Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests.

A three-step interlaboratory validation of alternative methods to the Draize eye irritation test (Draize test) was conducted by the co-operation of 27 organizations including national research institutes, universities, cosmetic industries, kit suppliers and others. Twelve alternative methods were evaluated using 38 cosmetic ingredients and isotonic sodium chloride solution. Draize tests were conducted according to the OECD guidelines using the same lot of test substances as was evaluated in the alternative tests. Results were as follows. (1) Variation in Draize scores was large near the critical range (maximal average Draize total scores (MAS)=15-50) for the evaluation of cosmetic ingredients. (2) Interlaboratory variation was relatively small for the alternative tests. The mean coefficients of variation (CV%) were less than 50 for all assays except for the hen's egg-chorioallantoic membrane test (HET-CAM), chorioallantoic membrane-trypan blue staining test (CAM-TB) and haemoglobin denaturation test (HD). The CV% of these three methods came into the same range as the other tests when non-irritants were excluded from the data analysis. (3) Results for acids (pH of 10% solution <2.5), alkalis (pH of 10% solution >11.5) and alcohols (lower mono-ol) in cytotoxicity tests clearly deviated from the other samples in the comparison of cytotoxicity with Draize results. (4) Pearson's correlation coefficients (r) between results from cytotoxicity tests using serum and MAS were -0.86 to -0.92 for samples excluding acids, alkalis and alcohols. (5) When the samples were divided into liquids and powders, r of CAM-TB increased from 0.71 for all samples to 0.80 and 0.92, respectively. (6) Spearman's rank correlation coefficients between the results of alternative methods and MAS were relatively high (r>0.8) in the case of HET-CAM and CAM-TB. Those for cytotoxicity tests were high if the data for acids, alkalis and alcohols were excluded (SIRC-CVS: r=0.945, SIRC-NRU: r=0.931, HeLa-MTT: r=0.926, CHL-CVS: r=0.880). Exclusion of data for powdered samples also increased the coefficient of HET-CAM and CAM-TB to 0.831 and 0.863, respectively. These results suggest that no single method can constitute an evaluation system applicable to all types of test substances by itself. However, several methods will be useful for the prediction of eye irritation potential of cosmetic ingredients if they are used with clear understanding of the characteristics of those methods.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test.

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC(50) value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC(50) values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC(50) value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearman's rank correlation between the HC(50) value and maximum average Draize total score (MAS) were -0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Interlaboratory Validation of the In Vitro Eye Irritation Tests for Cosmetic Ingredients. (6) Evaluation of MATREX((TM)).

MATREX(TM) is a test system for evaluating eye irritation potential, using the living dermal model (LDM). The LDM consists of normal human dermal fibroblasts in a contracted collagen lattice, which eventually forms a three-dimensional structure. This system has several advantages. It can be applied to insoluble substances and does not require sterile conditions for operation. In the present study, MATREX was introduced as an alternative to the Draize eye irritation test (Draize test) for cosmetics ingredients. MATREX was evaluated through a three-phase series interlaboratory validation as part of a joint project of the National Institute of Health Sciences (NIHS) and Japan Cosmetic Industry Association (JCIA). Toxicity for LDM was mainly evaluated by cytotoxicity, the indicator was EC(50) (concentration that inhibits the viability of the cell to 50% of control) value. Additionally, MATREX score indicating the grade of cytotoxicity was also introduced in the third phase of the validation study. Both test procedures were controlled under the same standard operating procedure (SOP), at all the participating laboratories. A total of 39 test substances both water-soluble and -insoluble were examined. LDM was applicable to almost all substances that could be evaluated by the Draize test. Furthermore interlaboratory variance was relatively low. The correlation coefficient between the EC(50) value and the maximal average Draize total score (MAS) was -0.672. The MATREX score was closely related to the EC(50) value. Moreover, the MATREX scoring method showed a similar prediction ability for eye irritation potential to the EC(50) method. Thus, the MATREX scoring method, a simplified EC(50) method, appears to be a viable alternative to the current EC(50) measurement method. The present results demonstrate the possibility that the MATREX system would form part of a prediction system of Draize test results.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (5) Skin(2TM) ZK1100 and tissue equivalent assay.

Skin(2TM) ZK1100 (ZK1100) assay and tissue equivalent assay (TEA, Skin(2TM) ZK1200) are human dermal models. These assays were evaluated as alternatives to the Draize eye irritation test (Draize test) in rabbits. Thirty-nine cosmetic ingredients were selected and used as test substances. The ZK1100 assay was conducted according to an original protocol provided by Advanced Tissue Sciences, a kit supplier. The TEA assay followed a protocol developed by Osborne et al., (1995a). Coefficients of variation (CV) ranged from 11.7 to 133 in results from the ZK1100 assay; three test substances showed the CVs more than 100. These were cetyltrimethylammonium chloride (S3-7), domiphen bromide (S3-11) and di(2-ethylhexyl) sodium sulfosuccinate (S3-14). Acid Red 92 (S2-3) was excluded from data analysis because its absorbance interfered with the endpoint of ZK1100 assay. The CVs from the TEA assay ranged from 31.8 to 119; two test substances showed the CVs more than 100. These were acetic acid and glycolic acid (S3-13). Butanol (S3-9) was excluded from the analysis because it was assumed to volatilize during a sample preparation. Pearson's coefficient of correlation with maximum average Draize total score (MAS) and 24hr score from the Draize tests were -0.71 and -0.72 for the ZK1100 results and -0.63 and -0.60 for the TEA results. When a MAS of 15 was set as a breakpoint for the classification of eye irritancy on Cooper's plots comparing the in vitro and the Draize data, the ZK1100 results showed five false positives and four false negatives; the TEA results showed three false positives and no false negatives.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (9). Evaluation of cytotoxicity test on HeLa cells.

The cytotoxicity test using MTT on HeLa cells (HeLa-MTT) was evaluated as an alternative method to the Draize eye irritation test (Draize test) by six to eight laboratories. The 50% inhibition concentration (EC(50)) for MTT reduction was calculated. A total of thirty-nine test chemicals were examined. The average interlaboratory coefficient of variation (CV) of the present assay was 25%. Comparison of the in vitro test results with those of the Draize test revealed a correlation coefficient of -0.799 between the log(EC(50)) value and the maximum average total score (MAS) for a 10% solution or suspension. The following characteristics of HeLa-MTT have become apparent through this validation: (1) HeLa-MTT could be applied to all substances including water-insoluble substances, esters, a colour additive, and a substance which is known to directly reduce MTT; (2) there is good interlaboratory reproducibility and strong correlation between HeLa-MTT and Draize MAS; (3) results for strong acids, alkanolamines and alcohols (lower mono-ol) clearly deviate from other samples with respect to the correlation between HeLa-MTT and Draize MAS. These results suggest that HeLa-MTT may be useful for predicting the Draize MAS if definite criteria can be established for applicable compounds.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (10) Evaluation of cytotoxicity test on CHL cells.

The present interlaboratory validation study was performed in order to evaluate the use of Chinese hamster lung cell lines that employs crystal violet staining (CHL-CVS) as an alternative cytotoxicity test to the Draize eye irritation test (Draize test) for cosmetic ingredients. Ten substances, nine of which were surfactants, were evaluated at seven laboratories in the first phase of the validation study; 15 substances including dyes and lipids were evaluated at seven laboratories in the second phase of the validation study; 14 substances including acids and alkalis were evaluated at four laboratories in the third phase of the validation study. The logEC(50) values obtained for CHL-CVS were compared with the maximal average Draize total score (MAS) for a 10% (w/v) solution of 38 cosmetic ingredients as well as isotonic sodium chloride solution. The interlaboratory coefficient of variation (CV) for EC(50)s was 35.6%, which was considered to be within a tolerable range. The correlation coefficient and the Spearman's rank correlation coefficient between the in vitro and in vivo tests were -0.729 and 0.709, respectively. The prediction ability of the proposed method was assessed from the linear regression line for a MAS cut-off point of 15. According to this analysis, four substances (two alcohols and two acids) were determined to be false negative. The present study revealed the following characteristic factors of this method: (1) CHL-CVS could be applied to all the test substances including dyes and lipids in this study; (2) The results for medium-insoluble substances varied according to the laboratory; (3) The correlation between the in vivo and in vitro data for acids and alcohols (lower mono-ol) differed from that of the other substances. These results suggested that the CHL-CVS might have a potential to predict the Draize MAS if definite criteria can be established for the compounds to be applicable.

Effects of X-ray irradiation on genomic DNA methylation levels in mouse tissues.

Effects of ionizing radiation on the level of genomic DNA methylation in liver, brain and spleen of mouse as well as in two kinds of cultured cells were examined by high-performance liquid chromatography. Ten Gy of whole body X-radiation reduced the 5-methyldeoxycytidine contents by about 40% within 8 hours after irradiation in liver. Similar effects were observed at 4 or 7 Gy of X-ray irradiation. However, no such change was detected in brain, spleen and cultured cells. The data indicate that radiation-induced alteration in genomic DNA methylation is not ubiquitous among different tissues and cells.

X-ray- and ultraviolet-radiation-induced mutations in Muta mouse.

To evaluate the usefulness of the transgenic Muta mouse for investigating radiation-induced mutations in vivo, we have examined the effects of whole-body X irradiation and compared them to the effects of ultraviolet light. The spontaneous mutation frequencies in young adults were about 7 x 10(-5) in the spleen, liver and skin. The mutation frequencies 1 week after a lethal dose of X radiation (8 Gy) were 3.2, 2.6 and 2.7 times the spontaneous levels in the spleen, liver and skin, respectively. When the skin was irradiated with 10 kJ m-2 of UVB, the mutation frequency increased about 6 times. The mutation frequencies induced by an acute dose of 4 Gy or by a fractionated dose of 11.7 Gy (0.15 Gy x 78 times, 3 times/week) in the spleen and liver were less than 2-fold the spontaneous levels at 16 weeks after irradiation. A comparison of X and UV radiation was also conducted with cultured cells derived from the mouse embryo. UVC of 5 J m-2 raised the mutation frequency to 15 times that of unirradiated cells, while 10 Gy X rays raised it 2.6 times. The findings indicate that the Muta mouse is less sensitive to X-ray-induced mutation than UV-induced mutation.

Cytogenotoxicities of sublimed urethane gas to the mouse embryo.

Urethane (ethyl carbamate) which has long been used for commonly used drugs and has proven to be useful in the formation of products in every-day use, is volatile, and small amounts sublime spontaneously. Pregnant ICR mice were maintained in the vinyl chamber (45 liter) which was ventilated 4 times per hour. To inhale urethane gas, air was passed first through a glass bottle containing 500 g of crystalline urethane and then into the vinyl chamber. Concentration of the sublimed urethane gas in the chamber was 1.28 +/- 0.08 mg/l, and sublimed urethane gas produced significantly high incidence of chromosomal aberrations in the cells of whole embryo, when mice inhaled it for 48 h from day 9 to day 11 of pregnancy. High and significant incidence of chromosomal aberrations (36.0%) was detected in the embryo 3 h after urethane gas inhalation, but decreased to 5.3% at 24 h after exposure and showed no significant differences from controls after 48 h, while the incidence in bone marrow cells from the adult (pregnant) mice was lower (21.5%) at 3 h after exposure but a significant increase remained until 72 h after exposure. A majority of chromosomal aberrations was chromatid types. As a consequence of cellular damages by urethane gas inhalation during pregnancy, significantly high incidence of fetal deaths and congenital malformations (cleft palate, polydactyly, tail anomaly etc.) was induced in the offspring. Thus, we must be aware of the risk of volatile chemicals, because it is difficult to perceive and avoid hazardous exposure via respiration.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic mouse increases with age.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic MutaTM mouse was examined at different ages. It was (3.2 +/- 1.3 (SD)) x 10(-5) at newborn and increased almost linearly with age up to (8.3 +/- 1.8) x 10(-5) at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.

High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of mice during pre- and postnatal development.

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5'-end with [gamma-32P]ATP. The cpm% of deoxycytidine 5'-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP)MspI,cpm/[(5mdCMP)MspI,cpm + (dCMP)MspI,cpm] x 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.

Effects of energy restriction on age-associated changes of DNA methylation in mouse liver.

DNA methylation is known to change with age in several mammalian species. Here we have examined the effect of dietary energy restriction on this age-associated change in liver DNA of C3H/SHN mice. The total 5-methyldeoxycytidine level in the genome decreased slightly soon after energy restriction started. The effect, however, diminished with time and no appreciable difference was detected at middle and old ages. The degree of methylation at the c-myc gene, on the other hand, was not affected by energy restriction at early periods, but the age-dependent alterations at later ages were repressed. This is a new finding to show that DNA methylation is one of the molecular indices of aging affected by energy restriction. It suggests an importance of DNA methylation in the aging process.

Prevention of radiation-induced bacteraemia by post-treatment with OK-432 and aztreonam.

The effects of combined treatment with OK-432, an immunomodulator prepared from Streptococcus haemolyticus, and aztreonam, a monobactum antibiotic, in the prevention of radiation-induced bacteraemia and mortality were examined in ICR-MCH mice irradiated with 9.5 Gy. The organisms recovered from the irradiated mice were Streptococcus faecalis and Proteus mirabilis. Treatment with aztreonam reduced the incidence of mice infected with Proteus mirabilis (p < 0.01), but it showed no efficacy on Streptococcus faecalis. OK-432 could reduce the frequency of bacteraemia attributed to both organisms (p < 0.05). Combined treatment with OK-432 and aztreonam further decreased the incidence of bacteraemia by both organisms; no organisms were recovered at 14 days following irradiation. The survival rate at 30 days following irradiation was 80% in mice treated with OK-432 plus aztreonam and 55% with OK-432 alone, while it was 0% in the groups treated with aztreonam or saline alone. These results indicated that combined treatment with OK-432 and a suitable antibiotic such as aztreonam is more effective than OK-432 or aztreonam alone.

Comparison of the age- and tumor-associated changes in the c-myc gene methylation in mouse liver.

Study of DNA methylation of the c-myc gene in liver tumors of BCF1 mice treated with various agents revealed frequent alteration from that in normal liver. The alteration, however, was complex and showed either an increase or a decrease of methylation to various degrees. On the other hand, the tumors obtained from middle-aged C3H/He mice, which develop liver tumors spontaneously at a high incidence rate starting in middle age, showed predominantly increases of methylation in the c-myc gene, while the large tumors found in older mice revealed decreases. The normal part of the liver showed a slight gradual age-dependent increase. These suggest that hypermethylation of the c-myc gene is common to aging and early tumor development in liver. Thus the alteration of DNA methylation seems to be a good clue to investigate why the tumor incidence rate increases rapidly as individuals grow older.

Biological significance of DNA methylation in the ageing process.

In order to understand the possible importance of DNA methylation in ageing, characteristics of its age-associated changes were examined in mouse and man. The total methylated deoxycytidine level in the genome decreased in the senescent period in mouse liver, but not in mouse brain and human liver. The examination of DNA methylation in each individual gene revealed that only a few genes showed alteration in the senescent phase while many genes change in the maturation period. The alterations were gene- and tissue-specific. Comparison of short-living mouse and long-living man for the age-associated changes of the c-myc gene methylation revealed that the rate of change in mouse was about 20 times faster than that in man. This suggests a deep involvement of DNA methylation in ageing. Further investigations into the causes and consequences of the changes would clarify a basic mechanism of ageing.

Post-irradiation protective effect of irsogladine maleate on intestinal crypt stem cells in mice.

Radioprotective effect of irsogladine maleate, an anti-ulcer drug, on the intestinal crypt stem cell survival was studied in mice using a crypt microcolony assay. Irsogladine maleate was injected intraperitoneally immediately after irradiation and then, daily for three days. A successive administration of the drug following 10 Gy of irradiation increased the survival of intestinal stem cells with a clear dose-related trend. In order to estimate the D0, survival curves were determined for X-ray plus placebo and X-ray plus 10 mg/kg of irsogladine maleate. The D0, for X-ray plus the drug was 2.2 Gy while it was 1.9 Gy for X-ray plus placebo. These findings suggest that isogladine maleate can be applied for the alleviation of intestinal damages in heavily irradiated people by radiation accidents.

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.

Teratogenic effects of methamphetamine in mice.

Eight-day pregnant Jcl:ICR mice were administered methamphetamine hydrochloride i.p. at doses of 11, 13, 14, 15, 17, 19 and 21 mg/kg. The number of animals that died after the treatment increased dose-dependently. The highest maternal mortality rate (50%) was observed in the 21 mg/kg group. On day 18 of gestation, the animals were killed and fetuses were examined for external and skeletal malformations. The mortality and malformation rates of fetuses increased with the increase in dose, but both rates reached their highest levels at a dose of 19 mg/kg. Exencephaly, open eyelids, cleft palate and rib anomaly were frequently observed malformations. The single teratogenic dose was estimated to be 19 mg/kg for both external and skeletal malformations. This amount was about ten times the daily intake of many Japanese abusers. The risk of malformed infants being born to female abusers was considered to be high. More crowded conditions have been reported to increase the fetal malformation rate. The higher malformation rate in the 19 mg/kg group than in the 21 mg/kg group was considered to be due to fewer surviving mice in the 21 mg/kg group, because of a higher mortality rate.

Effect of recombinant human granulocyte colony-stimulating factor on survival in lethally irradiated mice.

Repeated injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to lethally irradiated mice increased the rate of animal survival. Dose modification factor was 1.20 when 4.5 micrograms/mouse of rhG-CSF was given daily for 14 days after whole body irradiation. Haematological examinations revealed that rhG-CSF increased the number of blood-circulating leukocytes, neutrophils, monocytes and erythrocytes, but not that of lymphocytes and thrombocytes. Spleen weight and number of endogenous spleen colonies were also increased by rhG-CSF treatment compared with the values for mice irradiated only.

Post-irradiation treatment with OK432 can prevent radiation-induced bone marrow death.

The radioprotective effect of OK432, a Streptococcus haemolyticus preparation, on bone marrow death was examined in mice. The LD50 value was increased from 7.55 Gy in controls to 8.45 Gy in mice treated once with OK432 immediately after irradiation. Multiple administrations of the agent further elevated the LD50 value to 9.56 Gy. The radioprotective effect was also apparent when multiple treatments were commenced as late as 72 h after irradiation.