Correlation of chemical acute toxicity between the nematode and the rodent

Abstract

The utility of any non-rodent model system for chemical toxicity screening depends on the level of correlation between its responses and toxic reactions in rodents. Toxicity assays in the nematode Caenorhabditis elegans (C. elegans) can be fast and inexpensive; however few studies have been performed comparing toxic responses in the nematode with data on acute rodent toxicity. We assayed the acute toxicity of 21 types of chemicals in different toxicity categories using C. elegans. The nematodes were exposed to different concentrations of chemicals in 96-well plate for 24 h. The lethality rate was observed at 2, 4, 12 and 24 h, and median lethal concentration (LC₅₀) was calculated by the Probit method. The lethality rate was counted at 1, 8, 16 and 20 h additionally at the concentrations of 10.000, 21.500 and 46.400 mg ml⁻¹ to acquire median lethal times (LT₅₀). The results indicated that the chemical pH could affect the C. elegans LC₅₀ value. The pH toleration range for C. elegans was more than 2.75. Excluding 4 types of acidic chemicals, there were positive correlations between LC₅₀s of C. elegans and LD₅₀s of mouse/rat (r > 0.72, p < 0.01) after both 12 h and 24 h exposure. As to the LC₅₀ data following a 24 h exposure in C. elegans, the correlation of C. elegans LC₅₀s vs. rat LD₅₀s (r = 0.885) was greater than the correlation of mouse vs. rat LD₅₀s (r = 0.879), while the correlation of C. elegans LC₅₀s vs. mouse LD₅₀s (r = 0.741) was lower relative to that of mouse vs. rat LD₅₀s. The data were further compared with an in vitro cytotoxicity model utilizing human epidermal keratinocytes (NHK). The data indicate that the correlation of C. elegans LC₅₀s vs. rat LD₅₀s was equal to the correlation of mouse vs. rat LD₅₀s (r = 0.879), and was stronger than the correlation of NHK cell IC₅₀s vs. rat LD₅₀s (r = 0.844). In addition, LT₅₀ was significantly correlative with the LC₅₀ of C. elegans, indicating that both can be utilized as toxic effect index for further study on acute toxicity testing of chemicals. In summary, C. elegans may be a valuable model for predicting chemicals’ acute toxicity in rodents.

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Introduction

Traditional toxicity testing methods include acute, sub-chronic, and chronic tests as well as testing for mutagenicity, teratogenicity and carcinogenicity. Acute studies incorporating rodent animals serve as the basis for the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals,¹ as a guide to possible toxic modes of action, and in establishing a dosing regimen in sub-chronic toxicity studies. Yet acute toxicity testing often utilizes large numbers of rodents, and is extremely expensive and time consuming, which has resulted in a backlog of over 10 000 compounds that are in need of prioritization for further testing.² The development of a new model for chemical toxicity screening aims to predict the chemical toxicity in rodents and shorten the list of chemicals for further rodent testing, thereby prioritizing testing of high toxicity chemicals. Consequently in vitro basal cytotoxicity models or alternative in vivo animal models^3–6 are being explored to improve toxicity characterization, increase efficiency, reduce cost, as well as refine, reduce, or replace vertebrate animal testing.⁷

A number of studies have shown a correlation between in vitro cytotoxicity and in vivo acute rodent toxicity since the 1980s. The limitations of the in vitro methods are largely due to the differences between whole animal and cell culture systems with respect to chemical delivery, distribution, metabolism and excretion. In recent years, the nematode Caenorhabditis elegans (C. elegans) has been utilized in toxicity testing as an alternative in vivo animal model. Advantages of this animal model include its short and prolific life cycle, small body size, ease of maintenance, invariant and fully described developmental program, and well-characterized genome. These features have led to an increase in utilizing C. elegans in toxicology, not only for mechanistic studies but also for toxicity screening approaches. As with any model system, C. elegans will only prove useful in toxicity screening if results are predictive of toxic responses in other organisms, yet very few studies have been performed to evaluate correlations between toxicity responses in the nematode and mammals.⁸ In one such study, ranking of median lethal concentration (LC₅₀) values in C. elegans for eight metal salts paralleled ranking for rat median lethal dose (LD₅₀) values, and the LC₅₀ values from C. elegans were found to be also equally as predictive of relative toxicity in rats as LD₅₀ values were from mice to rats.⁹ Another study analyzing motility in adult C. elegans found that for fifteen organophosphate compounds, the toxicity ranking order was significantly correlated with rat LD₅₀s.¹⁰ Yet both of these studies used chemicals with similar molecular structures, which will likely not predict the toxicity of unrelated chemicals. Recently some studies have been performed using chemicals of diverse structures to determine correlations between toxicity responses in C. elegans and toxicity ranking in mammals. In one study seven toxicants were tested in C. elegans using the Complex Object Parametric Analyzer and Sorter (COPAS) system.⁸ The study determined that the chemical toxicity on C. elegans reproduction was highly correlative with rodent lethality. The growth and development were also assayed when C. elegans was exposed to (in order of decreasing toxicity) sodium arsenite, sodium fluoride, caffeine, valproic acid, sodium borate or DMSO for 72 h.¹¹ 5 of the 6 compounds tested (83.3%) were correctly ranked according to their toxicity based on oral rat LD₅₀ data. These results suggest that C. elegans is useful in predicting the potential toxicity of new chemicals in rodents.

In this report, the acute toxicity of 21 chemicals in different toxicity categories were assayed in C. elegans after 2, 4, 12 and 24 h exposure, and the LC₅₀ was calculated by the Probit method. The correlations between C. elegans toxicity and mouse/rat oral toxicity of these chemicals were determined and were compared with the correlation between mouse and rat acute oral toxicities. Correlations between in vitro cytotoxicity (using median inhibition concentration, IC₅₀ data),¹² C. elegans, mouse and rat acute toxicity were also compared to determine if the C. elegans toxicity test may be feasible to predict acute rodent toxicity similar to the in vitro basal cytotoxicity test method. In addition, median lethal time (LT₅₀) was calculated using the data collected at the concentrations of 10.000, 21.500 and 46.400 mg ml⁻¹. The correlation was also analyzed comparing C. elegans LC₅₀s and LT₅₀s of these chemicals. Our results show that the C. elegans toxicity assay may be useful in predicting the potential acute toxicity of chemicals in rodent animals.

Material and methods

Chemicals

Twenty-one test chemicals (>99% pure) of known rodent toxicity were selected for acute toxicity testing using C. elegans. These chemicals and the fluorescent dye propidium iodide (PI) were obtained from Sigma-Aldrich. Of all the chemicals tested, 6 chemicals (sodium dichromate dihydrate, dichlorvos, sodium fluoride, cadmium chloride, diquat dibromide, manganese chloride) are in category III (50 < LD₅₀ ≤ 300 mg kg⁻¹), 2 chemicals (cupric sulfate (pentahydrate), atropine sulfate) in category IV (300 < LD50 ≤ 2000 mg kg⁻¹), 5 chemicals (potassium chloride, boric acid, acetonitrile, lactic acid, sodium chloride) in category V (2000 < LD₅₀ ≤ 5000 mg kg⁻¹) and 8 chemicals (isopropyl alcohol, trichloroacetic acid, dimethylformamide, citric acid, ethylene glycol, methyl alcohol, ethanol, glycerol) as not classified (LD₅₀ > 5000 mg kg⁻¹). The classification for oral toxicity of chemicals was based on reference values obtained from the Globally Harmonized System of Classification and Labeling of Chemicals.¹

Strain preparation

C. elegans used in this study was wild-type Bristol (N2), originally obtained from the Caenorhabditis Genetic Center (Minneapolis, MN). They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli (E. coli) OP₅₀ at 20 °C as previously described.¹³ Gravid nematodes were washed off the plates into conical tubes, and were lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl). Age synchronous populations of L4-larval nematodes were obtained by the collection as described.¹⁴

Preparation of chemical dosing solutions

The stock dosing solution was prepared at twice the desired concentration in sterile K-medium (51 mM NaCl, 32 mM KCl). Individual dosing solutions were prepared by serially diluting 2.15 times the stock solution with K-medium and at twice the desired final concentration. For ethylene glycol, methyl alcohol and glycerol, the highest concentration was the original stock solution. The final concentrations of the tested chemicals are as follows: sodium dichromate dihydrate (0.215, 0.464, 1.000, 2.150, 4.640 mg ml⁻¹), dichlorvos (0.100 × 10⁻³, 0.215 × 10⁻³, 0.464 × 10⁻³, 1.000 × 10⁻³, 2.150 × 10⁻³, 4.640 × 10⁻³, 10.000 × 10⁻³ mg ml⁻¹), sodium fluoride (0.464, 1.000, 2.150, 4.640 mg ml⁻¹), cadmium chloride (0.046, 0.100, 0.215, 0.464, 1.000, 2.150, 4.640, 10.000, 21.500, 46.400, 100.00 mg ml⁻¹), diquat dibromide, manganese chloride, cupric sulfate pentahydrate (0.010, 0.022, 0.046, 0.100, 0.215, 0.464, 1.000, 2.150, 4.64, 10.000, 21.500 mg ml⁻¹), atropine sulfate (4.640, 10.000, 21.500, 46.400 mg ml⁻¹), potassium chloride and sodium chloride (2.150, 4.640, 10.000, 21.500, 46.400, 100.000 mg ml⁻¹), boric acid (2.150, 4.640, 10.000, 21.500 mg ml⁻¹), acetonitrile, isopropyl alcohol, dimethylformamide (4.640, 10.000, 21.500, 46.400, 100.000, 215.000 mg ml⁻¹), lactic acid (1.000, 2.150, 4.640, 10.000 mg ml⁻¹), trichloroacetic acid (0.046, 0.100, 0.215, 0.464, 1.000 mg ml⁻¹), citric acid (0.100, 0.215, 0.464, 1.000, 2.150, 4.640, 10.000 mg ml⁻¹), ethylene glycol (46.400, 100.000, 215.000, 464.000, 556.600 mg ml⁻¹), methyl alcohol (21.500, 46.400, 100.000, 215.000, 395.650 mg ml⁻¹), ethanol (21.500, 46.400, 100.000, 215.000 mg ml⁻¹), glycerol (46.400, 100.000, 215.000, 464.000, 630.500 mg ml⁻¹).

Acute toxicity assay

The age synchronous L4-larval nematodes were quickly collected in K-medium. The nematode suspension was allowed to settle for 10 minutes, and the supernatant containing the E. coli was aspirated. The remaining suspension was diluted to approximately 40–60 L4-larval nematodes per 100 μl with K-medium. 100 μl of this dilution and 2 μl PI (40 mg ml⁻¹) was added to each well of a 96-well plate and mixed. Live and dead nematodes were counted manually under an Olympus IX51 inverted microscope and recorded as N₀ and N. A worm was considered dead when it was motionless for at least 10 seconds. 100 μl of the individual dosing solutions of chemicals were pipetted into each of four wells of a 96-well plate to a final volume of 200 μl. For the LC₅₀ C. elegans experiment, the dead nematodes were counted at 2, 4, 12, 24 h after exposure and recorded as N₂, N₄, N₁₂, N₂₄. In the experiment of C. elegans LT₅₀, at the concentrations of 10.000, 21.500 and 46.400 mg ml⁻¹, the dead worms were also further counted at 1, 8, 16 and 20 h and recorded as N₁, N₈, N₁₆, N₂₀. The lethal rate was calculated as: lethality rate (%) = [N₁ (N₂, N₄, N₈, N₁₂, N₁₆, N₂₀, N₂₄) − N₀]/N × 100%. In addition, the dead nematodes demonstrated red fluorescence after PI staining whereas almost all of the living nematodes did not,¹⁵ which helped to judge the viability of nematodes.

pH measurements and the calculation of pH₁₀ and pH₅₀

Pre-exposure pH measurements were made at all final concentrations of dichlorvos, lactic acid, trichloroacetic acid and citric acid using F-33 pH/ion analyzer (Yiyuan Electronic Instrument Technology Company, Beijing, China). The pH of the other chemicals was only measured at the highest concentration. The pH producing 10% or 50% of the lethality rate is referred to as pH₁₀ or pH₅₀ respectively, which was calculated by Probit analysis.

Statistical analysis

All analysis was conducted using SPSS for Windows version 13.0. Probit analysis was performed to calculate the LC₅₀ with 95% confidence intervals (West, Inc., & Gulley, 1994) after 2, 4, 12 and 24 h exposure. The LC₅₀ derived from the concentration–response was supported by at least two responses, one on each side of the IC₅₀. LT₅₀ with 95% confidence intervals at the concentration of 10.000, 21.500, 46.400 mg ml⁻¹, pH₁₀ and pH₅₀ was also calculated in this way. The Spearman non-parametric correlation analysis was used when correlating C. elegans LC₅₀s vs. mouse/rat LD₅₀s, mouse vs. rat LD₅₀s, NHK cell IC₅₀s vs. rat LD₅₀s respectively. The correlation between LC₅₀ and LT₅₀ of C. elegans was also analyzed using this method. Differences between groups were analyzed statistically with one-way ANOVA followed by a Dunnett's t-test for multiple comparisons with p < 0.05 considered statistically significant (*p < 0.05; **p < 0.01). Graphs were generated using Microsoft EXCEL (Microsoft Corp., Redmond, WA).

Results

Nematode LC₅₀s following exposure to selected chemicals

The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and European Centre for the Validation of Alternative Methods (ECVAM) designed a multi-laboratory validation study to evaluate the performance of two standardized in vitro basal cytotoxicity test methods for estimating acute oral toxicity in rodents using 72 reference substances in 2006.¹² In this study, we used 20 chemicals selected from the 72 reference substances. In addition we assayed the toxicity of manganese chloride, a known human neurotoxicant.

Following exposure to the different chemical concentrations, C. elegans viability was recorded at 2 h, 4 h, 12 h and 24 h. Probit analysis was performed to obtain the LC₅₀ with 95% confidence intervals (Table 1).

Table 1 LC₅₀ with 95% confidence intervals of nematodes after various time of exposure to chemicals^a,b

Chemicals	2 h LC50 (mg ml^−1)	95% confidence intervals	4 h LC50 (mg ml^−1)	95% confidence intervals	12 h LC50 (mg ml^−1)	95% confidence intervals	24 h LC50 (mg ml^−1)	95% confidence intervals
a —: LC50 could not be achieved at the highest concentration that the chemicals could be dissolved.b /: 95% confidence intervals could not be achieved at this concentration.
Sodium dichromate dihydrate	—	/	—	/	3.02	2.45–3.97	0.59	0.49–0.69
Dichlorvos	2.12 × 10^−3	1.73 × 10^−3–2.61 × 10^−3	1.71 × 10^−3	1.40 × 10^−3–2.09 × 10^−3	0.76 × 10^−3	0.64 × 10^−3–0.91 × 10^−3	0.49 × 10^−3	0.43 × 10^−3–0.57 × 10^−3
Sodium fluoride	—	/	—	/	0.99	0.89–1.10	0.54	0.51–0.57
Cadmium chloride	61.37	/	60.84	/	2.73	2.08–3.79	0.85	0.62–1.19
Diquat dibromide	—	/	—	/	29.83	/	13.6	/
Manganese chloride	—	/	—	/	27.49	/	12.29	9.53–16.63
Cupric sulfate (pentahydrate)	7.42	5.45–10.34	2.08	1.82–2.40	0.05	0.03–0.07	3.36 × 10^−3	1.15 × 10^−3–6.41 × 10^−3
Atropine sulfate	44.31	31.30–87.24	30.85	21.81–55.07	24.01	18.54–33.34	20.72	15.83–28.90
Potassium chloride	54.15	33.85–119.24	35.70	22.43–72.97	27.24	17.09–54.07	24.62	15.33–49.57
Boric acid	—	/	—	/	—	/	16.89	12.30–29.58
Acetonitrile	61.66	/	58.69	/	56.25	/	55.68	/
Lactic acid	5.09	4.25–6.09	4.39	3.41–5.60	3.58	2.60–4.89	2.79	1.39–7.37
Sodium chloride	28.12	/	25.28	12.98–65.23	23.24	13.19–48.68	17.38	11.15–28.89
Isopropyl alcohol	85.06	/	64.14	34.62–122.65	32.68	21.33–53.95	24.47	16.72–40.52
Trichloroacetic acid	0.84	0.54–2.40	0.65	0.44–1.47	0.54	0.40–0.88	0.44	0.35–0.58
Dimethylformamide	52.65	/	51.99	/	50.14	31.61–99.68	44.70	30.32–75.21
Citric acid	5.62	3.53–11.66	3.11	1.76–6.76	1.92	1.04–3.94	1.58	0.94–2.85
Ethylene glycol	—	/	397.31	/	202.25	144.24–275.69	124.51	/
Methyl alcohol	259.34	/	175.97	121.76–382.91	127.00	/	71.36	52.77–100.18
Ethanol	130.05	/	64.12	37.12–108.67	58.90	26.39–185.70	52.62	43.13–65.37
Glycerol	457.36	/	445.30	/	300.54	243.14–394.88	214.05	184.21–251.19

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Nematodes LT₅₀s following exposure to selected chemicals

Three concentrations (10.000, 21.500, 46.400 mg ml⁻¹) of the chemicals were selected to determine the nematode LT₅₀. C. elegans viability was determined at 1, 2, 4, 8, 12, 16, 20 and 24 h. LT₅₀ and 95% confidence intervals were calculated according to the Probit method (Table 2). Fig. 1 shows a nematode lethality rate following exposure to a representative chemical – atropine sulfate. The nematode lethality rate is greater in dose-dependent and time-dependent manners.

Fig. 1 Nematode lethality rate following exposure to atropine sulfate. Time-response curve of a representative experiment based on observed mean lethality rate following exposure to 3 concentrations of atropine sulfate. One-way ANOVA followed by a Dunnett's t-test indicated that the difference between 0 h and each of the other hours with p < 0.05 considered statistically significant (*p < 0.05; **p < 0.01).

Table 2 LT₅₀ with 95% confidence intervals of nematodes after exposure to various concentrations of chemicals^a,b

Chemicals	LT50 (h)
	10.000 mg ml^−1	95% confidence intervals	21.500 mg ml^−1	95% confidence intervals	46.400 mg ml^−1	95% confidence intervals
a /: The experiment was not performed because the chemical was not soluble at this concentration.b —: There were no 95% confidence intervals at this concentration; <1: LT50 was achieved in an hour or less. >24: LT50 could not be achieved in 24 h.
Sodium dichromate dihydrate	/	—	/	—	/	—
Dichlorvos	<1	—	<1	—	<1	—
Sodium fluoride	2.98	2.84–3.12	2.95	2.82–3.09	/	/
Cadmium chloride	7.35	6.83–7.89	7.64	7.18–8.12	6.98	6.61–7.35
Diquat dibromide	>24	—	17.59	16.82–18.37	16.70	16.09–17.32
Manganese chloride	>24	—	12.86	10.87–15.14	7.46	7.17–7.77
Cupric sulfate (pentahydrate)	2.94	2.73–3.18	2.94	2.71–3.21	2.94	2.71–3.22
Atropine sulfate	>24	—	26.66	23.49–31.71	<1	—
Potassium chloride	>24	—	>24	—	2.64	1.48–3.48
Boric acid	>24	—	16.87	15.85–17.98	/	—
Acetonitrile	>24	—	>24	—	>24	—
Lactic acid	<1	—	<1	—	<1	—
Sodium chloride	>24	—	23.88	20.56–29.86	<1	—
Isopropyl alcohol	>24	—	>24	—	9.02	8.07–9.98
Trichloroacetic acid	<1	—	<1	—	<1	—
Dimethylformamide	>24	—	>24	—	>24	—
Citric acid	1.24	0.54–1.65	<1	—	<1	—
Ethylene glycol	>24	—	>24	—	>24	—
Methyl alcohol	>24	—	>24	—	>24	—
Ethanol	>24	—	>24	—	>24	—
Glycerol	>24	—	>24	—	>24	—

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Relationship of pH and nematodes lethality rate

Dichlorvos, lactic acid, trichloroacetic acid and citric acid are acidic chemicals. We assayed the pH of each desired concentration of these chemicals at room temperature (20 °C) and determined the lethality rate following a 12 h and 24 h exposure (Fig. 2). When the pH was between 2 and 3, the nematode lethality changed from approximately zero to 100%. The pH was tested at the highest concentrations of other chemicals used in this paper and the pH values were all more than 3.66 (Table 3).

Fig. 2 The relationship of pH (dichlorvos, lactic acid, trichloroacetic acid and citric acid) and the lethality rate of C. elegans following a 24 h exposure. Increasing concentrations of the four chemicals resulted in a decrease in pH and lethality rate.

Table 3 The pH of the highest concentration of chemicals

Chemicals	Concentration (mg ml^−1)	pH
Sodium dichromate dihydrate	4.640	3.66
Sodium fluoride	4.640	6.06
Cadmium chloride	100.000	5.01
Diquat dibromide	21.500	6.22
Manganese chloride	21.500	6.20
Cupric sulfate (pentahydrate)	21.500	4.51
Atropine sulfate	46.400	6.20
Potassium chloride	100.000	6.20
Boric acid	21.500	4.44
Acetonitrile	215.000	6.31
Sodium chloride	100.000	6.03
Isopropyl alcohol	215.000	6.50
Dimethylformamide	215.000	6.42
Ethylene glycol	556.600	6.62
Methyl alcohol	395.650	7.18
Ethanol	215.000	7.01
Glycerol	630.500	4.36

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pH₁₀ and pH₅₀ of nematodes were obtained by Probit analysis using the pH data of the four acidic chemicals at different concentrations and the lethality rate after a 12 h/24 h exposure (Table 4). pH₁₀ was between 2.75 and 3.08 when the nematodes were treated with chemicals after 12 h, and between 2.76 and 3.17 after a 24 h exposure. pH₅₀ was lower than pH₁₀. pH₅₀ was between 2.59 and 2.65 when the nematodes were treated with chemicals after 12 h, while 2.65 and 2.79 after 24 h.

Table 4 pH₁₀ and pH₅₀ following exposure to dichlorvos, lactic acid, trichloroacetic acid and citric acid

Chemicals	12 h		24 h
	pH10	pH50	pH10	pH50
Dichlorvos	2.96	2.65	3.14	2.79
Lactic acid	2.75	2.59	2.76	2.65
Trichloroacetic acid	3.08	2.59	3.17	2.68
Citric acid	2.91	2.63	2.94	2.68

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Correlation analysis of C. elegans LC₅₀s vs. mouse/rat LD₅₀s

Acute oral LD₅₀ data of mouse and rat were derived from internet-accessible databases or published literature as shown in Table 5. The GHS toxicity classification of chemicals was based on rat oral LD₅₀ values here.

Table 5 Acute oral LD₅₀ (mouse and rat) of tested chemicals^a

Chemicals	CAS number	Mouse LD50 (mg kg^−1)	Rat LD50 (mg kg^−1)	GHS toxicity category
a All adopted rat LD50 data were from ICCVAM^12 except that of manganese chloride, which was from the ChemIDplus database.^16 All mouse LD50 data were cited from the ChemIDplus database if not specified. The other chemicals and their mouse LD50 data were as follows: sodium dichromate dihydrate,^17 diquat dibromide,^18 cupric sulfate (pentahydrate),^19 atropine sulfate,^20 trichloroacetic acid (HSDB database).^21
Sodium dichromate dihydrate	7789-12-0	180	51	III
Dichlorvos	62-73-7	61	59	III
Sodium fluoride	7681-49-4	57	127	III
Cadmium chloride	10108-64-2	60	135	III
Diquat dibromide	6385-62-2	233	160	III
Manganese chloride	7773-1-5	1031	250	III
Cupric sulfate (pentahydrate)	7758-99-8	502	474	IV
Atropine sulfate	5908-99-6	609.2	819	IV
Potassium chloride	7447-40-7	1500	2799	V
Boric acid	10043-35-3	3450	3426	V
Acetonitrile	75-5-8	269	3598	V
Lactic acid	50-21-5	4875	3639	V
Sodium chloride	7647-14-5	4000	4046	V
Isopropyl alcohol	67-63-0	3600	5105	Not classified
Trichloroacetic acid	1976-3-9	4970	5229	Not classified
Dimethylformamide	68-12-2	2900	5309	Not classified
Citric acid	77-92-9	5040	5929	Not classified
Ethylene glycol	107-21-1	5500	7161	Not classified
Methyl alcohol	67-56-1	7300	8710	Not classified
Ethanol	64-17-5	3450	11 324	Not classified
Glycerol	56-81-5	4090	19 770	Not classified

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The Spearman correlation analysis was conducted between C. elegans LC₅₀s of 12 h/24 h and mouse/rat LD₅₀s (Table 6). The results show that if all the 21 chemicals were considered, C. elegans LC₅₀s following a 24 h exposure is significantly correlated with mouse/rat LD₅₀s (r = 0.474 and r = 0.697 respectively, both of p < 0.05) while significant correlation also exists between C. elegans LC₅₀s of 12 h and rat LD_50s (r = 0.639, p = 0.002).

Table 6 Correlation analysis between C. elegans LC₅₀s of 12 h/24 h and mouse/rat LD₅₀s

Correlation analysis	12 h treatment using C. elegans		24 h treatment using C. elegans
	r-Value	p-Value^c	r-Value	p-Value^c
C. elegans vs. mouse	0.424	0.062	0.474	0.030*
C. elegans vs. rat	0.639	0.002**	0.697	0.000**
C. elegans vs. mouse (excluding 4 acidic chemicals)	0.724^a	0.002**	0.741^b	0.001**
C. elegans vs. rat (excluding 4 acidic chemicals)	0.832^a	0.000**	0.885^b	0.000**

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	16 chemicals		17 chemicals
	r-Value	p-Value	r-Value	p-Value
a There were 16 chemicals in all analyzed after 12 h treatment using C. elegans, not including the 4 acidic chemicals and boric acid. Because LC50 of boric acid at 12 h was not obtained even at the highest concentration, when correlation analysis of 12 h C. elegans LC50s vs. mouse LD50s was done, SPSS software automatically filtered the missing value and boric acid was not analyzed. In order to make the analysis consistent, when compared with the correlation of LD50s between mouse and rat, boric acid was also excluded.b There were 17 chemicals in all analyzed at 24 h treatment using C. elegans, not including the 4 acidic chemicals.c *p < 0.05, **p < 0.01.
Mouse vs. rat (excluding 4 acidic chemicals)	0.891^a	0.000**	0.879^b	0.000**

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If acidic chemicals (dichlorvos, lactic acid, trichloroacetic acid and citric acid) are eliminated and only other tested chemicals are considered, the correlation coefficient r-value for C. elegans LC₅₀s of 12 h/24 h vs. mouse/rat LD₅₀s (r > 0.72) increases (Table 6). Correlation of C. elegans LC₅₀s with mouse/rat LD₅₀s was further compared with the correlation of mouse LD₅₀s with rat LD₅₀s. The results show that at 12 h treatment, the correlation of mouse vs. rat (r = 0.891) is stronger than that of C. elegans vs. mouse/rat (r = 0.724 and 0.832 respectively). But at 24 h treatment, the correlation of C. elegans vs. rat (r = 0.885) becomes stronger, higher than the correlation of mouse vs. rat (r = 0.879), while the correlation of C. elegans vs. mouse (r = 0.741) is lower than that of mouse vs. rat. All of the correlations are statistically significant (p < 0.01).

Correlation comparisons between cytotoxicity, C. elegans toxicity and mouse/rat toxicity

In vitro cytotoxicity methods have been evaluated as means of reducing and refining the use of animals. In 2006, the three laboratory validation study designed by NICEATM and ECVAM was implemented to evaluate the performance of two standardized in vitro basal cytotoxicity test methods using 72 reference substances for predicting starting doses for acute oral systemic toxicity test methods. Based on the procedures described in the Guidance Document,²² the validation study used two mammalian cell types (i.e., BALB/c 3T3 mouse fibroblasts (3T3) and primary normal human epidermal keratinocytes (NHK)) for cytotoxicity testing with a neutral red uptake (NRU) cell viability endpoint to get IC₅₀. Table 7 provides the geometric mean IC₅₀ combined across laboratories using NHK cells.¹² There were 16 chemicals listed out of the 21 tested chemicals, after excluding 4 kinds of acidic chemicals and manganese chloride. The IC₅₀ data of 3T3 cells are not listed here and were not used in this paper because the 3T3 cell IC₅₀ of methyl alcohol, which was one of the 16 chemicals, was not available.

Table 7 NHK NRU IC₅₀ (geometric means) of 16 chemicals^a

Chemicals	Geometric mean^b IC50 (μg ml^−1)	Chemicals	Geometric mean^b IC50 (μg ml^−1)
a In 2006, NICEATM and ECVAM designed a multi-laboratory validation study to evaluate the performance of two standardized in vitro basal cytotoxicity test methods for estimating acute oral toxicity in rodents using 72 reference substances.b This table shows the NHK IC50 values as geometric means of 3 laboratories.
Sodium dichromate dihydrate	0.721	Acetonitrile	9528
Sodium fluoride	49.8	Sodium chloride	1997
Cadmium chloride	1.84	Isopropyl alcohol	5364
Diquat dibromide	4.48	Dimethylformamide	7760
Cupric sulfate (pentahydrate)	197	Ethylene glycol	10 489
Atropine sulfate	81.8	Methyl alcohol	1529
Potassium chloride	2237	Ethanol	10 018
Boric acid	421	Glycerol	6198

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The Spearman non-parametric correlation was analyzed using data of C. elegans LC₅₀s following a 24 h exposure, NHK cell IC₅₀s and mouse/rat LD₅₀s of the 16 chemicals listed in Table 7. The correlation analysis of C. elegans LC₅₀s vs. rat LD₅₀s, NHK cell IC₅₀s vs. rat LD₅₀s and mouse vs. rat LD₅₀s was performed and the corresponding r-values were 0.879, 0.844, 0.879 with p-value all equal to 0.000.

Correlation analysis of C. elegans LT₅₀s vs. LC₅₀s

Correlation analysis between LT₅₀s at each concentration of 10.000, 21.500 and 46.400 mg ml⁻¹ and LC₅₀ of 12 h/24 h was performed (see Table 8). When exposed to chemicals such as lactic acid, the majority of C. elegans were dead within an hour, therefore the LT₅₀s of these chemicals were defined as 1 h; as for chemicals such as methanol and ethanol, LC₅₀ could not be obtained at 46.400 mg ml⁻¹ and the LT₅₀s at corresponding concentrations were defined as 24 h. All the correlations were statistically significant (p < 0.01).

Table 8 Correlation analysis between C. elegans LT₅₀s and LC₅₀s of tested chemicals^a

LT50	LC50 at 12 h treatment		LC50 at 24 h treatment
	r-Value	p-Value	r-Value	p-Value
a **p < 0.01.
LT50 at 10.000 mg ml^−1	0.816	0.000**	0.803	0.000**
LT50 at 21.500 mg ml^−1	0.794	0.000**	0.859	0.000**
LT50 at 46.400 mg ml^−1	0.853	0.000**	0.753	0.000**

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Discussion

The toxicity assessment and risk management of chemicals is immensely important for the safety of human health. The conventional rodent toxicity tests can no longer meet the increasing requirements in evaluating chemical toxicity. Studies utilizing in vitro basal cytotoxicity test methods or in vivo model organisms are at the frontier of research in the field of chemicals toxicity risk assessment.

A number of studies have been conducted and a positive relationship has been established between in vitro cytotoxicity and in vivo acute lethality in rodents. An international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) was initiated in 1983 to evaluate this relationship between in vitro cytotoxicity and acute human toxicity. Tests of 50 substances in 61 in vitro assays by multiple laboratories led to the identification of a battery of three human cell line assays whose cytotoxicity responses were highly correlated to human lethal blood concentrations.^23–25 The Registry of Cytotoxicity (RC), initially published in 1998, is a database of 347 substances that currently consists of acute oral toxicity data from rats and mice and in vitro cytotoxicity data from studies using various mammalian cell types with a number of different toxic endpoints. A regression formula, the RC millimole regression, constructed from these data was proposed by ZEBET, the German National Centre for the Documentation and Evaluation of Alternative Methods to Animal Experiments, as a method to reduce animal use by identifying the most appropriate starting doses for acute oral toxicity tests.²⁶ To investigate the usefulness and limitations of standardized cytotoxicity tests for estimating starting doses for acute oral toxicity tests, NICEATM and ECVAM sponsored and organized an international validation study using 72 substances tested against the 3T3 or NHK cell system by three laboratories. According to the results of the study, ICCVAM and ECVAM recommended that the RC regression model using an IC₅₀ value from an in vitro basal cytotoxicity test could be used to predict an LD₅₀ value for use as a starting dose for the Acute Toxic Class (ATC) method (TG 423) or the Up-and-Down Procedure (UDP) (TG 425) Test Guideline. Simulations for the reference substances showed that using in vitro cytotoxicity assays to estimate an LD₅₀ as a starting dose could potentially reduce animal use by up to 28% for acute oral toxicity testing, and as much as 50% for non-classified substances. In July 2010, the Organization for Economic Co-operation and Development (OECD) published guideline No. 129 “Guidance Document on Using Cytotoxicity Tests To Estimate Starting Doses For Acute Oral Systemic Toxicity Tests”.²⁷

Nevertheless, the in vitro cytotoxicity model showed a general trend to underpredict the toxicity of the most toxic chemicals, and to overpredict the toxicity of the least toxic chemicals. The limitations of the in vitro methods are largely due to the differences between whole animal and cell culture systems. Animal and cell culture systems are different with respect to how a chemical is delivered to the cell and how it is distributed within the cell, metabolized, and excreted. In a cell culture system, the test chemical is applied directly to the target cells and the only membranes that must be traversed are those of the target cell and its sub-cellular organelles. But in whole animals, the most critical target organs may not be exposed to chemicals or its active metabolite, or be exposed for only a limited time or to a relatively small fraction of the administered dose. As 3T3 and NHK cells have little to no capacity to metabolize xenobiotic compounds,^12,28 it was anticipated that the toxicity of chemicals metabolized in vivo to active compounds would be underpredicted. In addition, animals and cell culture systems may also differ with respect to the target on which a toxicant acts. Toxic mechanisms that include specific actions on the central nervous system (CNS) or the heart are not expected to be active in the 3T3 or NHK cells.

As an emerging in vivo model organism, C. elegans has a number of features that make it not just relevant but quite powerful as a model for toxicological research. First of all, C. elegans is easy and inexpensive to maintain in laboratory conditions with a diet of E. coli. The short, hermaphroditic life cycle and large number of offspring of C. elegans allow large-scale production of animals within a short period of time. Since C. elegans has a small body size, in vivo assays can be conducted in a 96-well microplate. The transparent body also allows clear observation of all cells in mature and developing animals. Furthermore, many of the basic physiological processes, stress responses and intermediary metabolism enzymes that are observed in higher organisms are conserved in C. elegans. Depending on the bioinformatics approach used, C. elegans homologues have been identified for 60–80% of human genes,²⁹ and 12 out of 17 known signal transduction pathways are conserved in C. elegans and human. In addition, though C. elegans lacks the structure of a cardiovascular system, it is an excellent model organism for the study of neurotoxicity effect. Its adult has 302 neurons and 56 neuronal support cells, constituting more than 1/3 of its total cells. The neurons communicate through approximately 6400 chemical synapses, 900 gap junctions, and 1500 neuromuscular junctions. Additionally, the main neurotransmitter systems (cholinergic, γ-aminobutyric acid (GABA)ergic, glutamatergic, DAergic, and serotoninergic) and their genetic networks (from neurotransmitter metabolism to vesicle cycling and synaptic transmission) are phylogenetically conserved from nematodes to vertebrates.⁶ These unique features suggest that C. elegans may be a more promising and valuable model than in vitro cytotoxicity model for toxicity screening and assessment of new chemicals.

In recent years, the utilization of C. elegans in toxicology has largely been used to evaluate heavy metal and pesticide toxicity testing and their role in ecotoxicology.^30–34 In this study, C. elegans LC₅₀s of 21 chemicals were determined by the nematode acute exposure assay. The correlation between C. elegans LC₅₀s and mouse/rat oral LD₅₀s of tested chemicals was analyzed. The correlations of mouse LD₅₀s, NHK cell IC₅₀s with rat LD₅₀s were calculated based on the published database. The result of comparison of the correlations suggests that C. elegans may be useful in predicting the potential toxicities of chemicals in mammals.

The pH of chemicals is one of the factors that affect C. elegans LC₅₀

Previous studies demonstrated that exposing C. elegans to low pH leads to decreased survival and movement.^10,35 Many chemicals are known to decrease the pH that could potentially confound the observed effects. Dichlorvos, lactic acid, trichloroacetic acid and citric acid are strongly acidic. The rat LD₅₀s from the strongest to the weakest toxicity rank 2nd, 12th, 15th, and 17th out of the 21 chemicals, while their C. elegans toxicity was at 1st, 3rd, 7th and 8th. We suspect that in addition to the specific interactions with their biological targets, the pH of the 4 chemicals may also contribute to the toxicity and lethality in the nematode.

The pHs of desired final concentrations of these chemicals were assayed and the results show that when the pH was between 2 and 3, the lethality rates of C. elegans increased from approximately zero to 100%. The pH₁₀ at 12 h and 24 h exposure was 2.75–3.14, indicating that 90% of the nematode would die below this range. For each acid, pH₁₀s and pH₅₀s were similar suggesting that there was a critical pH range below which nematode death would occur. Previously it was demonstrated that C. elegans can tolerate a wide pH range (pH 3.2–11.8) when raised in K-medium or when raised in moderately hard reconstituted water (MHRW).³⁵ Our results are consistent with this pH range.

Except for dichlorvos, lactic acid, trichloroacetic acid and citric acid, the pH of the other chemicals at its highest concentration ranged from 3.5 to 7.5. Because the chemicals were 2.15 times diluted, the greater the dilution ratio, the closer the pH to 7. So the pH of other chemicals at their various concentrations could not exert toxic effects on C. elegans.

Correlation analysis corroborates that there is intrinsic relationship between C. elegans toxicity LC₅₀s and mouse/rat acute toxicity LD₅₀s

If acidic chemicals (dichlorvos, lactic acid, trichloroacetic acid, citric acid) were excluded in the analysis, we found that there were positive correlations between C. elegans LC₅₀s and mouse/rat LD₅₀s after 12 h and 24 h exposure (p < 0.01) with correlation coefficient r-values more than 0.72. At 24 h, the correlation between C. elegans LC₅₀s and rat LD₅₀s (r = 0.885) was better than the correlation between mouse and rat LD₅₀s (r = 0.879). The above correlation analysis confirmed that there was an intrinsic relationship between C. elegans toxicity and mouse or rat acute toxicity. Overall, C. elegans appears to be an excellent predictor of rodent lethality. However, more chemicals should be tested to verify this conclusion.

C. elegans is a potentially valuable approach to predict acute oral systemic toxicity

Comparisons were further performed after correlating cytotoxicity, C. elegans, and mouse toxicity with rat toxicity. Rat LD₅₀s were used as standard index for comparison in our study due to two considerations, the current acute oral toxicity test guidelines recommend using rats^36–39 and the great majority of acute oral systemic toxicity testing performed in rats. The results in this study showed correlation of C. elegans LC₅₀s vs. rat LD₅₀s was equal to correlation of mouse vs. rat LD₅₀s (r = 0.879), stronger than correlation of NHK cell IC₅₀s vs. rat LD₅₀s (r = 0.844). The above results suggested that C. elegans as a multicellular model organism may have greater advantages in predicting the rodent acute toxicity than in vitro cytotoxicity model.

The recommended indicator and exposure concentration range of chemicals in C. elegans acute toxicity testing

We further studied whether LT₅₀ (h) could be applicable for observation indicators of C. elegans acute toxicity test. As there was a positive correlation between LC₅₀s (mg ml⁻¹) and LT₅₀s (h) with r-values between 0.753 and 0.859 after 12 h and 24 h treatment (p < 0.01), it was recommended that they could be used as acute toxicity test indicators in our follow-up study about chemical toxicity.

Based on these results, we recommend that in future studies the exposure concentration range of chemicals for 12/24 h exposure in various GHS toxicity category as follows:

0.215–46.400 mg ml⁻¹ (GHS III), 1.000–46.400 mg ml⁻¹ (GHS IV), 2.150–100.000 mg ml⁻¹ (GHS V), 10.000–464.000 mg ml⁻¹ (unclassified).

Limitations of this study are summarized as follows: first, this study did not cover the chemicals in all the categories based on GHS of Classification and Labeling of Chemicals, lacking highly toxic chemicals in category I and II, which were hard to access during our study process. Second, the chemicals used in this paper are all water-soluble chemicals because for water insoluble chemicals such as Busulfan it was hard to achieve LC₅₀ even at its maximum solubility. In recent years, more sophisticated and sensitive sublethal end points for toxicity testing have been developed including the use of transgenic strains with specific biomarkers,^40,41 growth and reproduction,⁴² and behavior.⁴³ Our next goal will be to test more high toxicity chemicals and develop sublethality test methods to further verify the feasibility of the C. elegans for predicting acute toxicity in rodents.

Conclusion

The unique features of C. elegans indicate that the nematode is an excellent model to complement mammalian models in toxicology research. Experiments with C. elegans do not have the experimental complexity as in vertebrate models, yet still permit the testing of hypotheses in vivo. Thus C. elegans may provide a rapid, efficient and cost effective method for predicting the acute toxicity in rodents of a large number of chemicals in a relatively short time.

Funding

This study was partly supported by the “National Natural Science Foundation of China” Grant (#81273108), “The capital health research and development of special” Project (# 2011-1013-03), and Beijing Health Bureau Project (2011–2013).

Conflict of interest statement

None of the authors have any conflicting interests.

Acknowledgements

We thank Dr Richard Nass from Indiana University School of Medicine for helpful discussions and assistance with the manuscript.

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