Evaluation of Harmful Substances and Health Risk Assessment of Mercury and Arsenic in Cosmetic Brands in Nigeria

Forty two different cosmetic samples consisting of 16 facial cosmetics, 6 soaps, 1 shower gel, 12 emulsions, 2 underarm cosmetics, 3 nail cosmetics and 2 perfumes were purchased from department stores and cosmetic shops within Onitsha Main Market and Eke-Awka in Anambra, Nigeria. Seven of these cosmetic (16.67%) were locally manufactured in Nigeria while thirty five (83.33%) were imported into Nigeria. The cosmetics were ashed before digestion and filtration. The filtrates were assayed for mercury and arsenic with AAS SearchTech AA320N. Hydroquinone presence was identified by chromatographic test while steroids, nitrite and N-nitrosamines were identified by colour test and together were assayed by UV-spectrophotometer (Spectrulab 21). The health risk assessment methods developed by the United States Environmental Protection Agency (US EPA) were employed to explore the potential human health risk of Mercury and Arsenic in cosmeticsamples. Results showed that two (2) of the cosmetic samples contained mercury ( 0.003 + 0.000mg/kg and 0.07 + 0.00mg/kg) while three cosmetic samples contained arsenic (0.002 + 0.000, 0.002+0.000 and 0.005 +0.000 mg/kg). Hydroquinone concentration ranged from 1.14 + 0.00 – 1.83 + 0.03 mg/kg (1.14E-02 – 1.83E-02 %).Steroid was found in only two samples with concentration of 16.70 + 0.74 mg/kg and 17.63 + 0.74 while N-nitrosamines and nitrite occurred in nine and eleven samples in the range of 4.66 + 0.09 – 43.52 + 0.47 and 0.87 + 0.02 – 13.42 + 2.90 respectively. The total cancer and non-cancer risk results indicated that although the chances of cancer risk and non-cancer risk resulting from the use of these cosmetic products were unlikely, build up of these heavy metals overtime on continuous usage could be detrimental.


Introduction
Cosmetic products are articles or preparations designed to be used on various parts of the human body for the purpose of cleaning, perfuming, protecting, beautifying, promoting attractiveness or altering appearance without changing the body's structure and operation (Adepoju-Bello et al., 2012;Oyedeji et al., 2011).Cosmetics are of diverse types and may include any material intended for use as a component of a cosmetic product; these include skin moisturizers, perfumes, lipsticks and lip glosses, finger nail polishes, eye and facial makeup preparations, shampoo, hair colours and deodorant.Certain chemicals that are a part of cosmetic formulations have been found to be harmful and use of cosmetic products containing such chemicals portends danger for human health.Notable examples of such chemicals, though not exhaustive, include heavy metals, hydroquinone, steroids and nitrosamines (Adepoju-Bello et al., 2012;SCCS,2012;WHO,2011;Oyedeji et al., 2011;Nnoruka and Okoye, 2006;Nnorom et al., 2005;Adebajo, 2002 ).It is unfortunate that despite regulations put in place to prevent/minimize the presence of such ingredients in cosmetics brands, heavy metals, organic and inorganic chemicals substances are still present in them.Some of the reasons advanced for their presence in cosmetic products include their existence as components of the major raw material used in cosmetic product manufacture (Adepoju-Bello et al., 2012) and their deliberate inclusion in cosmetic products (WHO, 2011).Cosmetic products and their ingredients are not subjected to clinical trials/laboratory testing(s) by regulatory bodies in Nigeria before pre-market approval.This is evident from Section C (2) of National Agency for Food and Drug Administration and Control (NAFDAC) Directorate of Registration and Regulatory Affairs' Guidelines for Registration of imported cosmetics in Nigeria which states that the requirement for cosmetic product registration is the attachment of a comprehensive certificate of analysis to the application for registration.Therefore, product safety and quality are monitored through post-market surveillance (PMS) activity.The implication is that the need for laboratory/clinical testing of cosmetics products by NAFDAC arises only when such products have been used by end-users and are discovered to be defective or have side effects on the consumers.Also in the same guideline, it is stated that mercury and its compounds as well as corticosteroids are not permitted in cosmetic products (Nigeria Nursing World, 2012).This is because mercury is reported to cause dermatitis and its cumulative toxicity causes damage to kidneys which could manifest as hypertension and fatal kidney failure.Prolonged use of cosmetics products containing corticosteroids on the skin causes recalcitrant acne, red striae, excessive hairiness, proneness to infections and absorption through skin which could manifest as severe hypertension, diabetes and cataract (Nigeria Nursing World, 2012).In addition, creams containing hydroquinone greater than two per cent (2%) are banned as they are reported to cause exogenous ochronosis which manifests as a dirty brown pigmentation on sun exposed areas and loss of skin elasticity (Nigeria Nursing World, 2012).
This study aims to evaluate some harmful organic and inorganic constituents and human health risk assessment of mercury and arsenic in cosmetic brands in Nigeria.

2.1Materials
The cosmetic samples used for this study were purchased from department stores and cosmetic shops in Onitsha Main Market and Eke-Awka in Anambra State, South-East Nigeria.

2.2Methods
The identification tests for hydroquinone was carried out on forty-two (42) cosmetic samples using the procedure of isolation and identification of drugs described by Clark(1975) while its thin-layer chromatographic determination and quantitative test were performed using the procedure described by Odumosu and Ekwe (2010).
The Liebermann-Burchard test was employed for the detection of steroid in which 0.5g of the test sample was dissolved in 10 cm 3 of analytical grade chloroform and mixed properly.To 2cm 3 of the sample solution, 6 drops of acetic anhydride was added gently in a test tube with gentle mixing.Then 0.5cm 3 of concentrated sulphuric acid was cautiously added down the side of the tube without mixing.The appearance of a blue-green colour indicates the presence of steroids.For quantitative determination, 1g of the test sample was dissolved in 10ml analytical grade methanol, mixed thoroughly and filtered with Whatman No. 41 filter paper, pre-washed with analytical grade methanol.To 2cm 3 of the sample solution in a 10cm 3 graduated test tube, 2cm 3 of 8M sulphuric acid was added followed by 2cm 3 of 0.5% w/v of Iron (III) Chloride and then 0.5cm 3 of 0.5% w/v potassium haxacyanoferrate (III) solution.
The mixture above was heated in a water bath maintained at 70 o C for 30 minutes with occasional shaking for colour to develop.This was then diluted to the mark with distilled water and absorbance was measured at 780nm against reagent blank.
A 1% solution (standard cholesterol in methanol) was prepared and serially diluted to give 10, 20, 30, 40 and 50 mg/mL solution whose absorbance were measured and together were used to prepare a calibration curve.Actual values of steroids present were extrapolated from the calibration curve.
Nitrosamine was determined using the spectrophotometric method as described in Okafor and Nwogbo (2005) while nitrite was determined by the spectrophotometric method described by Follet and Ratcliff (1963).
Samples were digested to determine mercury and arsenic were by the method described as follows: to a 1g of the sample 20cm 3 mixture of concentrated acid (Nitric acid, perchloric acid and hydrofluoric acid in 4:1:1 ratio) was added and heated in a fume cupboard till fumes stopped evolving.The hot beaker with its content was allowed to cool, then filtered into a 50 cm 3 measuring cylinder and made up to the 50 cm 3 with distilled deionised water.Mercury and arsenic were assayed using Search Tech AA320N Atomic Absorption Spectrometer.

2.3Risk Assessment Methods
The human health risk models including carcinogenic and non-carcinogenic ones established by the United States Environmental Protection Agency (US EPA) were adopted.These models and their threshold values were employed to assess the potential human health risks posed by arsenic and mercury.Human beings could be exposed to mercury and arsenic toxicity via dermal contact with cosmetic particles.
The calculations for the daily exposure dose of contaminants via dermal absorption pathway and the detailed explanation for all the parameters are shown below: Table1.Formula for calculating dermal absorption
In this work, the risk effect is made up of carcinogenic risk assessment for arsenic and mercury through dermal exposure pathway.Cancer risk can be evaluated from: Where cancer risk represents the probability of an individual lifetime health risks from carcinogens; CDI is the chronic daily intake of carcinogens (mg kg -1 d -1 ); SF is the Slope factor of hazardous substances (mg kg -1 d -1 ), Slope factor was calculated using the formula -slope factor = 1/6 (ED 10 ) (US EPA, 2011).
The cumulative cancer risk can be calculated from: Where CDI k is the chronic daily intake (mg kg -1 d -1 ) of substance k (i.e. in a given cosmetic), SF k is the slope factor for substance k (kg d -1 mg -1 ) (i.e. the sum of the calculated slope factor for the heavy metals detected in a given cosmetic).
The acceptable or tolerable risk for regulatory purposes is within the range of 10 -6 -10 -4 .
The non-carcinogenic risk from individual heavy metal can be expressed as the hazard quotient: Where the non-cancer hazard quotient (HQ) is the ratio of exposure to hazardous substances and RFD is the chronic reference dose of the toxicant (mg kg -1 d -1 ).
Where the chronic hazard index (HI) is the sum of more than one hazard quotient for multiple substances, CDI k is the daily intake of heavy metal (k) and RFD k is the chronic reference dose for the heavy metal k.HI values ˃ 1 shows that there is the chance that non-carcinogenic risk may occur and when HI ˂ 1 the reverse applies (Liu et al., 2013).

Results
Table 2.Shows the sample coding and data introduction of cosmetics products manufactured in Nigeria including manufacturing and expiry date, batch number and regulatory authorities (NAFDAC) number where available.[Key: ND -Not detected by method employed,] Table 6.Shows that Royal Gold Cortex of all locally made cosmetics does not pose non-cancer and carcinogenic risks arsenic's hazard quotient of 1.56E-08 is less than one(1), so non cancer risk is unlikely while cancer risk of 2.59E-14 is a minimal value.

Statistical Analysis
Student's t-test was used for the comparison while f-test/ levene's test for equality of variances evaluates the basic assumption of the t-test that the variance of the two groups are approximately equal; t (12) = 0.893, p = 0.390.All differences were considered significant at 5% level, therefore, a p-value ≤ 0.05 was considered statistically significant.Since p-value for the comparison of Nigerian-made cosmetics with imported cosmetics is greater than 0.05 (p = 0.390), it can be concluded that the place of production has no significant effect on the standard (in terms of safety) of cosmetic products sold in Nigeria.Therefore, preference for imported cosmetics is just as a result of mere perception.
Descriptive statistics were calculated using the Statistics for Social Sciences (SPSS) software version 16.The data was displayed using the parameter of the mean value.

Discussion
No mercury was discovered in any of the cosmetic samples (0 %) manufactured in Nigeria.Royal Gold Cortex Remover ® had an arsenic content of 0.002mg/L (2μg/L), which is below the 0.01mg/L (10μg/L) drinking water standard set by World Health Organization (WHO, 2010).Increased levels of skin cancer have been associated with arsenic exposure, even at levels below the 10 part per billion (10μg/L) drinking water standards (Knobeloch et. al., 2006).
Two samples (28.57%) of the cosmetics manufactured in Nigeria, namely, Royal Gold Cortex Remover ® and Classic White Whitening Cream ® , had hydroquinone content of 1.33mg/L(1.33E-04%)and 1.83mg/L(1.83E-04%)respectively, which is below the Maximum Acceptable Limit (MAL) of two per cent (2%) set by NAFDAC (Nigeria Nursing World, 2012).This is indicative that the hydroquinone in these cosmetics may have been used as an antioxidant rather than for skin lightening purposes (Oyedeji et. al., 2011).
None of the locally manufactured cosmetic sample contained steroid.This may suggest compliance with the National Agency for Food and Drug Administration and Control's (NAFDAC) total prohibition of corticosteroid usage in cosmetic products (Nigeria Nursing World, 2012).Only one sample (14.29 %), Beauty Fair Multi Active Toning Cream ® , had Nitrosamines content of 8.37mg/L.A proper classification and identification of the type of nitrosamine compound present in this cosmetic product becomes necessary as studies show that animal species tested with N-nitroso compounds were not resistant to their carcinogenic effect, irrespective of the mode of application.N-nitroso compounds are known to have similar metabolic effect in humans as they have in animals, hence may be carcinogenic to humans as well (Scientific Committee on Consumer Safety, 2012).Furthermore, available epidemiological evidence from case studies on nitrite and nitrosamine supports a positive association with gastric cancer while other evidence supports a positive association between nitrite and nitrosamine intake and oesophageal cancer (Jacksyn and Gonzalez, 2006).
Thirty three foreign cosmetic samples (94.29 %) did not contain mercury.However, two samples (5.71%) namely: Crusader Medicated Soap ® and LenthergicAntiperpirant Roll-on ® , had, mercury contents of 0.07mg/kg and 0.003mg/L respectively.The use of these two cosmetics calls for caution as case studies have shown effects such as tremors, impaired cognitive skills and sleep disturbance in workers with chronic exposure to mercury vapour even at low concentrations in the range 0.7 -42μg/m 3 (equivalent of 7.0 X 10 -10 mg/L(kg) -4.2 X 10 -8 mg/L(kg)) (Ngim et. al., 1992;Liang et. al., 1993).Also, Nigeria's National Agency for Food and Drug Administration and Control (NAFDAC) prohibits inclusion of mercury as ingredients in cosmetics (Nigeria Nursing World, 2012) 5).It is suggested that one of the ways by which humans can protect themselves from any negative effect of nitrosamines and its co-chemicals such as secondary amines and nitrite in cosmetics is to ensure that they are not used in the formulation (Lautenschläger, 2006).Only Beauty Fair Multiactive Toning Cream ® (14.29 %) out of the seven locally manufactured cosmetic sample contained nitrite with a concentration of 8.47 mg/kg which is above the WHO's recommended daily intake (ADI) value of 5.00mg/kg body weight(Table 5).Ten (28.57%) of the imported cosmetic samples contained nitrite with Empire Fade Cream Lotion ® having the least nitrite concentration of 0.87mg/kg while Fair and White Exfoliating Soap ® had the highest nitrite concentration of 12.43 mg/kg.Only four out of the ten imported cosmetic samples containing nitrite, namely Femtight Antiseptic Vaginal Wash ® , Cucumber Extract Facial Toner ® , Black Opal Crème Stick Foundation ® and Empire Fade Crème Lotion ® having concentrations of 4.15 mg/kg, 3.20mg/L, 2.88 mg/kg and 0.87mg/L respectively; had nitrite content below the WHO's recommended daily intake (ADI) value of 5 mg/kg (Table 5).Nitrite is approximately ten (10) times more toxic than nitrate (Schneider, 2012) and when nitrite enters the blood stream; it interacts with the haemoglobin and forms methaemoglobin, a compound which reduces the blood's oxygen carrying capacity (Lenntech, 2014;Okafor and Nwogbo, 2005).

Human Health Risk Assessment
The non-carcinogenic risk and cancer risk values of the elements present in each cosmetic product were determined (Tables 6 and 7).The results showed that for cosmetic products manufactured in Nigeria, only arsenic in Royal Gold Cortex ® with calculated non carcinogenic risk value of 1.56E-08 constituted a non-cancer risk while arsenic in St. Ives Body Cream ® (non-cancer risk value =4.68E-07), Bioclaire Body Lightening Lotion ® (non-cancer risk value =1.17E-06) and mercury in Crusader Medicated Soap ® (non-cancer risk value =5.47E-09) constituted non-cancer risks for cosmetic products manufactured outside Nigeria.Only arsenic is carcinogenic among the two elements examined (Liu et al., 2013), although the US EPA has determined mercuric chloride and methyl mercury as possible human carcinogen (Das et al., 2011).For cosmetic products manufactured in Nigeria, its calculated cancer risk value in Royal Gold Cortex ® (cancer risk =2.59E-14) constituted the only cancer risk while for cosmetic products manufactured outside Nigeria, its cancer risk value in St. Ives Body Cream (cancer risk =7.18E-18) and Bioclaire Body Lightening Lotion ® (cancer risk =1.95E-12) constituted the cancer risks.These values also translated into the total non-cancer risk values (Hazard Indices [HI]) and cancer risk values for each product.The values fell below the non-cancer risk (Hazard Index) and cancer risk threshold values of one (1) and 10 -6 -10 -4 respectively set by USEPA (Liu et al., 2013;US EPA 2001).These imply that the chance of non-cancer and cancer risk resulting from exposure to these metals at these concentrations is unlikely; however, heavy metals are cumulative in nature due to their long half lives and therefore build up of these elements on continuous usage of these cosmetic products could be detrimental.Mercury is also associated with kidney damage (Scragg, 2006).The US EPA has determined that mercuric chloride and methyl mercury are possible human carcinogens.The nervous system is very sensitive to all forms of mercury.Exposure to high levels can permanently damage the brain, kidneys, and developing foetuses.Effects on brain functioning may result in irritability, shyness, tremors, changes in vision or hearing and memory problems (Das et al., 2011).Inorganic arsenic is a known carcinogen and can cause cancer of the skin, lungs, liver and bladder.Ingestion of very high levels can possibly result in death.Long term low level exposure to arsenic can cause a darkening of the skin and the appearance of small "corn" or "warts" on the palms and soles (Das et al., 2011).Some major clinical effects of mercury and arsenic include tremor, gingivostomatitis, peripheral neuropathy, acrodynia, acute tubular necrosis, gastroenteritis, CNS effects and birth effects (Das et al., 2011).Despite the awareness of health hazards associated with organic and inorganic chemical compounds as well as heavy metal exposures, medical diagnosis of patients who may be suffering from heavy metal poisoning are highly reported in Nigeria hospitals.Although serious evidence that non communicable diseases (NCDs) such as cardiovascular diseases, diabetes mellitus, cancer and respiratory diseases which may be associated with heavy metal exposures and which were, before now, the major causes of morbidity and mortality in the developed world are now emerging as an important component of the disease burden in developing countries like Nigeria (Gill, 1999).In their work, Unachukwu et al., (2008), showed that out of 3294 total medical admissions at the University of Port-Harcourt Teaching Hospital (UPTH) Nigeria, from June, 2000 to June, 2004June, , 56.2% (1853) ) were cases of various non-communicable diseases.The breakdown showed that diseases of the cardiovascular, endocrine and renal systems occurred most accounting for 35.7% (662), 18.5% (342), and 18.6% (311) respectively.Others were neurological 14.5% (269), gastrointestinal 5.0% (93), haematological 4.9% (911), respiratory problems 2.4% (44), musculoskeletal 1.82% (33) while dermatological diseases accounted for 0.4% (7) of non-communicable admissions (Unachukwu et al., 2008), hypertension, diabetes mellitus and chronic renal failure were the most common cardiovascular, endocrine and renal disorders respectively.Various heavy metals have been implicated in most of these non-communicable diseases.

Conclusion
The existence of these hazardous substances in most of these cosmetic products examined calls for caution in their use by consumers as these cosmetic products may constitute significant route of exposure (most especially via the dermis) to these hazardous substances thereby constituting health concerns over extensive use.

Table 2 .
Sample Coding of Cosmetic Products Manufactured in Nigeria [Key:NA -Not Available, NAFDAC -National Agency for Food and DrugAdministration and Control]

Table 3 .
Shows the sample coding and data information of cosmetics products manufactured outside Nigeria including manufactures, country of manufacture, date of manufacture and expiration, batch number and regulatory authority's (NAFDAC) number, if any.

Table 3 .
Sample Coding of Cosmetic Products Manufactured Outside Nigeria.

Table 4 .
Shows that all the cosmetic products manufactured in Nigeria had no detectable mercury by the method employed while only Royal Gold Cortex Remover ® , of products made in Nigeria had 0.002mg/kg of arsenic.1.83 ±0.03mg/kg and 1.33 ± 0.03mg/kg of hydroquinone were contained in classic White Whitening Cream ® and Royal Acrylic Nail Remover.Steroids were not detected in any of the locally made products.8.38 ± 0.04 and 8.47 ±0.01mg/kg (mg/l) of nitrosamine and nitrites were determined in Beauty Fair Multiactive Toning Cream.

Table 4 .
Levels of Hazardous Substances in Cosmetic Products Manufactured in Nigeria.

Table 5 .
Levels of Hazardous Substances in Cosmetic Products Manufactured Outside Nigeria.

Table 6 .
Non-carcinogenic Risk (Hazard Quotient [HQ]) and Cancer Risk for Individual Mercury and Arsenic Detected in Cosmetic Products Manufactured in Nigeria.

Table 7 .
Non-carcinogenic Risk (Hazard Quotient [HQ]) and Cancer Risk for Individual Heavy Metal Detected in Cosmetic Products Manufactured outside Nigeria.