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Hair dyes and the chromosomes
Articles of general interest--Fd work involved isotopic labelling of DNA with [6-JH]uracil or [2-14C]adenine over five or six generations followed by conversion of the cells to spheroblasts (swollen cells), either immediately (for controls) or after treatment with formaldehyde, or after further incubation of control or formaldehyde-treated cells. In some cases the formation of spheroblasts (carried out to facilitate subsequent lysis) preceded the formaldehyde treatment. These procedures were followed by lysis of the spheroblasts on the top of alkaline sucrose gradients through which the DNA was subsequently sedimented. Analysis of the sedimentation profiles obtained under the various experimental conditions showed that formaldehyde caused a dose-dependent incidence of single-strand breaks in the DNA of exponentialphase cells of the wild-type yeast through a metabolic process, a finding in line with that in E. coli reported by Poverenny rr uI. (lot. cit.). Further evidence was also obtained in support of the indications of this process presented by Chanet ef al. (1976, lot. cit.). Mutant strains of S. cereoisiae defective in excision repair of pyrimidine dimers induced by ultraviolet irradiation showed a reduced capacity to undergo single-strand breaks after treatment with formaldehyde, but as is the case with ultraviolet irradiation. they were also more sensitive than the wild-type cells to the lethal effect of the treatment. These findings suggest a common step in the mechanisms for the repair of damage induced by irradiation and by formaldehyde. Incubation of wild-type cells in growth
HAIR
DYES
AND
TtiE
The controversial debate over the safety-in-use of commercially available hair dyes continues apace. Ever since Ames and his colleagues (Proc. mtn. Acad. Sci. 1975. 72, 2423) produced evidence that certain hair-dye constituents were mutagenic when tested in short-term laboratory tests designed to detect mutagens and potential carcinogens, these materials have been subjected to an intensive programme of toxicological examination. Already a number of these compounds, including 2,4-toluenediamine (2,4-TD). 2.4-diaminoanisole (4-methoxy-m-phenylenediamine; 2.4-DAA). Direct Blue 6. Direct Black 38, Direct Brown 95 and 4.4’-thiodianiline have been shown to have a carcinogenic effect in studies that involved the feeding of each material to rats (and mice in some cases) at high dose levels (Fd hem. News 1978, 19 (46), 60; Federal Register 1978, 43, 19923 & 20562). Whilst the use of some ingredients has been voluntarily abandoned by some manufacturers, the United States authorities have now proposed a label warning for hair dyes containing ingredients that have been shown to cause cancer in laboratory animals (Fd them. News 1978, 19 (43), 38). Although the findings of Ames et al. (Ioc. cit.) have since been substantiated by the experimental animal studies, efforts to refine and demonstrate the validity of short-term predictive tests have led to an examination of the effects of various hair-dye components in eukaryotic systems. Using L5178Y mouse lymphoma cells. Palmer er al. (J. enuir. Path. Toxicol.
Cosmer. Toxicol.
Vol. 17. No. 3
301
medium after exposure to formaldehyde showed a lag in cell division during which rejoining of single-strand breaks occurred, leading to a low recovery of DNA of normal molecular weight. Single-strand breaks are not the only lesion produced in yeast DNA by formaldehyde. Other work by this group (Magaiia-Schwencke & Ekert, lot. cit.). involving conversion of labelled cells of a wild-type haploid yeast to protoplasts (cells denuded of the cell wall), before or after treatment with formaldehyde. and subsequent lysis and sedimentation on a neutral sucrose gradient, demonstrated a dose-related induction of cross-links between DNA and proteins by formaldehyde. Ultraviolet irradiation also produced cross-links between DNA and proteins in this strain. However, the degree of cell survival for a comparable degree of cross-linking was much higher after formaldehyde treatment than after radiation, suggesting either that formaldehyde-induced cross-links were repaired more efficiently than those due to radiation exposure or that another type of damage, such as the pyrimidine dimers induced by radiation, were responsible for the observed differences. These studies shed useful additional light on the biochemical mechanisms underlying the genetic effects of formaldehyde in simple organisms. knowledge that may, in the long run, help to establish the extent to which effects of this type may be relevant to the mammalian situation. [P. Cooper-BIBRA]
CHROMOSOMES 1977, 1, 87) were able to assess chemically-induced mutations at the thymidine kinase (TK) heterozygous locus. The test system was exposed to 2,5-diaminoanisole sulphate (2.5-DAA) at 3.125, 4.69 and 6.25 pg/ml. to 2,4-DAA at 12.5, 18.75 and 25 pg/ml, to 2-nitro-pphenylenediamine (2-NPPD) at 25, 50 and 75 pg/ml. to rn-phenylenediamine (MPD) at 25, 50 and IO0 pg/ml, and to 4-nitro-o-phenylenediamine (CNOPD) at 50, 100 and 200pg/ml, in each case for a period of 24 hours. All three phenylenediamines produced dose-related increases in the total number of mutants, in mutation frequency and in the ratios of induced to spontaneous mutation frequency. MPD being the most potent and 4-NOPD the least. Although the diaminoanisoles produced similar effects. the results proved rather more difficult to interpret because of the narrow range of dose levels used. As a result of this, the investigators suggested that 2.4-DAA had produced a questionable mutagenic response while 2.5-DAA had evoked a negative response. They also concluded that, in the absence of an activation component for the system, enzymes actually present in the cells were probably involved. during the long exposure period, in the production of mutagenic intermediates responsible for the positive response. Alternatively they suggested that the compounds underwent a structural alteration to pr@ duce the active compound during the long incubation period at 37°C. The responses to 2,4-DAA and 2,5-DAA seemed to indicate that the enzymes that
302
Articlesof general interest-Fd Cosmet.Toxicol. Vol.
17,
No. 3
activated these latter compounds were different from extended to involve human cell types. Seafle et al. those that activated the phenylenediamines. A simi- (Nature, Lmd. 1975, 255, 506) have looked into the larly long exposure period also ve rise to a positive events following exposure of cultures of human peripheral blood lymphocytes to 2-NPPD. At concenresponse to 2-NPPD and 4-N crPD in a morphologitrations between 50 and lOO~g/ml a considerable cal transformation system used by Benedict (Nature, Lond. 1976, 260, 368). number of chromosome and chromatid gaps and The same investigator (lot. cit.) also examined chro- breaks were evident. Mitotic delay and toxicity were mosomal aberrations in a hamster cell line, evident at lOO/~g/ml but it was still possible to A(T,)Cl-3, being developed as a rapid screen for demonstrate that up to 45% of the cells contained chemical carcinogens. Both 2-NPPD and 4-NOPD damaged chromosomes. Cultures treated with 25 pg produced chromatid breaks. In addition, chromo- 2-NPPD/ml did not differ significantly from controls. Similar experiments with CNOPD at concentrations somal aberrations of a type considered by the author to be indicative of malignant transformation and up to lOO~g/ml did not produce any chromosomal oncogenesis resulted from treatment with 2-NPPD damage. More recently such investigations of chromosomal but not 4-NOPD. Unfortunately the scoring of chromosomal aber- damage have been utilized in an epidemiological rations has a number of disadvantages. It is a highly study of the potentially genotoxic effects of hair dyes skilled and subjective procedure and very tedious at (Kirkland et a/. Lancer 1978, II, 124). Sixty hair low concentrations of the chemical. Recently, how- tinters and 36 control individuals were investigated. ever, new cytological techniques have been develaped The tinters were volunteers from salons, whilst conwhich permit an assessmentof chromosomal damage trols were drawn both from within the hairdressing by scoring the frequency of sister chromatid profession and from other occupations. All volunteers exchanges (SCEs). When Chinese hamster cells are had to provide details of smoking habits, alcohol conallowed to pass through two cell cycles in the pres- sumption, medical history, use of drugs, infections, ence of 5bromodeoxyuridine and are then stained, vaccinations, X-ray exposure and whether or not their SCEs, showing a dose-dependent response following own hair was dyed. Information on occupational exposure to a range of chemical mutagens, may be exposure was also gathered. Results from the study observed. Using this method, Perry & Searle indicated that (i) there were no significant differences (Mutation Res. 1977, 56, 207) examined the induction in chromosomal damage in the cultured lymphocytes of SCEs by the hair-dye components 2-NPPD and from the peripheral blood of tinters and controls CNOPD. Exponentially-growing cultures of a near closely matched for age and sex, (ii) when agediploid Chinese hamster cell line growing in McCoy’s matched women were re-grouped according to 5A medium with HEPES buffer were exposed to 1 whether their own hair was dyed or not, a significant 10 LIM 5-bromodeoxvuridine together with varying excess of chromatid and chromosome gaps and concentrations of the dye components. Ethyl methI breaks was observed in women with dyed hair, (iii) anesulphonate (EMS) was used as a positive control. men whose hair was not dyed (mean age 31-5 years) Both dye components at each concentration produced had significantly more chromosome breaks than men high frequencies of SCEs and were somewhat more with dyed hair (mean age 22.9 years). Discussing these potent than EMS. The 2-NPPD proved to be slightly findings, the investigators pointed out that tinters more potent than 4-NOPD, but the latter exhibits normally wear gloves during the dyeing operation a poor solubility in tissue-culture media. and, even in the absence of gloves, percutaneous Negative results were obtained by Hossack & absorption of dye components may be impeded by Richardson (Experientia 1977, 33, 377) in micronuc- the horny surface of the hands and the lack of sebaleus tests on I2 hair-dye constituents. The compounds ceous glands in the palms. They added that dye conexamined were 2,4-DAA, p-phenylenediamine, stituents are more readily absorbed through the scalp 4-NOPD. 2-NPPD, paminophenol, m-aminophenol, and would therefore be more likely to induce chromo2-amino-4-nitrophenol, 2,5-toluenediamine sulphate, somal aberrations by this route. The authors felt that resorcinol, 4-chlororesorcinol, 4-amino-2-hydroxyan age effect might be responsible for the lower fretoluene and 1-naphthol. Groups comprising five male quency of chromosome aberrations in the young men and five female rats were dosed by gastric intubation with dyed hair compared that recorded in slightly with the test compounds in 0.5% (w/v) gum traga- older men without dyed hair. Furthermore, it was canth containing O*OS’%sodium sulphite. The total suggested that the frequency of hair-dye use by the dosages were close to the lethal’doses and were ad- younger men was too low to reverse this age effect. ministered in two equal parts separated by an interval In conclusion it was emphasized that the excess of of 24 hours. Six hours after the second dose the ani- cytogenetic damage in women with dyed hair could mals were killed and bone-marrow smears were pre- not be considered definitive at this stage. pared. The incidence of micronucleated cells per 2ooO The study by Kirkland et al. (lot. cit.) has given polychromatic erythrocytes was compared with the rise to some controversy. In a brief comment on the values for the control group and with laboratory stan- paper (ibid 1978, II, 271), Van Abbe extracted the dard values. The mean values and ranges of mic- data for subjects who had recently been exposed to ronucleated cells found were essentially similar to X-rays or viral infections, whether or not their hair those of the control group and were within the labora- had been dyed. On analysis, it appeared that exposure tory standard range. None of the compounds to X-rays or a recent viral infection significantly inshowed evidence of mutagenicity in this test. creased the proportion of damaged cells. In contrast. Understandably the examination of effects of hair- the effect of the dyeing of the subject’s own hair was dye components on chromosomal material has been not significant nor was there any significant interac-
Articles
or general
interest-
Ftl Cmrmq.
tion between dyeing and recent cxposurc to X-rays or virus infection. On the basis of these findings, Van Ahhc suggested that :I lady who had her hair tinted 90 times with a permanent hair dye and ten times with a semi-permanent enc. was unlikely to suffer grcatcr chromosomal damngc than if she underwent routine dental X-rays or occasionally caught a cold. In their reply. Kirkland and his colleagues (ihid 1978. II. 272) pointed out that their definition of X-ray exposure did not cover procedures like dental X-rays; they were referring to diagnostic X-ray of considcrablc dosage. and the viral exposure considcrcd were recent influenza. chicken pox. heavy vaccination or glandular fever but not the common cold. Van Abbe acknowledged these points in a subsequent lcttcr (ihid 1978. II. 368). commenting that if the chromosome damage associated with hair dyeing was similar in degree to that caused by routine dental or chest X-rays or common-cold infection. the risk would not seem to be unreasonable. In the same issue of the Lancct. the statistical methods used by both sides in this discussion were the subject of further questioning. with a suggestion that the data would bc better analyscd using a G-test (Price. ibid 1978. II. 368). USC of this method confirmed that there was ;I statistically significant excess of damaged cells in the women with dyed hair as well as in those exposed to X-rays or recent viral infection. but that there was no interaction between the two factors. A detailed criticism of the study was added to the debate by Fcinstcin (ihid 1978. II, 627) but all his points were answered by Kirkland PI (I/. (ihid 1978. II. 628). who reiterated their original statement that their findings were “not definitive” and that further studies were needed. This important point seemed in dimgcr of becoming lost in the lengthy arguments. An important comment comes from Burnett (ihid 1978. II. 685). Accepting these data at face value, there remain serious doubts about their significance in terms of human hazard from cancer or mutation. Observations of chromosome gaps and breaks alone
Tosic~ol.
Vol.
17. No.
3
303
cannot be taken as an indication of heritable damage. nor is there any strong evidence that they arc closely associated with malignancy. Both statistical and risk analyses arc an important aspect of any interpretation of the safety-in-use of ;I chemical. whether it be a food additive. a cosmetic or an industrial chemical. Already the relevance for man or the results from the animal carcinogenicity studies on 2.4-DAA has been questioned (Fd chm News 1978,20 (9). 32). Dr. H. J. Eiermann of the FDA Division of Cosmetics Toxicology has indicated that normal use of hair dyes would be equivalent to ii dietary dose of 2.4-DAA of only 003033 mg/kg/day. The CTFA. which claims that even the proposed label warning is unjustified and should be withdrawn. has estimated the risks of the various dyes at bctwccn I in I6 million and I in 2400 million. FDA estimates arc considerably higher. ranging from I in 31.000 to I in a million. All of the projected risk assessments arc worded in a rather conservative fashion and admit the need for better data to make more reasoned analyses. In an attempt to resolve the difficulties of making risk assessments on such materials. an interagency workshop on percutaneous toxicology has now been set up in the United States. The results from epidemiological studies have an important contribution to make to the debate. In this respect, the IARC has already stated that the cpidcmiological evidence currently available suggests an elevated risk for both users of hair dyes and those (barbers and hairdressers) occupationally exposed to hair preparations (this issue, p. 294). The report goes on to suggest that epidemiological studies, which should include workers employed in the production of hair dyes, arc required before any firm conclusions can be drawn. These could in turn be supplemented by a further examination of exposed individuals for gcnotoxic effects. along the lines of the studies undertaken by Kirkland CI ul. (lot. cif.). [S.
P. Johnson-BIBRA]
HAIR
DYES
AND
TtiE
The controversial debate over the safety-in-use of commercially available hair dyes continues apace. Ever since Ames and his colleagues (Proc. mtn. Acad. Sci. 1975. 72, 2423) produced evidence that certain hair-dye constituents were mutagenic when tested in short-term laboratory tests designed to detect mutagens and potential carcinogens, these materials have been subjected to an intensive programme of toxicological examination. Already a number of these compounds, including 2,4-toluenediamine (2,4-TD). 2.4-diaminoanisole (4-methoxy-m-phenylenediamine; 2.4-DAA). Direct Blue 6. Direct Black 38, Direct Brown 95 and 4.4’-thiodianiline have been shown to have a carcinogenic effect in studies that involved the feeding of each material to rats (and mice in some cases) at high dose levels (Fd hem. News 1978, 19 (46), 60; Federal Register 1978, 43, 19923 & 20562). Whilst the use of some ingredients has been voluntarily abandoned by some manufacturers, the United States authorities have now proposed a label warning for hair dyes containing ingredients that have been shown to cause cancer in laboratory animals (Fd them. News 1978, 19 (43), 38). Although the findings of Ames et al. (Ioc. cit.) have since been substantiated by the experimental animal studies, efforts to refine and demonstrate the validity of short-term predictive tests have led to an examination of the effects of various hair-dye components in eukaryotic systems. Using L5178Y mouse lymphoma cells. Palmer er al. (J. enuir. Path. Toxicol.
Cosmer. Toxicol.
Vol. 17. No. 3
301
medium after exposure to formaldehyde showed a lag in cell division during which rejoining of single-strand breaks occurred, leading to a low recovery of DNA of normal molecular weight. Single-strand breaks are not the only lesion produced in yeast DNA by formaldehyde. Other work by this group (Magaiia-Schwencke & Ekert, lot. cit.). involving conversion of labelled cells of a wild-type haploid yeast to protoplasts (cells denuded of the cell wall), before or after treatment with formaldehyde. and subsequent lysis and sedimentation on a neutral sucrose gradient, demonstrated a dose-related induction of cross-links between DNA and proteins by formaldehyde. Ultraviolet irradiation also produced cross-links between DNA and proteins in this strain. However, the degree of cell survival for a comparable degree of cross-linking was much higher after formaldehyde treatment than after radiation, suggesting either that formaldehyde-induced cross-links were repaired more efficiently than those due to radiation exposure or that another type of damage, such as the pyrimidine dimers induced by radiation, were responsible for the observed differences. These studies shed useful additional light on the biochemical mechanisms underlying the genetic effects of formaldehyde in simple organisms. knowledge that may, in the long run, help to establish the extent to which effects of this type may be relevant to the mammalian situation. [P. Cooper-BIBRA]
CHROMOSOMES 1977, 1, 87) were able to assess chemically-induced mutations at the thymidine kinase (TK) heterozygous locus. The test system was exposed to 2,5-diaminoanisole sulphate (2.5-DAA) at 3.125, 4.69 and 6.25 pg/ml. to 2,4-DAA at 12.5, 18.75 and 25 pg/ml, to 2-nitro-pphenylenediamine (2-NPPD) at 25, 50 and 75 pg/ml. to rn-phenylenediamine (MPD) at 25, 50 and IO0 pg/ml, and to 4-nitro-o-phenylenediamine (CNOPD) at 50, 100 and 200pg/ml, in each case for a period of 24 hours. All three phenylenediamines produced dose-related increases in the total number of mutants, in mutation frequency and in the ratios of induced to spontaneous mutation frequency. MPD being the most potent and 4-NOPD the least. Although the diaminoanisoles produced similar effects. the results proved rather more difficult to interpret because of the narrow range of dose levels used. As a result of this, the investigators suggested that 2.4-DAA had produced a questionable mutagenic response while 2.5-DAA had evoked a negative response. They also concluded that, in the absence of an activation component for the system, enzymes actually present in the cells were probably involved. during the long exposure period, in the production of mutagenic intermediates responsible for the positive response. Alternatively they suggested that the compounds underwent a structural alteration to pr@ duce the active compound during the long incubation period at 37°C. The responses to 2,4-DAA and 2,5-DAA seemed to indicate that the enzymes that
302
Articlesof general interest-Fd Cosmet.Toxicol. Vol.
17,
No. 3
activated these latter compounds were different from extended to involve human cell types. Seafle et al. those that activated the phenylenediamines. A simi- (Nature, Lmd. 1975, 255, 506) have looked into the larly long exposure period also ve rise to a positive events following exposure of cultures of human peripheral blood lymphocytes to 2-NPPD. At concenresponse to 2-NPPD and 4-N crPD in a morphologitrations between 50 and lOO~g/ml a considerable cal transformation system used by Benedict (Nature, Lond. 1976, 260, 368). number of chromosome and chromatid gaps and The same investigator (lot. cit.) also examined chro- breaks were evident. Mitotic delay and toxicity were mosomal aberrations in a hamster cell line, evident at lOO/~g/ml but it was still possible to A(T,)Cl-3, being developed as a rapid screen for demonstrate that up to 45% of the cells contained chemical carcinogens. Both 2-NPPD and 4-NOPD damaged chromosomes. Cultures treated with 25 pg produced chromatid breaks. In addition, chromo- 2-NPPD/ml did not differ significantly from controls. Similar experiments with CNOPD at concentrations somal aberrations of a type considered by the author to be indicative of malignant transformation and up to lOO~g/ml did not produce any chromosomal oncogenesis resulted from treatment with 2-NPPD damage. More recently such investigations of chromosomal but not 4-NOPD. Unfortunately the scoring of chromosomal aber- damage have been utilized in an epidemiological rations has a number of disadvantages. It is a highly study of the potentially genotoxic effects of hair dyes skilled and subjective procedure and very tedious at (Kirkland et a/. Lancer 1978, II, 124). Sixty hair low concentrations of the chemical. Recently, how- tinters and 36 control individuals were investigated. ever, new cytological techniques have been develaped The tinters were volunteers from salons, whilst conwhich permit an assessmentof chromosomal damage trols were drawn both from within the hairdressing by scoring the frequency of sister chromatid profession and from other occupations. All volunteers exchanges (SCEs). When Chinese hamster cells are had to provide details of smoking habits, alcohol conallowed to pass through two cell cycles in the pres- sumption, medical history, use of drugs, infections, ence of 5bromodeoxyuridine and are then stained, vaccinations, X-ray exposure and whether or not their SCEs, showing a dose-dependent response following own hair was dyed. Information on occupational exposure to a range of chemical mutagens, may be exposure was also gathered. Results from the study observed. Using this method, Perry & Searle indicated that (i) there were no significant differences (Mutation Res. 1977, 56, 207) examined the induction in chromosomal damage in the cultured lymphocytes of SCEs by the hair-dye components 2-NPPD and from the peripheral blood of tinters and controls CNOPD. Exponentially-growing cultures of a near closely matched for age and sex, (ii) when agediploid Chinese hamster cell line growing in McCoy’s matched women were re-grouped according to 5A medium with HEPES buffer were exposed to 1 whether their own hair was dyed or not, a significant 10 LIM 5-bromodeoxvuridine together with varying excess of chromatid and chromosome gaps and concentrations of the dye components. Ethyl methI breaks was observed in women with dyed hair, (iii) anesulphonate (EMS) was used as a positive control. men whose hair was not dyed (mean age 31-5 years) Both dye components at each concentration produced had significantly more chromosome breaks than men high frequencies of SCEs and were somewhat more with dyed hair (mean age 22.9 years). Discussing these potent than EMS. The 2-NPPD proved to be slightly findings, the investigators pointed out that tinters more potent than 4-NOPD, but the latter exhibits normally wear gloves during the dyeing operation a poor solubility in tissue-culture media. and, even in the absence of gloves, percutaneous Negative results were obtained by Hossack & absorption of dye components may be impeded by Richardson (Experientia 1977, 33, 377) in micronuc- the horny surface of the hands and the lack of sebaleus tests on I2 hair-dye constituents. The compounds ceous glands in the palms. They added that dye conexamined were 2,4-DAA, p-phenylenediamine, stituents are more readily absorbed through the scalp 4-NOPD. 2-NPPD, paminophenol, m-aminophenol, and would therefore be more likely to induce chromo2-amino-4-nitrophenol, 2,5-toluenediamine sulphate, somal aberrations by this route. The authors felt that resorcinol, 4-chlororesorcinol, 4-amino-2-hydroxyan age effect might be responsible for the lower fretoluene and 1-naphthol. Groups comprising five male quency of chromosome aberrations in the young men and five female rats were dosed by gastric intubation with dyed hair compared that recorded in slightly with the test compounds in 0.5% (w/v) gum traga- older men without dyed hair. Furthermore, it was canth containing O*OS’%sodium sulphite. The total suggested that the frequency of hair-dye use by the dosages were close to the lethal’doses and were ad- younger men was too low to reverse this age effect. ministered in two equal parts separated by an interval In conclusion it was emphasized that the excess of of 24 hours. Six hours after the second dose the ani- cytogenetic damage in women with dyed hair could mals were killed and bone-marrow smears were pre- not be considered definitive at this stage. pared. The incidence of micronucleated cells per 2ooO The study by Kirkland et al. (lot. cit.) has given polychromatic erythrocytes was compared with the rise to some controversy. In a brief comment on the values for the control group and with laboratory stan- paper (ibid 1978, II, 271), Van Abbe extracted the dard values. The mean values and ranges of mic- data for subjects who had recently been exposed to ronucleated cells found were essentially similar to X-rays or viral infections, whether or not their hair those of the control group and were within the labora- had been dyed. On analysis, it appeared that exposure tory standard range. None of the compounds to X-rays or a recent viral infection significantly inshowed evidence of mutagenicity in this test. creased the proportion of damaged cells. In contrast. Understandably the examination of effects of hair- the effect of the dyeing of the subject’s own hair was dye components on chromosomal material has been not significant nor was there any significant interac-
Articles
or general
interest-
Ftl Cmrmq.
tion between dyeing and recent cxposurc to X-rays or virus infection. On the basis of these findings, Van Ahhc suggested that :I lady who had her hair tinted 90 times with a permanent hair dye and ten times with a semi-permanent enc. was unlikely to suffer grcatcr chromosomal damngc than if she underwent routine dental X-rays or occasionally caught a cold. In their reply. Kirkland and his colleagues (ihid 1978. II. 272) pointed out that their definition of X-ray exposure did not cover procedures like dental X-rays; they were referring to diagnostic X-ray of considcrablc dosage. and the viral exposure considcrcd were recent influenza. chicken pox. heavy vaccination or glandular fever but not the common cold. Van Abbe acknowledged these points in a subsequent lcttcr (ihid 1978. II. 368). commenting that if the chromosome damage associated with hair dyeing was similar in degree to that caused by routine dental or chest X-rays or common-cold infection. the risk would not seem to be unreasonable. In the same issue of the Lancct. the statistical methods used by both sides in this discussion were the subject of further questioning. with a suggestion that the data would bc better analyscd using a G-test (Price. ibid 1978. II. 368). USC of this method confirmed that there was ;I statistically significant excess of damaged cells in the women with dyed hair as well as in those exposed to X-rays or recent viral infection. but that there was no interaction between the two factors. A detailed criticism of the study was added to the debate by Fcinstcin (ihid 1978. II, 627) but all his points were answered by Kirkland PI (I/. (ihid 1978. II. 628). who reiterated their original statement that their findings were “not definitive” and that further studies were needed. This important point seemed in dimgcr of becoming lost in the lengthy arguments. An important comment comes from Burnett (ihid 1978. II. 685). Accepting these data at face value, there remain serious doubts about their significance in terms of human hazard from cancer or mutation. Observations of chromosome gaps and breaks alone
Tosic~ol.
Vol.
17. No.
3
303
cannot be taken as an indication of heritable damage. nor is there any strong evidence that they arc closely associated with malignancy. Both statistical and risk analyses arc an important aspect of any interpretation of the safety-in-use of ;I chemical. whether it be a food additive. a cosmetic or an industrial chemical. Already the relevance for man or the results from the animal carcinogenicity studies on 2.4-DAA has been questioned (Fd chm News 1978,20 (9). 32). Dr. H. J. Eiermann of the FDA Division of Cosmetics Toxicology has indicated that normal use of hair dyes would be equivalent to ii dietary dose of 2.4-DAA of only 003033 mg/kg/day. The CTFA. which claims that even the proposed label warning is unjustified and should be withdrawn. has estimated the risks of the various dyes at bctwccn I in I6 million and I in 2400 million. FDA estimates arc considerably higher. ranging from I in 31.000 to I in a million. All of the projected risk assessments arc worded in a rather conservative fashion and admit the need for better data to make more reasoned analyses. In an attempt to resolve the difficulties of making risk assessments on such materials. an interagency workshop on percutaneous toxicology has now been set up in the United States. The results from epidemiological studies have an important contribution to make to the debate. In this respect, the IARC has already stated that the cpidcmiological evidence currently available suggests an elevated risk for both users of hair dyes and those (barbers and hairdressers) occupationally exposed to hair preparations (this issue, p. 294). The report goes on to suggest that epidemiological studies, which should include workers employed in the production of hair dyes, arc required before any firm conclusions can be drawn. These could in turn be supplemented by a further examination of exposed individuals for gcnotoxic effects. along the lines of the studies undertaken by Kirkland CI ul. (lot. cif.). [S.
P. Johnson-BIBRA]