

NON-NANO CeO₂

960

1.994

92.87

> 1440

0.03

1200

1.975

97.36

1440

1.729

85.23

POSITIVE CONTROL 1% TX₁₀₀

240

1.035

61.42

260.10

<0.18

360

0.212

12.58

480

0.095

5.64

REFERENCE MATERIAL 20% SLS

0.983

58.34

20.68

1.00

0.710

42.14

0.273

16.20

120

0.100

5.93

The ET₅₀values for the nano cerium oxide, 1% (w/v) Triton X-100 the positive control and 1% of 20% (w/v) SLS a standard reference material were 1517.18, 260.10 and 20.68 minutes respectively. The non-nano cerium oxide was assayed at a later time than the nano cerium oxide material and the corresponding ET₅₀values for non-nano cerium oxide, the positive control and the reference material were > 1440, 410.60 and 46.22 minutes respectively.

Using the equation below the Mean Irritation Potential (MIP) for both the nano- and non-nano cerium oxide test materials was calculated and determined as 0.01 for nano cerium oxide and 0.03 for non-nano cerium oxide.

MIP calculation equation : MIP = ET 50 ( reference material ) ET 50 ( test material ) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacPC6xNi=xI8qiVKYPFjYdHaVhbbf9v8qqaqFr0xc9vqFj0dXdbba91qpepeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGacaGaaiaabeqaaeqabiWaaaGcbaGaeeyta0KaeeysaKKaeeiuaaLaeeiiaaIaee4yamMaeeyyaeMaeeiBaWMaee4yamMaeeyDauNaeeiBaWMaeeyyaeMaeeiDaqNaeeyAaKMaee4Ba8MaeeOBa4MaeeiiaaIaeeyzauMaeeyCaeNaeeyDauNaeeyyaeMaeeiDaqNaeeyAaKMaee4Ba8MaeeOBa4MaeiOoaOJaeeyta0KaeeysaKKaeeiuaaLaeyypa0tcfa4aaSaaaeaacqqGfbqrcqqGubavdaWgaaqaaiabiwda1iabicdaWaqabaGaeeiiaaIaeiikaGIaeeOCaiNaeeyzauMaeeOzayMaeeyzauMaeeOCaiNaeeyzauMaeeOBa4Maee4yamMaeeyzauMaeeiiaaIaeeyBa0MaeeyyaeMaeeiDaqNaeeyzauMaeeOCaiNaeeyAaKMaeeyyaeMaeeiBaWMaeiykaKcabaGaeeyrauKaeeivaq1aaSbaaeaacqaI1aqncqaIWaamaeqaaiabbccaGiabcIcaOiabbsha0jabbwgaLjabbohaZjabbsha0jabbccaGiabb2gaTjabbggaHjabbsha0jabbwgaLjabbkhaYjabbMgaPjabbggaHjabbYgaSjabcMcaPaaaaaa@8773@

Since both nano and non-nano cerium oxide test materials had MIP values which were < 0.8 neither was considered to have the potential to be an in vivo skin irritant 10.

Based upon a recent statement from the European Centre for the Validation of Alternative Methods (ECVAM) the Epiderm model reliably identifies skin irritants 11 but negative results may require further testing in line with current guidance for this endpoint as described in the OECD TG 404.

Cytotoxicity assay

The mean cytotoxicity/reactivity grades, related to the concentration and toxicity of soluble components diffusing from the test sample, are illustrated in Table 3. The negative control (polypropylene pipette tips) had a cytotoxicity grade of 0 and the positive control (tin-impregnated PVC strips) showed evidence of a moderate cytotoxicity with a grade 3 score; thus indicating that the assay system is reliable. Both non-nano particulate cerium oxide and nanoparticulate cerium oxide showed no evidence of cytotoxicity and each had a cytotoxicity grade of 0.

Table 3

Results of the cytotoxicity testing

TEST MATERIAL

MEAN CYTOTOXICITY/REACTIVITY GRADE

Culture medium (Control)

Negative control (polypropylene pipette tips)

Positive control (tin-impregnated PVC strips)

'Non-nano' particulate CeO₂

Nano-particulate CeO₂

Ames test

Neither of the two cerium oxide particulate samples (non-nano cerium oxide and nano-cerium oxide) were toxic to Salmonella strain TA100 when tested in a preliminary toxicity assay and nor did either of the two test materials cause any visible reduction in the growth of the bacterial background lawn at any dose level. Therefore the two cerium oxide particulate test materials were tested up to the maximum recommended dose level of 5000 μg/plate. Non-nano cerium oxide precipitation was not observed on the plates at any of the doses tested in either the presence or absence of S9-mix. A cream-coloured film was observed at 1500 μg/plate and above with an associated precipitate^{at 5000 μg/plate in the case of the nanoparticulate cerium oxide tested material (the observation of a precipitate of a test sample is a routine means of ensuring that the test has been performed adequately). These observations did not, however, prevent the scoring of revertant colonies and confirmed that the samples were tested up to a maximal dose level.}

There were no significant increases in the frequency of revertant colonies recorded for any of the strains of Salmonella, at any dose level, either with or without metabolic activation, for either the non-nano cerium oxide or nanoparticulate cerium oxide (Table 4, 5, 6, 7, 8, 9). All of the positive control chemicals used in the study induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table 4

Results of bacterial gene point mutation assay. Cerium oxide – non-nanoparticulate. Summary of mean revertant colonies obtained

Experiment 1

Substance

Dose Level (μg/plate)

TA100

TA1535

TA102

TA98

TA1537

Mean ± S.D.

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

DMSO

100 μl

83 ± 7

64 ± 5

40 ± 2

12 ± 2

309 ± 24

333 ± 29

21 ± 5

26 ± 4

10 ± 2

17 ± 3

Cerium Oxide Non-nano

85 ± 12

73 ± 11

37 ± 5

13 ± 1

333 ± 28

361 ± 29

19 ± 4

29 ± 2

12 ± 2

19 ± 6

150

96 ± 7

70 ± 9

34 ± 3

11 ± 2

341 ± 21

359 ± 7

19 ± 3

29 ± 3

13 ± 3

18 ± 3

500

80 ± 12

76 ± 10

35 ± 7

11 ± 2

345 ± 9

367 ± 11

22 ± 7

29 ± 6

13 ± 3

20 ± 4

1500

69 ± 10

75 ± 11

34 ± 2

13 ± 6

338 ± 10

350 ± 26

16 ± 2

25 ± 3

12 ± 5

21 ± 5

5000

84 ± 7

73 ± 9

36 ± 3

10 ± 2

313 ± 21

354 ± 14

16 ± 4

31 ± 4

11 ± 1

22 ± 1

Table 5

Results of bacterial gene point mutation assay. Cerium oxide – non-nanoparticulate. Summary of mean revertant colonies obtained

Experiment 2

Substance

Dose Level (μg/plate)

TA100

TA1535

TA102

TA98

TA1537

Mean ± S.D.

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

DMSO

100 μl

93 ± 16

105 ± 13

27 ± 7

9 ± 1

345 ± 17

374 ± 13

20 ± 3

27 ± 7

8 ± 3

13 ± 4

Cerium Oxide Non-nano

87 ± 10

115 ± 10

32 ± 2

11 ± 7

349 ± 42

352 ± 31

16 ± 7

23 ± 2

15 ± 4

19 ± 4

150

95 ± 4

91 ± 8

28 ± 7

14 ± 2

343 ± 20

397 ± 21

20 ± 3

23 ± 2

8 ± 4

19 ± 5

500

94 ± 13

89 ± 4

37 ± 5

9 ± 1

349 ± 27

364 ± 23

18 ± 2

29 ± 3

9 ± 1

17 ± 3

1500

82 ± 2

99 ± 2

33 ± 12

9 ± 3

351 ± 22

380 ± 10

17 ± 2

28 ± 4

8 ± 4

21 ± 6

5000

107 ± 2

97 ± 13

26 ± 4

10 ± 2

350 ± 49

382 ± 21

18 ± 4

23 ± 6

15 ± 5

19 ± 3

S.D. Standard deviation

Table 6

Results of bacterial gene point mutation assay. Cerium oxide - nanoparticulate. Summary of mean revertant colonies obtained

Experiment 1

TA100

TA1535

TA102

TA98

TA1537

Substance

Dose Level (μg/plate)

Mean ± S.D.

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

DMSO

100 μl

118 ± 7

97 ± 13

39 ± 3

32 ± 6

347 ± 29

351 ± 4

28 ± 3

36 ± 7

13 ± 5

22 ± 2

Cerium Oxide Nano

100 ± 3

109 ± 17

37 ± 2

29 ± 13

356 ± 10

363 ± 25

23 ± 5

36 ± 2

11 ± 5

18 ± 4

150

88 ± 13

115 ± 9

40 ± 3

36 ± 1

360 ± 13

381 ± 20

25 ± 6

35 ± 6

12 ± 5

15 ± 5

500

84 ± 16

114 ± 20

33 ± 3

36 ± 3

377 ± 12

350 ± 17

26 ± 6

35 ± 7

14 ± 5

17 ± 4

1500 (F)

101 ± 13

115 ± 8

28 ± 4

32 ± 9

343 ± 38

330 ± 17

22 ± 3

35 ± 1

14 ± 4

24 ± 2

5000 (F+M+P)

101 ± 4

113 ± 7

39 ± 3

38 ± 2

343 ± 16

332 ± 14

23 ± 4

37 ± 4

12 ± 2

20 ± 5

Table 7

Results of bacterial gene point mutation assay. Cerium oxide - nanoparticulate. Summary of mean revertant colonies obtained

Experiment 2

Substance

Dose Level (μg/plate)

TA100

TA1535

TA102

TA98

TA1537

Mean ± S.D.

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

DMSO

100 μl

97 ± 14

132 ± 8

28 ± 6

30 ± 11

394 ± 9

380 ± 25

21 ± 5

38 ± 7

12 ± 10

15 ± 6

Cerium Oxide Nano

99 ± 19

123 ± 17

32 ± 2

29 ± 5

359 ± 28

397 ± 39

21 ± 4

33 ± 10

12 ± 1

13 ± 6

150

98 ± 20

127 ± 10

35 ± 6

40 ± 6

361 ± 32

391 ± 32

22 ± 4

32 ± 2

14 ± 7

12 ± 4

500

84 ± 6

126 ± 11

35 ± 11

32 ± 4

382 ± 29

350 ± 7

21 ± 4

32 ± 5

14 ± 4

16 ± 4

1500 (F)

92 ± 8

133 ± 7

36 ± 8

36 ± 7

361 ± 43

313 ± 29

19 ± 5

37 ± 4

9 ± 3

12 ± 3

5000 (F+P+M)

95 ± 3

121 ± 22

28 ± 5

29 ± 3

371 ± 24

353 ± 36

19 ± 3

26 ± 3

9 ± 1

17 ± 2

S.D. Standard deviation

F Film

P Precipitate

M Manual Count

Table 8

Results of bacterial gene point mutation assay. Positive control data.

Experiment 1

TA100

TA1535

TA102

TA98

TA1537

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

Compound

ENNG

2AA

ENNG

2AA

MMC

DAN

4NQO

9AA

2AA

Dose Level

3 μg

1 μg

5 μg

2 μg

0.5 μg

10 μg

0.2 μg

5 μg

80 μg

2 μg

Mean ± S.D.

716 ± 35

669 ± 23

731 ± 71

212 ± 4

1338 ± 103

717 ± 38

238 ± 17

213 ± 8

789 ± 79

305 ± 7

Table 9

Results of bacterial gene point mutation assay. Positive control data.

Experiment 2

TA100

TA1535

TA102

TA98

TA1537

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

(-S9)

(+S9)

Compound

ENNG

2AA

ENNG

2AA

MMC

DAN

4NQO

9AA

2AA

Dose Level

3 μg

1 μg

5 μg

2 μg

0.5 μg

10 μg

0.2 μg

5 μg

80 μg

2 μg

Mean ± S.D.

687 ± 88

1055 ± 181

706 ± 38

304 ± 49

1370 ± 86

832 ± 53

247 ± 8

353 ± 84

1909 ± 192

207 ± 44

S.D. Standard deviation

ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine

MMC Mitomycin C

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

2AA 2-Aminoanthracene

DAN 1,8-dihydroxyanthraquinone

BP Benzo-a-Pyrene

Daphnia magna immobilisation study

There were no toxic effects observed i.e. no immobilisation occurred at any time period during this test.

The 48-hour EC₅₀for nano and non-nano cerium oxide to Daphnia magna magna based on nominal test concentrations was greater than 100% (v/v) saturated solution (see Table 10) and correspondingly the No Observed Effect Concentration (NOEC) was 100% (v/v) saturated solution.

Table 10

Cumulative immobilisation data in definitive Daphnia test

Nominal concentration (% v/v saturated solution)

Cumulative immobilised Daphnia (initial population: 10 per replicate)

24-hour

48-hour

Number per replicate

Total

Number per replicate

Total

Control:

100% Nano cerium oxide:

100% Non nano cerium oxide:

R₁

R²

R₁

R₂

Positive control (mg/l) (potassium dichromate):

0.32

0.56

1.0

100

R1 – R4 = Replicates 1 to 4

Activated sludge respiration inhibition study

The 3-hour EC₅₀for inhibition of respiration of activated sewage sludge bacteria for both nano cerium oxide and non-nano cerium oxide test materials was greater than 1000 mg/l (see Table 11). There was no effect on the respiration of activated sewage sludge following a 3-hour exposure to either of the test materials at 1000 mg/l i.e. No Observed Effect Concentration (NOEC) = 1000 mg/l.

Table 11

Oxygen consumption rates and percentage inhibition values in the definitive ASRIT test after 3-h contact time

Nominal concentration (mg/l)

Initial O₂reading (mg O₂/l)

Measurement period (minutes)

Final O₂reading (mg O₂/l)

O₂consumption rates (mg O₂/l/min)

% inhibition

Control:

6.9

2.9

0.40

6.6

2.4

0.42

6.9

2.8

0.41

6.3

3.0

0.41

Nano cerium oxide:

1000 R1

6.7

2.5

0.42

[2]

1000 R2

6.8

2.8

0.40

1000 R3

6.5

2.9

0.40

Non-nano cerium oxide:

1000 R1

6.7