Genotoxicity studies in vitro and in vivo on carminic acid (natural red 4)

Fd Chem. Toxic. Vol. 30, No. 9, pp. 759-764, 1992 Printed in Great Britain. All rights reserved

0278-6915/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd

GENOTOXICITY STUDIES I N VITRO AND I N VIVO ON CARMINIC ACID (NATURAL RED 4) G. LOPRIENO,G. BONCRISTIANIand N. LOPRIENO Dipartimento di Scienze dell'Ambiente e del Territorio, Laboratory of Genetic Toxicology, The University, Via S. Giuseppe 22, 56123 Pisa, Italy (Accepted 24 April 1992) Abstract--Tge potential genotoxic activity of carminic acid (CAS no. 1260-17-9; EINECS no. 215-023-3; C.I. no. 75410), a component of natural red colouring products (cochineal:CAS no. 1343-78-8; EINECS no. 215~580-6; C.I. no. 75470), used in food, cosmetics and drugs, has been evaluated by means of a series of short-term tests in vitro and/n vivo, namely Salmonella reverse mutation, chromosome aberrations and sister chromatid exchanges/n vitro on Chinese hamster ovary ceils, and the mouse micronucleus test. All studies have produced negative results. The data obtained strongly support the non-mutagenic/non-carcinogenic activity of this compound. Genotoxicity data previously obtained for carminic acid, concerning the induction of a series of other genetic endpoints in different test systems, have also been considered, as have recent findings that indicate lack of carcinogenic activity in the cochineal preparation containing 29.8% carminic acid.

short-term tests both in vitro and in vivo on this chemical, namely the Salmonella reverse mutation Carminic acid, or Natural Red 4 (CAS no. test, the chromosome aberration test in cell culture, 1260-17-9; EINECS no. 215-023-3; Color Index the mouse bone marrow micronucleus test, and sister no. 75410) is normally contained in cochineal (CAS chromatid exchanges (SCEs) in cell culture. Samples no. 1343-78-8; EINECS no. 215-680-6; Color of two commercially available products, with differIndex no. 75470), a natural red dyeing agent obtained ent degrees of purity, were used in our studies. from an aqueous-alcoholic extract of the air-dried The recent paper by Mori et al. (1991) on the lack bodies of the pregnant female insect Dactylopius of carcinogenicity in a cochineal sample containing coccus Costa, a parasite of the plant Opuntia ficus 29.8% carminic acid has stimulated the publication in indica (Mori et al., 1991); it is widely used as a food this paper of some detailed data, which may be of colouring agent, in cosmetic and drug products. some interest in the context of a possible correlation Carminic acid, 7-D-glucopyranosyl-3,5,6,8-tetrahy- between short-term tests and long-term studies on droxyl- 1-methyl-9,10-dioxoanthracene-2-carboxylic animals. acid, has been reported to be non-mutagenic in different cell bacterial systems in vitro [e.g. Bacillus MATERIALS AND METHODS subtilis (Kada et aL, 1972), Salmonella typhimurium Test chemicals. Carminic acid of known purity (Barale and Loprieno, 1978; Brown and Brown, 1976; (Table 1) was obtained from Davide Campari Haveland-Smith and Combs, 1976), Saccharomyces (Milano, Italy). A sample of carminic acid from pombe and Saccharomyces cerevisiae (Barale and Aldrich Chemical Co. (Batch no. 304862) was obLoprieno, 1978)] and non-genotoxic when tested for tained by the courtesy of Dr Errol Zeiger, of the the induction of unscheduled DNA repair synthesis in National Institutes of Environmental Health Scirat hepatocyte primary culture in vitro (Mori et aL, ences, Research Triangle Park, NC, USA. The results 1988) or in rat liver assay in vivo (Kornbrust and of the chemical analysis performed as with the Barfknecht, 1985). previous sample are reported in Table 2. Cochineal, on the other hand, has been found Short-term test systems. Salmonella reverse mupositive for the induction of chromosome aberrations tation studies used strains TA1535, TA1537, TA98 in Chinese hamster ovary (CHO) cells grown in vitro and TA100, assayed in the presence and in the at a concentration of 12 mg/ml: the purity of the absence of an S-9 mix activation system obtained sample was, however, not specified (Ishidate, 1984). from Aroclor 1254-treated rats, at concentrations of Interest in a potential genotoxic (and carcinogenic) 10 and 30%; carminic acid was tested at levels of up hazard of carminic acid has prompted a series of to 2000/~g/plate in two independent experiments. The two carminic acid samples were tested in Abbreviations: CHO=Chinese hamster ovary; NCE= parallel. normochromatic erythrocytes; NTP =National ToxiChromosome aberrations. These were analysed in cology Program; PCE=polychromatic erythrocytes; SCE = sister chromatid exchange. CHO cells (two independent cultures) treated with INTRODUCTION

759

G. LOPRIENOet al.

760

Table I. Chemicalanalysis of carminic acid (Campari) Constituent Percentage Carminic acid content (spectrophotometric value) 87.50 Ammonia nitrogen 0.19 Non-ammonia nitrogen 0.27 Proteins 1.70 Volatile at 105°C 10.60 Ash at 800°C 0.76 (Ca = 210 ppm; Fe = 140 ppm; Cu = 7 ppm; Zn = 51 ppm; Cr III = 16 ppm; As=0.06 ppm; Cd =0.22 ppm; Pb = 3.9 ppm)

carminic acid (two samples) from 0.20 to 200 #g/ml in the presence and absence of S-9 mix (10%): cells were analysed 24 hr after treatment, when assayed in the absence of S-9 mix, and 12 and 24hr after treatment when assayed in the presence of S-9 mix. Sister chromatid exchanges. SCEs were evaluated in CHO cells (two independent cultures) treated with carminic acid (two samples) from 20.0 to 200/ag/ml, in the presence and the absence of S-9 mix. Micronucleus induction. This was evaluated in bone marrow cells of CD-1 mice treated orally for 24 and 48 hr with doses of carminic acid of 1250, 2500 or 5000 mg/kg (five males + five females + negative control + 5 mg mitomycin/kg). All results were subjected to statistical analysis. RESULTS The results of the Salmonella test from two independent experiments performed with the four strains (TA1535, TA1537, TA98 and TA100) in the absence and in the presence of two concentrations of S-9 mix (10 and 30%), with the Campari sample of carminic acid are reported in Table 3. The sample of carminic acid obtained from the Aldrich Chemical Company was tested on TAI00 only after this had been suggested by Dr E. Zeiger: the data obtained in the two independent experiments are reported in Table 4. All the results are negative. The induction of chromosome aberration in CHO cells was studied in the two samples of carminic acid, tested at concentrations of 20, 63 and 200 ~g/ml; the cells were analysed 12 and 24 hr after treatment with metabolic activation (3 hr), and 24 hr after treatment without metabolic activation. Positive controls were represented by mitomycin-C and cyclophosphamide for the two different conditions of treatment. The results are summarized in Table 5. No statistically significant increases in the number of cells showing aberrations were found either in the presence or the absence of S-9 metabolism at any level of treatment with carminic acid (Campari), compared with the solvent control cultures. In the case of the Aldrich sample of carminic acid, in the cultures treated in the presence of S-9 and harvested at 24hr, a statistically significant increase in the number of cells bearing aberrations was noted at the lowest dose level selected (20.0 # g/ml). As statistically significant increases were not obtained in both repli-

cate cultures at the dose level concerned, this increase did not meet the criterion for a positive result. Furthermore, the increase was not accompanied by significant toxicity. The induction of SCEs in CHO cells was studied for the two samples of carminic acid when tested at 20, 63 or 200/~g/ml, either with or without S-9 mix. Findings were negative; however, in the presence of S-9 mix, a statistically significant depression of SCE induction was observed for all three treatment doses with both samples of carminic acid (Table 6). Carminic acid (Campari) was tested on CD-I mice orally treated with 1250, 2500 and 5000 mg/kg for the induction of micronuclei in bone marrow cells: these doses produced in the same mouse strain respectively 20, 40 and 80% of the LDs0. Scoring was on two different groups of animals (five females + five males at each treatment level) after 24 and 48 hr (Table 7). The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test. No significant increases in the incidence of micronucleated polychromatic erythrocytes (compared with the vehicle control values) were observed at any dose level or sampling time in the carminic acid treatment groups. Marked increases in the normochromatic/polychromatic erythrocyte (NCE:PCE) ratio were observed following carminic acid treatment at both sampling times, suggesting that the test substance exerted a toxic effect on bone marrow erythropoietic cells. Statistically significant increases over control values were seen in the positive control group (5 mg mitomycin/kg). DISCUSSION Carminic acid has been tested in four short-term genotoxicity tests (Salmonella reverse mutations, chromosome aberrations and SCEs on CHO cells grown in vitro and the mouse micronucleus bone marrow test) for the evaluation of its potential genotoxic effect. In the present study a sample of 87.5% purity provided by the Davide Campari Company has been used. All the studies have demonstrated that carminic acid is not mutagenic: the results reported here complete the previous studies performed on carminic acid or its natural raw material, cochineal, which had reported negative results (Barale and Loprieno, 1978; Brown and Brown, 1976; Haveland-Smith and Combs, 1976; Kada et al., 1972; Kornbrust and Barfknecht, 1985; Mori et al., 1988). Ishidate (1984) has reported, Table 2. Chemical analysis of carminic acid (Aldrich; Batch 304862) Constituent Percentage Carminic acid 53.13 Ammonia nitrogen 0.34 Non-ammonia nitrogen 16.50 Volatile at 105"C 3.60 Ash at 800"C 5.19 (Pb = 1.50 ppm; As = 0.29 ppm; Fe = 86.3 ppm; Cu = 5.90 ppm; Zn = 21.8 ppm)

367 ± 17.3

22 18 17 27 29 25 21

497 + 14.3

15 24 24 25 26 26 23

TA1535

104 ± 4.0

15 13 17 13 12 12 12

65±2.3

14 12 15 13 14 13 13

TA1537

*Average no. of revertants from three plates. tWithout any treatment. :~Treated with vehicle alone.

Untreated 0 125 250 500 1000 2000 Sodium azide (1 ~g/plate) 2-Amino-anthracene (1 ~g/plate) 9-Amino-acridine (50 #g/plate) 2-Nitro-fluorene (2 ~g/plate)

Untreatedt 0~ 125 250 500 1000 2000 Sodium azide (I ~g/plate) 2-Aminoanthracene (1 ~g/plate) 9-Aminoacddine (50#g/plate) 2-Nitrofluorene (2 pg/plate)

Carminic acid (2 og/plate)

137 ± 3.0

41 31 28 36 37 44 49

128 + 11.6

46 51 46 50 53 47 59

TA98

Without metabolic activation*

626 ± 7.5

151 157 167 161 152 166 174

626 ± 6.6

173 153 155 161 167 172 177

TAI00

126+4.6

16 12 15 14 15 15 17

172 + 11.8

16 15 15 17 18 17 17

TAI535

98 __.3.2

18 21 18 18 14 19 21

2ndExpe~ment

102 ± 1.0

1~ Experiment 17 19 18 18 16 20 27

TA1537

616-t- 16.2

53 46 48 47 49 61 69

620 + 9.0

58 52 59 50 60 68 73

TA98

With S-9 mix* (10%)

1441 +32.0

164 163 180 168 170 158 172

1126 _ 6.7

182 164 172 169 177 156 163

TAI00

116+3.8

18 16 15 13 17 18 21

82 + 6.7

22 12 16 13 13 16 14

TAI535

54+ 1.7

22 20 18 25 23 25 25

48 + 3.8

12 9 11 18 21 25 17

TA1537

230_+20.5

50 48 55 55 65 67 61

199 + 7.2

52 43 51 49 54 49 58

TA98

With S-9 mix* (30%)

157 157 151 159 174 170 173

TA100

496+ 16.5

181 169 167 158 156 164 163

580 + 30.9

Table 3. Results of the Salmonella (strains TAI535, TA1537, TA98 and TA100) test of carminic acid (Campari) tested in the absence and in the presence of two concentrations of S-9 mix

~" ~.

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e~

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G . LOPRIENO el al.

762

Table 4. Results of the Salmonella (strain TAI00) test of carminic acid (Aldrich) tested in the presence and absence of S-9 mix (10%) Without metabolic activation* Carminic acid (#g/plate) Untreatedt 0~ 125 250 500 I000 2000 Sodium azide (1 ~ug/plate) 2-Aminoanthracene (1 ,ug/plate)

Experiment 1

With metabolic activation*

Experiment 2

149 ± 4.4 132 ± 7.8 147 ± 8.5 160 ± 7.2 165±0.9 166 ± 14.0 142 _+ 8.7

135 ± 133 ± 135 ± 121 ± 142± 151 ± 156 ±

853 _+ 65.0

862 ± 45.6

Experiment I 168 ± 154 ± 169 ± 172± 162 ± 166 ± 166 ±

12.9 5.5 5.7 9.0 11.3 6.4 4.2

Experiment 2

8.2 5.1 12.7 10.3 3.2 21.9 15.5

140 + 110± 131 ± 126 ± 143 ± 129 ± 133 ±

458 + 29.4

1.3 12.3 2.4 6.8 3.8 1.7 2.2

851 ± 29.5

*Average no. of revertants from three plates. tWithout any treatment. :~Treated with vehicle alone.

Table 5. Chromosome aberration test of carminic acid (Campari and Aldrich) on CHO cells in the absence ( - ) and in the presence ( + ) of S-9 mixt

Treatment

Dose level (,ug/m])

- S - 9 mix: 24hr

+S-9 mix: 12hr

+S-9 mix: 24hr

% C/A:~

% C/A

MI

% C/A

MI

4.1 9.2 6.9 7.4 6.5 6.3 NT

3.0 3.5 2.0 3.0 1.0 NT 7.5

10.4 10.0 10.9 11.0 13.6 NT 8.7

2.0 3.0 0.5 2.5 4.5 NT 25.0*

4.5 5.6 4.2 3.4 9.8 NT 6.0

10.1 11.4 11.1 11.9 10.2 4.7 NT

5.0 2.5 4.5 3.5 4.0 NT 18.5

18.6 15.5 16.1 16.5 14.7 NT 10.7"

4.5 4.5 10.5 5.0 7.5 NT 29.0*

13.0 12.3 11.3 13.6 14.6 NT 20.3

MI§

Campari Untreated Solvent Carminic acid Carminic acid Carminic acid Mitomycin-C Cyclophosphamide

-1% 20.0 63.0 200.0 0.10 6.60

1.0 1.5 1.5 2.0 2.5 18.5" NT

Untreated Solvent Carminic acid Carminic acid Carminic acid Mitomycin-C Cyclophosphamide

-1% 20.0 63.0 200.0 0.10 6.60

6.5 4.5 3.0 6.0 7.5 69.0* NT

Aldrich

NT = not tested tCombined values from two replicate cultures at each dose level (200 cells scored for each treatment). J;% C/A = percentage of cells bearing aberrations (excluding gaps). §MI = mitotic index expressed as percentage. *Statistically significant at P < 0.001.

Table 6. Sister chromatid exchange test of carminic acid (Campari and Aldrich) on CHO cells treated in the absence and in the presence of S-9 mix'l"

Treatment

Dose level (#g/ml)

- S - 9 mix Mean SCE/cell

+S-9 mix % M2~:

Mean SCE/cell

% M2

93.5 96.0 90.5 85.0 81.5 NT

8.56 6.16"*§ 7.30*§ 6.24**§ NT 15.56**

88.5 94.0 86.0 91.0 NT 84.5

99.0 97.5 97.0 99.0 92.5 87.5 NT

6.50 6.82 5.60**§ 6.20 5.96*§ NT 12.18"*

99.5 96.5 99.0 99.0 93.5 NT 87.5

Campari Solvent control Carminic acid Carminic acid Carminic acid Mitomycin-C Cyclophosphamide

1% 20.0 63.0 200.0 0.03 0.30

6.54 6.68 6.22 6.28 16.46* * NT

Untreated Solvent Carminic acid Carminic acid Carminic acid Mitomycin-C Cyclopbosphamide

-1% 20.0 63.0 200.0 0.03 0.30

6.22 6.00 5.84 6.06 5.48 22.26* * NT

Aldrich

NT = not tested tCombined values from two replicate cultures at each dose level (200 cells scored for each treatment). :~% M2 = percentage of cells in second post-treatment mitosis. ~SCE frequency significantly lower than solvent control values. *Statistically significant at P < 0.05. **Statistically significant at P < 0.01.

G e n o t o x i c i t y studies o f c a r m i n i c acid

763

the NTP studies carminic acid was classified positive after testing in TA100 in the absence of a metabolic activation, as a dose-related increase; however, it never reached a level double that of the control values. A sample of carminic acid from the same source as that tested in the NTP studies (Aldrich Chemical Co.) was therefore subjected to a series of short-term tests, as described above. The results are reported in Tables 4 (Salmonella TA100 strain), 5 (chromosome aberration, CHO cell line) and 6 (SCEs, CHO cell line). All studies, including that with the TAI00 Salmonella strain, gave negative results. A recent paper by Mori et al. (1991) reported the results of a 2-yr bioassay in B6C3F l male and female mice treated with dietary concentrations of 0, 3 or 6% of cochineal (CAS no. 1343-78-8), a natural extract containing 29.8% carminic acid (ashes = 8.2%; prot e i n s = l . 1 5 % ; C a = 1 1 7 . 6 ppm; F e = 4 0 . 8 ppm;

however, that cochineal has produced positive results for the induction of chromosome aberrations on CHO cells grown in vitro, treated with a dose of 12 mg/ml, and analysed 48 hr after treatment. In the present study, the maximum dose tested in the same system with carminic acid (200 pg/ml) has not produced any increase in the frequency of spontaneous chromosome aberrations or SCEs. In the Annual Plan of Fiscal Year 1986 of the National Toxicology Program (NTP), carminic acid was one of the chemicals selected for mutagenicity testing in Salmonella: the results of such testing was reported as a 'weak response' in the Annual Plan of Fiscal Year 1987 of the NTP. On the basis of information provided by Dr E. Zeiger, Head of the Environmental Mutagenesis Group of the National Institute of Environmental Health Sciences, Research Triangle Park, USA (personal communication), in

Table 7. Micronucleus test of carminic acid (Campari) on bone marrow ceils of CD-I mice analysed 24 hr after oral treatment Incidence of micronuclei per 1000 ceils Dose level (mg/kg)

Polychromatic Scored PCE

Cells NCE

NCE/PCE ratio Mean

SE

Normochromatic

Min

Max

Mean

SE

Min

Max

24 hr after oral treatment Males

0.00 1250 2500 5000 Mitomycin C 5.00

5012 4446 3445 4444

4202 5190 4464 5214

0.84 1.33 1.43 1.29

1.8 2.2 3.4 1.7

0.4 0.9 1.3 0.4

1.0 0.0 1.0 1.0

3.0 5.0 6.7 3.0

0.8 0.4 1.6 1.4

0.4 0.2 0.4 0.6

0.0 0.0 0.8 0.0

1.8 1.0 2.4 3.3

5000

5344

1.07

16.6

4.2

5.0

31.0

1.3

0.6

0.0

3.1

5000 4470 5000 4412

4805 4356 5075 4937

0.96 1.10 1.02 1.27

1.2 1.4 1.4 0.8

0.2 0.6 0.6 0.4

1.0 0.0 0.0 0.0

2.0 3.0 3.0 2.0

1.2 0.5 2.0 1.6

0.6 0.3 0.2 0.4

0.0 0.0 1.6 0.7

3.0 1.3 2.9 2.8

4470

5085

1.24

12.7

3.3

7.0

25.5

0.7

0.5

0.0

2.6

10,012 8916 8445 8856

9007 9546 9539 10,151

0.90 1.21 1.20 1.28

1.5 1.8 2.3 1.2

0.2 0.5 0.7 0.3

1.0 0.0 0.0 0.0

3.0 5.0 6.7 3.0

1.0 0.4 1.8 1.5

0.3 0.2 0.2 0.3

0.0 0.0 0.8 0.0

3.0 1.3 2.9 3.3

9470

10,429

1.16

14.7

2.6

5.0

31.0

1.0

0.4

0.0

3.1

Females

0.00 1250 2500 5000 Mitomycin C 5.00 Both sexes

0.00 1250 2500 5000 Mitomycin C 5.00

48 hr after oral treatment Males

0.00 1250 2500 5000 Mitomycin C 5.00

5021 3480 3171 4480

4468 5011 6230 5670

0.89 1.52 2.21 1.35

1.6 2.0 1.4 1.4

0.6 0.9 0.6 0.7

0.0 0.0 0.0 0.0

3.0 4.2 2.7 4.0

0.9 0.7 0.6 1.2

0.2 0.5 0.2 0.3

0.0 0.0 0.0 0.0

1.2 2.0 1.0 1.9

3775

5364

1.77

5.3

2.2

1.0

13.6

2.4

0.9

0.6

5.0

5000 5001 2917 3844

5246 4229 4427 4643

1.05 0.85 1.70 1.48

2.4 1.8 0.8 0.8

0.5 1.1 0.5 0.4

1.0 0.0 0.0 0.0

4.0 6.0 2.0 2.0

1.6 0.4 1.9 0.9

0.4 0.3 0.7 0.5

0.6 0.0 0.0 0.0

2.7 1.1 3.3 2.5

5000

4038

0.81

2.2

0.6

1.0

4.0

0.7

0.3

0.0

1.4

10,021 8481 6088 8324

9714 9240 10,657 10,313

0.97 1.15 1.98 1.41

2.0 1.9 1.1 1.1

0.4 0.7 0.4 0.4

0.0 0.0 0.0 0.0

4.0 6.0 2.7 4.0

1.3 0.5 1.2 1.1

0.2 0.2 0.4 0.3

0.0 0.0 0.0 0.0

2.7 2.0 3.3 2.5

8775

9402

1.29

3.8

1.2

1.0

13.6

1.6

0.5

0.0

5.0

Females

0.00 1250 2500 5000 Mitomycin C 5.00 Both sexes

0.00 1250 2500 5000 Mitomycin C 5.00

PCE = polychromatic erythrocytes NCE = normochromatic erythrocytes

764

G. LOPRIENOet aL

Mg = 30.4 ppm; AI = 22.4 ppm; As < 4 ppm; heavy metals < 16 ppm): in treated animals the frequency of tumours developed (hepatocellular adenomas or carcinomas, pulmonary adenomas or adenocarcinomas and lymphomas or lymphatic leukaemias) did not differ statistically from that observed in untreated animals. N o other signs of chronic toxicity were reported from the study. This study therefore may be considered adequate for the evaluation of the carcinogenic potential of carminic acid (administered at a dose of 0.89 and 1.79 in the diet for 2 yr) in mice and yielded a clear negative result. During the study each mouse was exposed to 1.70 or 3.41 g carminic acid. Thus, to summarize the results of the short-term genotoxicity studies on various samples of carminic acid in different laboratories and of the long-term carcinogenicity study with cochineal recently published, it may be assumed that carminic acid or cochineal do not display a carcinogenic or genotoxic activity. Acknowledgements--The authors are grateful to Davide

Campari Co., Milano, Italy and to Dr Errol Zeiger, Research Triangle Park, NC, USA, for providing samples of carminic acid, and to the Ministry of the University and of Technical and Scientific Research, Rome, Italy, for financial support. REFERENCES

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G. Vettorazzi. pp. 349-357. Elsevier, Amsterdam. Brown T. P. and Brown R. J. (1976) Mutagenesis by 9,10-anthraquinone derivatives and related compounds in Salmonella typhimurium. Mutation Research 40, 203-224. Haveland-Smith R. B. and Combs R. D. (1980) Screening of food dyes for genotoxicity activity. Food and Cosmetics Toxicology lg, 215-221. Ishidate M., Jr, Sofuni T., Yoshikawa K., Hayashi H., Nohmi T., Sawada M. and Matsuoka A. (1984) Primary mutagenicity screening of food additives currently used in Japan. Food and Chemical Toxicology 22, 623~36. Kada T., Tutikawa K. and Sadaie Y. (1972) In vitro and host-mediated 'Rec-assay' procedures for screening chemical mutagens, and phloxine, a mutagenic red dye detected. Mutation Research 16, 165-174. Kornbrust D. and Barfknecht T. (1985) Testing of 24 food, drug, cosmetic, and fabric dyes in the in vitro and in the in vivo/in vitro rat hepatocyte primary culture/ DNA repair assays. Environmental Mutagenesis 7, 101-120. Mori H., Yoshimi N., Iwata H., Tanaka T., Kawai K. and Sankawa U. (1988) Additional survey on genotoxicity of natural anthraquinones in the hepatocyte primary culture/DNA repair assay. Journal of Toxicological Sciences 13, 161-166. Mori H., Ivata H., Tanaka T., Morishita Y., Mori Y., Kojima T. and Okumura A. (1991) Carcinogenicity study of cochineal in B6C3Fj mice. Food and Chemical Toxicology 29, 585-588. National Toxicology Program (1986) Fiscal Year 1986 Annual Plan. p. 67. US Department of Health and Human Services, Research Triangle Park, NC, USA. National Toxicology Program (1987) Fiscal Year 1987 Annual Plan. p. 69. US Department of Health and Human Services, Research Triangle Park, NC, USA.