SCCNFP/0649/03, final

THE SCIENTIFIC COMMITTEE ON COSMETIC PRODUCTS AND NON-FOOD PRODUCTS
INTENDED FOR CONSUMERS

OPINION

CONCERNINGZINC OXIDECOLIPA n° S 76

Adopted by the SCCNFP during the 24th plenary meetingof 24-25 June 2003

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

1.

Terms of Reference

1.1

Context of the question

The adaptation to technical progress of the Annexes to Council Directive 76/768/EEC of 27 July
1976 on the approximation of the laws of the Member States relating to cosmetic products.
Request for inclusion of Zinc oxide in Annex VII, part 1 – List of permitted UV Filters which
Cosmetic Products may contain – to Council Directive 76/768/EEC.
1.2

Request to the SCCNFPThe SCCNFP is requested to answer the following questions :*Is Zinc oxide safe for use in cosmetic products as a UV filter up to 25 %?

*Does the SCCNFP propose any restrictions or conditions for its use in cosmetic products?

1.3

Statement on the toxicological evaluation

The SCCNFP is the scientific advisory body to the European Commission in matters ofconsumer protection with respect to cosmetics and non-food products intended for consumers.
The Commission’s general policy regarding research on animals supports the development ofalternative methods to replace or to reduce animal testing when possible. In this context, theSCCNFP has a specific working group on alternatives to animal testing which, in co-operation
with other Commission services such as ECVAM (European Centre for Validation of Alternative
Methods), evaluates these methods.
The extent to which these validated methods are applicable to cosmetic products and its
ingredients is a matter of the SCCNFP.
SCCNFP opinions include evaluations of experiments using laboratory animals; such tests are
conducted in accordance with all legal provisions and preferably under chemical law regulations.
Only in cases where no alternative method is available will such tests be evaluated and theresulting data accepted, in order to meet the fundamental requirements of the protection of
consumer health.

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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

2.

Toxicological Evaluation and Characterisation

2.1.

GeneralThroughout this document, it is assumed that the biological and toxicological effects are
determined by the zinc cation. As such, data from any inorganic zinc compound has been used toassess the toxicological potential of ZnO.
Zn2+ is generally considered a non-toxic metal. This view, as reviewed by Walsh et al., isbased on the knowledge that Zn 2+ (5) :
*
*
*
*
*
*

is an essential nutrient present in virtually every cell and critical for numerous
physiological functions;must be consumed in the diet and absorbed to maintain human health;does not appear to accumulate with age;is regulated such that deficiencies lead to an increase in absorption whereas excessive
intake is associated with decreased absorption and increased excretion ;administration for therapeutic reasons in man at doses above the recommended daily
allowance (RDA) has not produced significant pathology; andlong history of safe use in sunscreen products in US and other countries.

In contrast to states of zinc toxicity, there is much known about the consequences of Zn2+
deficiency and procedures for correcting such conditions. Acute and chronic Zn 2+
deficiency due to nutritional factors and several diseases produce clinical symptoms, which
are varied ranging from mild to severe and affecting several organ systems including skin (6).
Correcting such deficiencies is accomplished through dietary supplementation.
Zn2+ and many of its salts are ingredients in multivitamins. ZnO is available as a dietary
supplement and generally recognised as safe in most countries. Importantly, as a dietary
supplement there were no studies located that suggested any adverse effects in humans as aresult of excess Zn2+ consumed through the diet.

2.1.1.

Primary name

Zinc oxide (INCI name)2.1.2.

Synonyms

Zinc oxide (IUPAC)
Zinkoxid neutral H&H
Zinkoxid NDM
2.1.3.

Trade names and abbreviations

Zcote HPIMZO 350
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

2.1.4.

CAS no.

CAS n°EINECS

::2.1.5.

Structural formula1314-13-2
215-222-5/
2.1.6.

Empirical formula

Emp. Formula
Mol weight

:
:

ZnO
81.38

2.1.7.

Purity, composition and substance codes

Assay

:

≥ 95% (based on uncoated materials)≥ 92% (based on coated materials)

Coatings :Commonly used coatings for ZnO include dimethicone, vegetable oils, metalsalts and fatty acids. These materials when combined with ZnO can make the particle hydrophobic,
rendering it more compatible with waterproof or anhydrous systems and more convenient for theformulator. ZnO has been used as a coating to photostabilize other pigments.
Comment :2.1.8.

no information is given on metals and other impurities.

Physical properties

Substance code :Appearance:

Melting point
Boiling point
DensityRel. vap. dens.
Vapour Press.
Log Pow

2.1.9.

:
:
:
:
:
:

S76
ZnO is odourless, non-flammable, white or faintly yellowish,
amorphous powder. Microfine powders, used in sunscreen products,
have an average particle size of approximately 0.20 microns or lesswith a narrow distribution.
1975 °C/
5.47 g/cm²///

Solubility

Practically insoluble in water (0.00016 g/100 ml water), soluble in diluted mineral acids.

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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

2.2.

Function and uses

Requested use : up to 25% in sunscreen formulations.
Main industrial uses of ZnO are rubber, ceramics, optical glass, paint and plastics. Cosmetics &pharmaceuticals: ointments, baby and skin creams, toothpaste, deodorants, sunscreens. ZnO is added
to animal food and fertilisers, as essential trace element.

TOXICOLOGICAL CHARACTERISATION
2.3.

Toxicity

2.3.1.

Acute oral toxicity

SpeciesNumber of animals
Test substance
Dose
Results:::::

Conclusion:

2.3.2.

Wistar-II rats,
10 males and 10 females
ZnO
Single oral dose of 5000 mg/kg bw by gavage
LD50 > 5000 mg/kg. No clinical signs of toxicity were observed
during this study. No deaths occurred.
ZnO is non-toxic after a single oral ingestion.
Ref. : 7

Acute dermal toxicity

Method:Species:Number of animals:
Test substance:Concentration and vehicle :Dose:Observation period
Results::

Conclusion:

2.3.3.

According to the JO CEE recommendations, 19/09/1984(L251/103.105)
Sprague Dawley rats
5 males and 5 females
ZnO
The test substance was prepared as a 50% w/v paste in distilled
waterSingle dose of 2000 mg/kg applied uniformly on
approximately 10% of the total surface of the animals.
14 days
LD50 > 2000 mg/kg. No clinical signs of toxicity wereobserved during this study. No deaths occurred.
ZnO is non-toxic after a single dermal application
Ref. : 8

Acute inhalation toxicity

In Great Britain, the occupational exposure standards of ZnO fume are 5 mg/m³ (long term) and
10 mg/m³ (short term).
5

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________In the USA, the permissible exposure limit is 5 mg/m³ (long term) and the recommended
exposure limits are 5 mg/m³ (long term) and 15 mg/m³ (maximum; short term).
Ref. : 65
2.3.4.

Repeated dose oral toxicity

SpeciesNumber of animals
Test substance
Dose

:
:
:
:

Exposure period:

Sprague-Dawley female rats
64 in 4 groups of 16 rats each
ZnO
400 ppm ZnO in the diet (approx. 40 mg/kg bw/day) with orwithout co-administration of 184.000 µg Vitamin A equivalents/kg feed (basal diet as control group)
120 days

The study was designed to investigate the effects of chronic feeding of high levels of vitamin A
in combination with zinc on total lipids, cholesterol, vitamin A and zinc concentrations in theliver and serum cholesterol.
Results
The administration of ZnO oxide at a dose level of 40 mg/kg bw/d for a period of 120 days inthe diet did not lead to any noticeable clinical toxicity. In the high zinc groups a significant
elevation of serum cholesterol was observed as the only biologically relevant effect which was
independent from the vitamin A co-administration.
Ref. : 13
2.3.5.

Repeated dose dermal toxicity

No data
2.3.6.

Repeated dose inhalation toxicity

No data
2.3.7.

Sub-chronic oral toxicity

Animal study
MethodSpeciesNumber of animals
Test Substance
Dose

:
:
:
:
:

Exposure period:

standard toxicological study of 13-week duration
ICR mice and Wistar rats
12 animals of each sex
ZnSO4 .7 H2O
0, 300, 3000 and 30,000 ppm in the diet (mice: 42.7/46.4;
458/479 and 4927/4878 mg/kg/d for males/females; rats :23.2/24.5, 234/243 and 2514/2486 mg/kg/d for males/females)
13 weeks

The clinical signs of the animals were observed every day, they were weighed weekly and their dietand water intake measured twice a week. Dead and moribund animals were necropsied and samples
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

for histopathological observations were taken. Blood for haematological and biochemical
examinations was taken.
Results
ICR mice : at the high dose level 4 males and I female were found dead or killed in extremis. Highdose animals showed a moderate decrease in hematocrit, haemoglobin and leucocyte counts (onlymales). Total protein, glucose and cholesterol were reduced and alkaline phosphatase and urea
nitrogen were increased in high dose animals. High dose females showed reduced ALAT and
increased calcium levels, ASAT was increased in high dose males. Absolute and relative thyroid
weights of males and kidney weights of females were increased at the highest dose. Gross pathology
and histopathology showed changes in kidneys, thyroids, pancreas (degeneration/necrosis of acinar
cells, clarification of nucleoli), gastrointestinal tract, and spleen.
The NOEL for mice in this study was found at the mid dose at 3000 ppm (about 470 mg ZnSO4.7
H2O/kg bw (104 mg Zn2+/kg bw).
Wistar rats : at the high dose level a moderate reduction in leucocyte counts, slightly decreased
hematocrit and haemoglobin values (males only) and decreased total protein and cholesterol values
were recorded. Absolute and relative liver weights and relative kidney weights were decreased inhigh dose males. Histopathology showed pancreatic damage (degeneration, necrosis of acinar
cells, clarification of centroacinar cells and interstitial fibrosis) in high dose animals.
The NOEL for rats was found at the mid dose at 3000 ppm (about 240 mg ZnSO4.7 H2O/kg bw for
males and females, respectively (53.5 mg Zn2+/kg bw).
Ref. : 14
Human studies
Since zinc is an essential trace element for all mammalian species, its presence in the diet ofhumans and animals as well as in drinking water is required. In this regard, the mammalianorganism is used to handle varying quantities of externally administered zinc, such that Zn2+
homeostasis is regulated via control mechanisms of absorption and excretion. In humans, theaverage daily intake is estimated at about 15 mg of Zn2+. The available evidence from humanstudies points to a NOEL of about 0.5 mg/kg/day and thus confirms the safe exposure level,
which could be predicted from the animal data.
A series of human experimental studies have been published which report adverse effects fromoral administration of zinc salts :*47 healthy volunteers ingested ZnSO4 tablets three times a day for 6 weeks, resulting
in a total daily dose of 2 mg Zn 2+/kg bw. Headaches, nausea, vomiting, loss of appetite andabdominal cramps were seen in 26 of the volunteers.
Ref. : 15*
Oral doses of 160 mg ZnSO 4/day (2.3 mg Zn2+/kg bw/day) given to 12 healthy
adult men for 35 days resulted in decreased HDL cholesterol, total serum cholesterol
showed no change.
Ref. : 16
7

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________*ZnSO 4 tablets were administered twice daily to I I adult men for a total intake of 300
g/day (4.3 mg Zn2+/kg bw/day). There was a significant increase in serum zinc levels andreduction in lymphocyte stimulating response to PHA after 4 and 6 weeks of treatment. In
addition, a slight increase in LDL and a significantly reduced level of HDL cholesterol
were observed.
Ref. : 17*
After 6 weeks oral administration of 150 mg ZnSO4/day (2.1/2.5 mg Zn 2+/kg
bw/day for males/females, respectively), the total plasma and HDL cholesterol remained
unchanged, whereas the LDL cholesterol was significantly decreased in female volunteers.
Apart from these studies using dosages close to the threshold for adverse systemic effects,
the following reports confirm safe exposure levels in humans: In a double-blind trial, 13
humans received 200 mg zinc sulphate (corresponding to about 1.35 mg Zn2+) three times
a day for 18 weeks, while 14 humans received a placebo. No signs of toxicity associatedwith the zinc treatment were reported (18). In a study of Greaves and Skillen also no toxicity
was seen in 18 humans after administration of 220 mg zinc sulphate (about 150 mg Zn2+) 3
times a day for 16-26 weeks.
Ref. : 19
Finally, the administration of 50 mg Zn2+/day (as zinc gluconate) in a 10-week study withfemale volunteers is considered as a LOAEL for humans after repeated dosing, being 0.83
mg Zn 2+/kg bw/day (taking into account the dietary background level of 9.72 mg Zn2+/day
for females, the LOAEL would be 0.99 mg Zn 2+/kg bw/day) (Ref. : 55). Based on thishuman study, the Health Council of the Netherlands (1998) established in agreement withthe recommendation of the Commission of the European Communities (1993), a Maximal
Tolerable Daily Intake (MTDI) of 30 mg zinc/day (0.5 mg/kg/day).
2.3.8.

Sub-chronic dermal toxicity

No data
2.3.9.

Sub-chronic inhalation toxicity

No data

2.3.10.

Chronic toxicity

No data
2.4.

Irritation & corrosivity

2.4.1.

Irritation (skin)

MethodSpecies::

OECD 404
New Zealand rabbits
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Number of animals
Test Substance
Site of application
Concentration

:
:
:
:

Observation times:

3 malesZnO (purity unknown)
Right flank, shaved 24 hours before the test application for 4 hours.
The test substance was suspended 1:1 (w/w) in distilled water.
0.5 ml of this test article was applied with the help ofhydrophilic gauze.
1, 24, 48 and 72 hours after removal of the patch.

Results
Animal Number

Erythema
1h
24h
48h
72h
__________________________________________________
0280
0
0
0
0
0302
0
0
0
0
0309
0
0
0
0
Animal Number

Oedema
1h
24h
48h
72h
__________________________________________________
0280
0
0
0
0
0302
0
0
0
0
0309
0
0
0
0
Conclusion :ZnO is not a skin irritant.
Ref. : 20

Methods
Species::Number of animals
Test substance
Application
Site of application
Concentration

:
:
:
:
:

Application conditions:Observations:

Repeated patch test
New Zealand White rabbits, TO (outbred) and AG2 (inbred) mice,
Dunkin-Hartley white guinea pigs
8 rabbits, 6 mice, 8 guinea pigs
ZnO (98% purity)
Once daily on 5 consecutive days
Shaved dorsal skin sites of 5 cm 2
Application of 0.5 ml/day as a 20% aqueous suspension in 0.1%
Tween 80.
Rabbits, mice, Guinea pigs :open patch test.
Second rabbit study:
occlusive patch test
24 hours after last application, samples for histological evaluation
were taken

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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Results
Macroscopic observations in rabbit, mouse and Guinea-pig skin exposed to ZnO
for 5 consecutive days
Treatment

Rabbit (open
Rabbit (occlusive
Mouse
Guinea pig
patch test)
patch test)
N
Irritancy N
Irritancy
N
Irritancy N
Irritancy___________________________________________________________________________
ZnO
4
4
6
8
0.1 %Tween 80 44
6
8
Deionized H2O 4
4
68
ZnO exhibited no overt skin irritation or other histological effects in rabbit, mouse or Guinea pig.
Conclusion
ZnO is not a skin irritant after repeated application in different animal species.
Ref. : 21
2.4.2.

Irritation (mucous membranes)

MethodSpecies
Test substance
Site of application
Concentration

:
:
:
:
:

Observation times:

Directive 92/69/EEC B.5 and OECD 405
New Zealand White rabbits
ZnO (particle size unknown)
Conjunctiva sac of one eye
Approximately 64 mg of the test substance (0.1 ml) were
instilled into one eye of the rabbit, the other eye remained
untreated and served as control. After 24 hours both eyes of two
animals were rinsed with water.
1, 24, 48 and 72 hours after instillation

Results
No symptoms of systemic toxicity were observed and no mortality occurred. Slight iridial
irritation (grade 1) was observed in one animal, at 1 hour only. Slight irritation of the conjuctivae(grade 1-2) was seen as redness (mean scores over 24-72 hours 0.7, 1 and 1), which hadcompletely resolved at 72 hours in all animals. Chemosis (grade 2) and discharge (grade 1) werealso observed in all animals, but at 1 hour only. No corneal opacity or epithelial damage wasobserved in any of the animals.
Conclusion
According to EU criteria, ZnO is considered non irritating to the eyes.
Ref. : 22
MethodSpeciesNumber of animals
Site of application
Test substance

:
:
:
:
:

Draize test
Japanese white rabbits.
6 males
Into the eye lids
Ultra-fine ZnO, white powder
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Concentration:

Observation times:

The test substance was prepared as a 6% w/w aqueous suspension
and instilled at a volume of 0.1 ml. The eyelids were closed forapproximately one second. The eyes of 3 animals were rinsed withtap water after 30 seconds of the instillation.
1, 2, 3, 4 and 7 days after instillation of the test substance referring
to Draize criteria.

Results
Total Score of Ocular Lesions with ultra-fine ZnO (6% w/w aqueous suspension)
Animal No.
Not rinsed

RinsedTotal Score Rate
After 24 h. After 48 h. After 72 h. After 4 d.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

1
2
3
average
7
8
9
average

After 7 d.
0
0
0
0
0
0
0
0

Conclusion
Ultra-fine ZnO was evaluated as negative irritant for the eye according to the test result of a single
dose instillation eye irritation test in the rabbit.
Ref. : 23
2.5.

Sensitisation

MethodSpecies
Test substance
Number of animals
Induction

:
:
:
:
:

Challenge:

Directive 96/54/EC B.6 and OECD 406
Female Dunkin Hartley Guinea pigs
ZnO (purity 99.69%)
10 per test group, 5 in control group
Intradermal injection of 20% test substance in water and epidermal
exposure to a 50% concentration (highest practically feasible
concentration). Similar treatment of control group with water alone.
Approx. 24 hours before epidermal induction exposure all animals
were treated with 10% SDS (sodium dodecyl sulfate).
Two weeks after epidermal application all animals were challenged
with a 50% test substance concentration and the vehicle.

Results
In the first study, in response to the 50% test substance concentration skin reactions of grade 1
were observed in 4/10 experimental animals 24 hours after the challenge, while no skinreactions were evident in the controls. In the second study no skin reactions were evident in theexperimental animals, while a skin reaction grade I was seen in one control animal. Combining
both studies 4/20 animals (20%) showed skin reactions

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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Conclusion
According to EU criteria, ZnO is a non-sensitiser to the skin and, as such, does not have to beclassified for skin sensitisation.
Ref. : 24a, 24b
MethodSpeciesNumber of animals
Test substanceInduction

:
:
:
:
:

Challenge:

Results:

Directive 96/54/EC B.6 and OECD 406 (Guinea pig max. test)
Female Dunkin Hartley Guinea pigs
10 per test group, 5 per control group
ZnO (Zinkweiss Pharma A, purity 99.9%)
Intradermal injection of 2 % test substance in water (highest
reproducibly injectable concentration) and epidermal exposure to a
50% concentration (highest practically feasible concentration).
Similar treatment of control group with water alone. Approx. 24hours before epidermal induction exposure all animals were treatedwith 10 % SDS.
Two weeks after epidermal application all animals were challenged
with a 50% test substance concentration and the vehicle.
None of the treated animals showed a skin reaction.

Conclusion
According to EU criteria ZnO does not have to be classified for skin sensitisation and is a nonsensitiser to the skin.
Ref. : 25
Method:Number of volunteers :Test substance:Concentration
Induction::

Challenge:

Observations
Results::

Human Repeat Insult Patch Test.
60 (56 completed the test)
Microfine ZnO coated with 2.5% dimethicone [25% suspension of
the test substance in corn oil (dimethicone content of the test article:
0.625%)]
0.2 ml of a 25% suspension of the test substance in corn oil
9 occlusive 24-hour exposures every other day for 3 consecutive
weeks
After a 10-14 day rest period, a single treatment was applied to apreviously unexposed test site.
Reactions were scored 24 and 48 hours after application.
No adverse reactions were noted during the course of the study.

Conclusion
ZnO as tested was neither a skin irritant nor a skin sensitiser.
Ref. : 26
2.6.

Reproductive toxicity

2.6.1.

Teratogenicity

Species:

Female Wistar rats
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Number of animals :Test substance:

25 animals per group
ZnSO 4 (unspecified)
Aspirin (positive control)
Doses:
Zinc sulfate : 0, 0.4, 2.0, 9.1 and 42.5 mg/kg by gavage inwater; Aspirin : 250 mg/kg
Exposure period:
Day 6-15 of gestation
Procedure:
The effects of ZnSO4 were assessed using daily clinicalobservations in the pregnant dams, body weight determination
on day 0, 6, 11, 15 and 20 p.c., and food consumption throughout the experimental
period. Necropsy was performed on day 20 p.c. The pregnant uteri and ovaries were
examined for numbers of corpora lutea, implantations, viable foetuses and resorptions.
All foetuses were weighed, sexed and examined for gross external defects. In addition,skeletal examinations (in two-thirds of the foetuses) and soft tissue examinations (in theremaining one-third of foetuses) were performed.
Results
No compound-related effects were observed at any dose level. In particular, there was
no indication of any specific teratogenic activity of ZnSO4 under the conditions of thisexperiment.
Conclusion
The No Observed Effect Level (NOEL) with regard to maternal and developmental
toxicity of ZnSO4 was 42.5 mg/kg (corresponding to about 17 mg/kg if expressed as zinc).
Ref. : 27
Species:Number of animals :Test substance:Female Dutch rabbits
14-19 animals per group
ZnSO 4 (unspecified)
6-amino-nicotinamide (positive control)
Doses:
ZnSO 4 : 0, 0.6, 2.8, 13.0 and 60.0 mg/kg by gavage in water
6-amino-nicotinamide : 2.5 mg/kg by gavage
Exposure period:
ZnSO 4: day 6-18 of gestation (p.c.)6-amino-nicotinamide: single application at day 19 of gestation
Procedure:
The effects of ZnSO4 were assessed using daily clinicalobservations in the pregnant dams, body weight determination
on day 0, 6, 12, 18 and 29 p.c., and food consumption throughout the experimental
period. Necropsy was performed on day 29 p.c. The pregnant uteri and ovaries were
examined for numbers of corpora lutea, implantations, viable foetuses and resorptions.
Foetuses were weighed, sexed and examined for gross external defects, in addition
skeletal examinations and soft tissue examinations were performed in all foetuses.
Results
No compound-related effects were observed at any dose level. In particular, there was no
indication of any specific teratogenic activity of ZnSO4 under the conditions of thisexperiment. The positive control substance 6-aminonicotinamide induced the expected
embryotoxic effects (increased number of resorptions and birth defects).

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Conclusion
The No Observed Effect Level (NOEL) with regard to maternal and developmental
toxicity of Zinc Sulfate was at least 60.0 mg/kg (corresponding to about 24 mg/kg if
expressed as zinc).
Ref. : 28
SpeciesNumber of animals
Test substance:::Female Sprague Dawley: CFE rats
10 animals per group
ZnO

Doses and exposure period
7 groups received the test substance at levels of 2000 (about 100 mg/kg/d) or 4000 ppm (about
200 mg/kg/day) in the diet. 7 equally sized control groups received the basal diet without test
substance. 4 groups were fed the test diets at 4000 ppm from day 0 through day 15, 16, 18 or
20 of gestation. In a separate experiment, 3 dose groups (2 at dietary levels of 4000 and 1 at2000 ppm) received test diets starting 21 days prior to the mating until day 15 or 16 of
gestation.
Procedure
The effects of ZnO were assessed using reproduction and litter data as well as zinc, iron and copper
content in maternal and foetal tissues.
Results
The dietary level of 2000 ppm ZnO administered from day 21 prior to mating until day 15 ofgestation induced no adverse effects on foetal development. In contrast, marked embryotoxic(embryo-lethal) effects were observed after the different treatment regimens at the high dietarylevel of ZnO. In dose groups which received the test diet from day 21 prior to mating until day 15
or 16 of gestation, complete resorption (100% prenatal death) were observed in all treated dams.
Determination of tissue levels revealed a dose related increase in maternal whole body and liver
zinc concentration and decreased copper concentrations particularly in maternal livers. Similarchanges in electrolyte concentrations were observed in foetal whole body samples. These analyticalresults support the interpretation that the observed developmental effects of exaggerated high zincdoses are mediated by depletion of certain essential minerals such as copper.
Conclusion
ZnO, 2000 ppm, administered from day 21 prior to mating until day 15 of gestation induced noadverse effects on foetal development. This dietary level corresponds to a daily dose of about 100
mg/kg expressed as ZnO, or about 80 mg/kg expressed as zinc which represents the NOEL in thisstudy.
Ref. : 29
2.7.

Toxicokinetics (incl. Percutaneous Absorption)

MethodVolunteers:
Test substance
Site of application
Exposure

:
:
:
:
:

In vivo, human
6 healthy males
ZnO
Chest, upper and lower legs
Daily application of 40% ZnO in ointment for a of period 10 days
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Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Procedure:

Following the application, blood samples were drawn from eachsubject at 1-hr intervals for a total of 3 hours. Serum zincconcentrations were determined by flame atomic absorption
spectrometry.

ResultTopical application of the ZnO preparation had no statistically significant effect on serum zincconcentrations.
Conclusion
Topical application of ZnO in ointment does not affect systemic levels of zinc.
Ref. : 30
MethodVolunteers
Test substanceConcentration

:
:
:
:

Procedure:in vivo, human
31 subjects with chronic psoriasis
ZnO
ZnO -containing ointments were applied during 14 day at a daily
dose of 2.68 g Zn2+
Blood samples were obtained prior to treatment (i.e., baseline) and 7
and 14 days of treatment. Plasma Zn2+ concentrations were
determined using a flame-ionisation atomic absorption
spectrophotometer.

Results
There was no significant increase in plasma Zn2+ concentrations in any individual over the 2-week
study period.
Conclusion
Even in diseased skin, dermal Zinc oxide application did not increase plasma zinc levels.
Ref. : 31
MethodSkin samples
Test substance:::Concentration:in vitro, human skinObtained from the abdomen of surgery patients.
Mixtures of ZnO, ZnSO.4, ZnPC (zinc 2-pyrrolidone 5-carboxylate)and CuPC (copper 2-pyrrolidone 5carboxylate), CuSO4
5 formulations were used (3 emulsions and 2 ointments) :Emulsion A (water/oil, 0.25% (w/w) of ZnPC + 1.5% (w/w) ZnO + 0.5% (w/w) CuPC;
Emulsion B (water/oil, 0.25% (w/w) of ZnSO4 + 1.5% (w/w) ZnO + 0.5% (w/w) CuSO4;
Emulsion C (water/oil, 0.1% (w/w) of ZnSO4 + 7% (w/w) ZnO + 0.2% (w/w) CuSO4 ;Ointment D 0.1% (w/w) ZnSO4 + 10% (w/w) ZnO + 0.35% (w/w) CuSO4;
Ointment E 10% (w/w) ZnO + 0.2% CuSO4.
16 mg/cm2 (1.88 mg Zn2+/cm2) of each formulation was applied to the outer skin surface.

15

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Procedure
After 72hrs, any remaining formulation persisting on the treated skin surface was gently removed
using cotton swabs. The receptor solution (isotonic saline) was replaced after 2, 4, 12, 24, 48 and72hrs. The stratum corneum was stripped twice using adhesive tapes. Samples of dermis andwhole epidermis were taken from treated skin. Zinc and copper concentrations were determined inreceptor solution and in the skin preparations by atomic absorption spectrometry. Values werenormalized by dividing Zn2+ concentration in control skin (percent of Zn2+ increase) and thendivided by applied dose of Zn2+, mg/cm2.
Results
The maximum concentration of Zn2+ was 0.34 ± 0.22% of the applied dose of ZnO recoveredin the receptor fluid after 72 hr. The relative increase of Zn2+ was 0.5% in the epidermis and1.1'% in the dermis.
Conclusion
The systemic availability of dermally applied ZnO as tested (determined as fraction
analysed in the receptor fluid) was found to be 0.34%.
Ref. : 33
MethodTest substanceConcentration:::in vitro, pig skin
ZnSO4 x H2O and ZnO
Aqueous solutions of Zinc sulfate monohydrate and ZnO wereapplied to the skin preparations at a concentration of 40 mg/ml

Procedure
Skin preparations measuring 1 mm in thickness with stratum corneum, stratum germinativum andblood vessel containing parts of the dermis were obtained from pigs using a modified dermatome. In
two independent experiments for each compound 7 skin preparations were mounted in Teflon flowthrough diffusion chambers, which were continuously rinsed with physiological receptor fluid(0.9% NaCl). After checking the integrity using caffeine as a marker substance, each of the test
formulations was applied to 6 skins at a dose of 1 mg/cm² for 8 hours without occlusion andsubsequently washed off with a neutral shampoo. After 0, 2, 4, 6, 8, 16, 24, 40, 48, 64 and 72
hours, the cutaneous permeation was determined by quantifying zinc with atomic absorption
spectroscopy (detection limit 10 ng/ml) in the receptor fluid. The experiment was stopped at 72
hours. Furthermore, zinc was analysed in the skin preparations and the rinsing fluids. The amountsof zinc detected in receptor fluid and different layers of the skin were corrected for backgroundlevels of endogenous zinc.
Results
The following dermal absorption rates (% of applied dose) through pig skin in vitro within 72 hours
were determined (corrected for background levels of zinc)
Skin compartment
Zinc sulfate
ZnO______________________________________________
Receptor fluid
0.3 %
0.03%
Horny layer
1.3 %12.3 %
Residual skin0%
2.6 %Total recovery of applied zinc in both experiments ranged from 82.0-109%.
16

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Conclusion
Based on the amount recovered at 72 hours from the receptor fluid, the systemically available
amount of zinc can be considered to be < 1 % of the applied dose.
Ref. : 34
2.8.

Genotoxicity/Mutagenicity

Existing literature data on the in vitro and in vivo studies on mutagenicity and/or genotoxicity ofZinc Oxide and Zinc salts have not been included in the report.
In its general summary, the COLIPA submission I of 26 November 2001 includes a table thathas been prepared by TNO. This table reports the summary data on mutagenicity/genotoxicity
studies. From the table, it possible to conclude that Zinc Oxide and Zinc salts have mutagenic
and /or genotoxic in vitro activity (gene mutation, chromosome aberrations and UDS in
mammalian cell).
In vivo studies presented are not conclusive (some positive results have been reported : bonemarrow cytogenetic assay in rodents and host-mediated assay in mice).
We presume that all these studies refer to the non-micronized material. It is possible that zincoxide under the micronized form may display a genotoxic profile of some concern.
Ref. : 3
2.9.

Carcinogenicity

No data
2.10.

Special investigations

No data
2.10.1.

Photo-chemical properties

Method:Test substance :Light source:

Photostability as measured by UV absorption
15% microfine (< 0.2µm) ZnO (non-coated)
Oriel Xenon Arc Solar Simulator

A thin film, 2 mg/cm2, of the sunscreen product was applied to a synthetic substrate and, using ausing a solar simulator, irradiated. The absorption curves were measured after exposure to 0 and
30 joules /cm2 solar-simulated UV.

17

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Results
□0J

300

■30 J

310 320 340 350 360 370 380 390 400

ZnO based sunscreen before and after UV irradiation
Conclusion
As can be seen, the absorption curve for ZnO was unchanged following exposure to 30 J/cm2
solar simulated UV by pre-irradiation demonstrating complete photo-stability.
Ref. : 39
2.10.2.

Photo-reactivity

Photoreactivity can be defined as the propensity of a given molecule/crystal to react withneighboring molecules when irradiated. The photoreactivity of pigments (i.e. ZnO) can be easily
measured using a well-recognised assay. Briefly, the tests are performed in a small batch
photoreactor (200 cm³ capacity), held at a constant temperature (24 °C), and under constantfocused photon flux from a 500W medium pressure mercury arc lamp (Priel Products Ltd). The
photoactivity index is measured as the zero order rate of the photocatalytic oxidation (reactive
oxygen species) of liquid propan-2-ol to propanone, under oxygenated conditions for a fixed massof particulate. Photoactivities are expressed in terms of moles converted per gram of particulate perhour of irradiation. Propanone analysis was conducted at regular intervals using gas
chromatography (Perkin Elmer GC 1830).
The photoreactivity of microfine ZnO and a photocatalytic standard was measured using themethod described above. Non-coated, dimethicone coated and silica coated versions of bothpigments were measured.

18

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Results

Measurement of the photoreactivity of microfine ZnO and a photocatalytic standard.
Non-coated, dimethicone coated and silica coated versions of both pigments were measured

Conclusion
As shown in figure 2, microfine ZnO is virtually non-photoreactive. Note that the error on thetest is ~3.0.
Ref. : 39
Sunscreen formulations (2 mg/cm2) were placed upon a hydrated collagen substrate (Vitro-Skin).
Using a solar simulator (Oriel Xenon Arc Solar Simulator), the thin film was irradiated andsamples taken at 0, 10, 20 and 30 joules/cm2. The samples were digested and the percent octyl
methoxycinnamate (OMC) remaining was determined by HPLC. A 50:50 mix (coated:noncoated)
was used to test the hypothesis that ZnO does not need to be coated to be non-reactive.
Test substance
Two formulations were studied. One contained only 7.5% OMC as the sunscreen and the othercontained 7.5% OMC in combination with 10% microfine ZnO. The microfine ZnO used was a
50:50 mix of commercially available coated (dimethicone) and non-coated particles. The
dimethicone coating contributed 1 % of the weight of the powder.
Results
Lack of interaction of microfine ZnO and octyl methoxycinnamate (OMC) under UV irradiation
FormulaPercent OMC Remaining
10 J/cm2
20 J/cm2
30J/cm2_____________________________________________________________________
7.5 % OMC
85.1
80.7
78.1
7.5% OMC + 10% Microfine ZnO

87.5

85.8

83.8

Conclusion
ZnO was shown to be non-photoreactive.
Ref. : 39

19

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

2.10.3.

Phototoxicity / Photoirritation

Method:Cell type:
Test substance :Test vehicle:
Light source:

Neutral Red Uptake Phototoxicity Test in vitro (EU Directive
67/548/EEC, Annex V, B41)
Balb/c 3T3 mouse fibroblasts, Clon 31
Micronised ZnO (uncoated) Chlorpromazine (positive control)
ZnO in EBSS/ 1% (v/v) ethanol solubilised/dispersed in a concentration
range of 1.0 to 100.0 mg/l. Chlorpromazine in EBSS
Metal-halide lamp with cut-off filters according to Spielmann et al.
(40, 41). UVA 1667 mW/cm2 for 50 min (5.0 J/cm2), UVB: nondetectable.

Results and conclusion
Both ZnO irradiated and unirradiated had similar cytotoxic concentration response curves. Bothcurves were similarly shaped with nearly identical NR50 values at approximately 9 mg/L. The ratioof [NR50 (-UV)/ NR50 + UV)] PIF equals 1, demonstrating that ZnO does not exhibit a phototoxic
potential in this test.
Ref. : 42
MethodVolunteers
Test substanceConcentration
Application

:
:
:
:
:

Irrad. Cond.

:

Observ. Period :Occlusive patch test on humans
30
Micronised ZnO
25% test substance in cosmetic w/o emulsion
Test articles were applied for 24 hours to the skin of the back of the volunteer
by using HAYES-test chambers (provide occlusive patch effect),consisting of a plastic capsule 10 x 10 mm, providing a test area of 100
mm² and holding a volume of approx. 50 µ1. These chambers werearranged in strips of 2 x 5 patches forming a patch set.
One duplicate patch site on the right back or arm within 10 min after
patch removal with 18 J/cm² UV-A.
Skin reactions were read 0, 24 and 48 hours after patch removal in thepretest for phototoxicity.

Results
No phototirritant skin reaction in any volunteer was observed.
Conclusion
ZnO as tested was not photo-irritant.
Ref. : 43
Method:Volunteers:
Test substance :Occlusive patch test in humans
10
25% ZnO (dimethicone coating)

Application and irradiation conditions
Test sites were tape stripped with hypo-allergenic tape 3 times to remove several layers ofcornified epithelium. 0.2 g or 0.2 ml of the test material or hydrophilic ointment USP (negative
control) was placed onto a 2 cm x 2 cm occlusive patch, then applied to the non-irradiated20

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

control site (left arm) and allowed to remain in place for 24 hours. Concurrently the test material
or negative control was applied to the right arm directly onto the skin. The site was thenirradiated with non-erythemogenic UV-A irradiation at a distance of 10 cm of the source
and receiving a UV-A light dosage of greater than 4.4 mW/cm2 UV-A. The test site was thencovered with occlusive patch containing additional test material (0.2 g or 0.2 ml).
Observation times
Skin reactions were read 0, 24, 48 hours and I week after patch removal.
Results
No adverse effects were observed.
Conclusion
ZnO as tested was not photo-irritant.
Ref. : 44
2.10.4.

Photosensitisation / Photoallergy

MethodVolunteers
Test substanceConcentration

:
:
:
:

Photo maximisation/photoallergy test
30
micronised ZnO
25% test substance in a cosmetic emulsion (w/o)

Application:
Test articles were applied to the skin of the back of the volunteer by using
HAYES-test chambers (provide occlusive patch effect), consisting of a plastic capsule 10 x 10mm, providing a test area of 100 mm² and holding a volume of approx. 50 µl. These chambers
were arranged in strips of 2 x 5 patches forming a patch set.
Pretest:Testing of irritant and photo-irritant properties by applying the test articles
once for 24 hours in duplicate and irradiating with UV-A immediately after patch removal.
Induction:
6 times under occlusion for 24 hours within three weeks and irradiation
with UV-B immediately after patch removal. The patch site on the right back or arm was exposed
to approximately twice the MED (minimal erythema dose) of UV-B within 10 min. after patch
removal. If the skin became tanned after several exposures, irradiation time was increased in order
to induce sufficiently intense erythema. A non-irradiated patch site was deemed unnecessary during
induction and was not applied.
Challenge:
One application under occlusion for 24 hours after 10 to 14 days induplicate. UV-A irradiation of one duplicate immediately after patch removal. Patch areas on an
untreated site were irradiated with 18 J/cm2 of UVA within 10 min. after patch removal.
Skin Evaluations
Pretest:0, 24 and 48 hours after patch removal.
Induction :0, 24, 48 and 72 hours after patch removal, “Thursday”-patches immediately
and 72 hours after patch removal.
Challenge :0, 24, 48 and 72 hours after patch removal.
Results
ZnO did not produce any photoallergic skin reaction.
21

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Conclusion
ZnO can be considered as non-photosensitising under the conditions of this study.
Ref. : 43
MethodVolunteers
Test substance
Irrad. System

:
:
:
:

Photo maximisation/photoallergy test
25
25% ZnO (dimethicone coating)
150 W xenon arc solar simulator (Solar Light Co., Philidelphia PA)

Determination of Minimal erythema dose (MED) :Prior to the testing of sunscreen
products, the sensitivity of the unprotected skin of each subject was determined in seconds, basedon the length of exposure, which first elicits a slight reddening of the skin, as observed 24 hour
following exposure.
Induction :Duplicate test areas (2.5 x 2.5 cm), to which 62.5 µl of the test material was
evenly applied (density 10 mg/cm2), were delineated on the subject's back. Sites were then covered
with 2 cm x 2 cm occlusive patches. After 2 hours the test sites were wiped dry and one set was
exposed to 3 MED's (minimum erythema dose) of solar simulating radiation while the alternate set
was again left non-irradiated. This sequence was repeated for a total of 6 exposures twice
weekly.
Challenge :10-14 days after the final induction exposure. Similar occlusive applications are
made for 2 hours to a previously unexposed untreated, area of normal skin; the sites were thenirradiated with 4.0 J/cm2 of UVA radiation.
Skin Evaluation : 48 and 72 hours post challenge irradiation.
Results
No adverse effects or unexpected reactions of any kind were observed on any of the subjects.
Conclusion
ZnO can be considered as non-photosensitising under the conditions of this study,
Ref. : 45
2.10.5.

Photo-mutagenicity

Bacterial Reverse Mutation Test
Guideline
Species/strain::

Replicates
Test substanceBatch noConcentrations

:
:
:
:

Notes of Guidance 2000
Salmonella typhimurium, TA98, TA100, TA1537,
E. coli WP2
Triplicate plates, only 1 experiment performed
HR96/104702 dissolved in 10 % emulsion in 3 % Tego Care 450(Batch 50108745, purity 97.2 %)direct plate incorporation assay without S9 mix.
TA 98 : 2, 6.32, 20, 63.25 and 200 mg/plate
TA 1537 : 20, 63.25, 200, 632.5 and 2000 mg/plate
TA 100 and WP2 : 50, 158.1, 500, 1581 and 5000 mg/plate
22

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Positive Control :UV irradiation :
:
GLP

:

According to the strains + 8-MOP
Heraeus Suntest CPS light
Dose of UV :UVA 35/41 mJ/cm2/min
UVB 2.3/2.6 mJ/cm2/min
Quality Assurance statement included

Results
Without UV
TA 98:
TA 100:TA 1537 :E. coli WP2 :From 0 to 200 µg/plate no increase in the number of revertants was observed.
From 0 to 5000 µg/plate no increase in the number of revertants was observed.
From 0 to 200 µg/plate no increase in the number of revertants was observed.
From 0 to 5000 µg/plate no increase in the number of revertants was observed.

With UVUVA 8 mJ/cm2/min
TA 98:
From 0 to 200 µg/plate no increase in the number of revertants was observed.
UVA 4 mJ/cm2/min
TA 100:From 0 to 5000 µg/plate no increase in the number of revertants was observed.
TA 1537 :From 0 to 200 µg/plate no increase in the number of revertants was observed.
UVA 90 mJ/cm2/min
E. coli WP2 :From 0 to 5000 µg/plate no increase in the number of revertants was observed.
8-MOP at dose of 500 µg/plate and irradiated with UVA 90 mJ/cm2/min gives an increase of
more 10 times in the number of revertants
Conclusions
Based on the reversion rate, the test agent HR96/104702 dissolved in 10 % emulsion in 3 %Tego Care 450 at different concentrations, with or without UV irradiation, it is concluded thatno mutation were induced in Salmonella typhimurium and E. coli tester strains.
Micronized material has no mutagenic activity on bacteria cells; it is not photomutagenic in thesame cell system.
Ref. : 50
In vitro Mammalian Chromosomal Aberration Test
Guideline:
Species/strain:
Replicates:
Test substance :Batch no:Positive Control :Concentrations :UV irradiation ::Notes of Guidance 2000
Chinese Hamster Ovary Cells
Duplicate cultures, independent experimentHR96/104702 dissolved in 10 % emulsion in 3 % Tego Care 450
Batch 50108745, purity 97 %4-NQO and 8-MOP.
7 doses from 95.74 - 813.8 µg/ml
Heraeus Suntest CPS light
Dose of UV :23

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Doses of UV
GLP

:
:

UVA 35/34 mJ/cm2/min
UVB 2.1/2.2 mJ/cm2/min
375 and 750 mJ/cm2/min
Quality Assurance statement included

Exposure and concentrations*
Samples were exposed during 3 hours with or without UV.
*
Cultures were prolonged during 17 hours before harvest.
Results
Structural chromosome aberrations
Without UV
A dose dependence trend of the number of aberrations was found (chromatid and chromosome
deletions, chromosome and chromatid exchanges).
With UV (375 and 750 mJ/cm2/min)
A higher (3 - 4 x) dose dependence trend of the number of aberrations was found than without
UV(chromatid and chromosome deletions, chromosome and chromatid exchanges).
Results were repeated in the independent experiment
Statistical analysis of the data indicates that the frequency of aberrant cells excluding gaps is
statistically significant at the highest 2 doses in the absence of UV light as compared to controls.
In the presence of UV light, in the first experiment, statistical significance was observed for thehighest 3 doses.
In the second experiment with or without UV light, only the highest doses induced a statisticallysignificant increase in the percentage of cells displaying structural aberrations.
Polyploidy
Into account that no specific positive control agent has been used in this assay, and that only
metaphases with 19 - 23 chromosomes were considered for scoring, polyploidy means a neartetraploid karyotype.
While not statistically significance has been determined, a biologically relevant increase in thenumber of poliyploid and endoreduplicated metaphases was observed.
Conclusions
Based on the structural and/or numerical chromosome aberrations observed in the absence and in
the presence of UV light, the test agent HR96/104702 dissolved in 10 % emulsion in 3 % Tego
Care 450 at different concentrations showed positive effects in cultured Chinese Hamster Ovary(CHO) cells under the conditions of the study.
Micronized material has clastogenic activity on mammalian cells cultured in vitro; it has been
also shown, that the test agent displays aneugenic activity; it is also photoclastogenic andpossibly photoaneugenic in the same cell system.
Ref. : 51
In vitro Mammalian Chromosomal Aberration Test
Guideline:

Notes of Guidance 2000
24

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Species/strain:
Replicates:
Test substance :Batch no:Positive Control :Concentrations :Chinese Hamster V79 Cells
Duplicate cultures, independent experiment
HR99/104702 dissolved in culture medium
Batch 50376240, purity > 95 %4EMS and 8-MOP.
7 doses
Experiment #1
0.4, 1.1, 3.3, 10, 30, 90, 150 µg/ml with or whithout irradiation
Experiment # 2 – Without irradiation
2, 4, 7, 10, 13, 16 µg/ml
Experiment # 2 – With irradiation
0.5, 1, 2, 3, 6, 12 µg/ml
UV irradiation :Xenon lamp Suntest CPS light
Doses of UV:Intensity of irradiation UVA/UVB
0.3/0.015 mW/cm2
Irradiation period : 20 min 50 sec
Dose UVA/UVB : 375/18.75 mJ/cm2
Total treatment time with the chemical : 3 hours
Recovery:
15 hours.
GLP:
Quality Assurance statement included
Exposure and concentrations*
Samples were exposed during 3 hours with or without UV.
*
Cultures were prolonged during 18 hours before harvest.
Results
Cytotoxicity
Experiment # 1 : the maximum dose gave a reduction of survival of 76 % without irradiation and
61 % with irradiation.
Experiment # 2 : survival was in the range of the control with or without irradiation
Structural chromosome aberrations
Experiment # 1
Without UV:
A dose dependence trend of the number of aberrant cells was found(chromatid breaks and chromatid exchanges; a few cells displayed chromosome typeaberrations).
With UV:A higher dose dependence trend of the number of aberrant cells wasfound than without UV (chromatid breaks and chromatid exchanges; a few cells displayed
chromosome type aberrations). Statistical analysis showed that the difference was significant ascompared to the controls.
Experiment # 2
The results confirmed clearly the results found in the first experiment.
Polyploidy
No difference in the percentage of polyploid cells have been observed as compared to thecontrols in both experiment.

25

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Conclusions
Based on the structural chromosome aberrations observed in the absence and in the presence of
UV light in both experiment, the test agent HR99/104702 dissolved in culture medium at
different concentrations, displayed positive effects in cultured Chinese Hamster V79 cells under
the conditions of the study. It should be noted that the observed effects were induced with low
doses.
This micronized material has clastogenic activity on mammalian cells cultured in vitro; it is also
photoclastogenic in the same V79 cell system.
Ref. : 49
Photocomet assay in vitro
Guideline
Species/strain:::
Replicates:
Test substance :Batch no:Positive Control :Treatment time :Concentrations:

UV irradiation

:

GLP

:

/
HaCaT cells (human keratinocytes)
Chinese Hamster V79 Cells
Duplicate cultures, only 1 experiment
HR00/106407 dissolved in deionized water
Batch 50508802, purity : 93.3 %EMS and Chlorpromazine
Not indicatedThree sets of data were presented for HaCaT cells:#1 : Without irradiation#2 : Directly after irradiation (with and without pre-incubation in thedark).
#3 : 20 hours after irradiation (with pre-incubation in the dark).
One set of data was presented for V79 cells:#1: Directly after irradiation (with pre-incubation in the dark).
5 doses
HaCat - without irradiation
3.9 – 62.5 µg/ml
HaCat – directly and 20 hours after irradiation
1.95 – 31.3 µg/ml
V79 cells - directly after irradiation
1.95 – 31.3 µg/ml
Heraeus lamp Suntest CPS light
HaCaT cells with and without pre-incubation
450/20 mJ/cm2 UVA/UVB
V79 cells with and without pre-incubation
980/40 mJ/cm2 UVA/UVB
HaCaT cells “variation 20 hours”
230/10 mJ/cm2 UVA/UVB
Quality Assurance statement included

Results
In the presence of UV, V79 cells indicate the induction of DNA damage in both experiments asevidenced by the mean tail length.
HaCaT cells did not showed any induction of DNA damage in the absence of UV irradiation andin all conditions after UV irradiation.
26

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

Conclusions
Based on the mean tail length, the test agent HR00/106407 dissolved in deionized water isconsidered photogenotoxic in cultured Chinese Hamster V79 cells under the conditions of thephotocomet assay in vitro.
Ref. : 54
Overall conclusions
The micronized material has been found clastogenic, possibly aneugenic and inducing DNA
damages in cultured mammalian cells in vitro, under the influence of UV light.

2.11.

Safety evaluation
NOT APPLICABLE

2.12.

Conclusions

A considerable part of the investigations and their results submitted have been performed 15 ormore years ago and consequently cannot fulfil modern requirements. However, there is a broad
basic knowledge on Zn2+ and its compounds, e.g. ZnO.
The physico-chemical specifications of ZnO used in many of the studies are incomplete, thepurity/impurities not specified. On the other hand, ZnO is practically insoluble in water. Thus, ingeneral, ZnO may be considered as a non-toxic substance, including when used in cosmetic
products.
The main concern of the present evaluation is related to the risk assessment of micronised(approximately 0.2 µm) ZnO, which may be coated by other compounds, and which is used as aningredient in sunscreen formulations.
Micronised ZnO has been demonstrated to be photoclastogenic, possibly photo-aneugenic, and aphoto-DNA damaging agent in mammalian cells cultured in vitro. The relevance of thesefindings needs to be clarified by appropriate investigations in vivo.
There is a lack of reliable data on the percutaneous absorption of micronised ZnO and thepotential for absorption by inhalation has not been considered.
As to a safety assessment of an use of UV-filters by children over the age of 1 year, the SCCNFP issued
a position statement (SCCNFP/0557/02).

2.13.

OpinionBased on the conclusions (point 2.12), the SCCNFP is of the opinion that there more informationis needed to enable a proper safety evaluation of micronised Zinc oxide for use as a UV filter incosmetic products. Consequently, an appropriate safety dossier on micronised ZnO itself,
including possible pathways of cutaneous penetration and systemic exposure, is required.
27

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

2.14.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.

References
Landsdown, A.B.G., Zinc in the healing wound, Lancet (1996), 347, 706-707
Fergusson, J., Addo, H.A., Jones, S., Johnson, B.E. and Frain Bell, W., A study of
cutaneous photosensitivity induced by amiodarone, (1985) Br. J. Dermatol., 113, 537-549
Risk assessment zinc oxide, Draft of 21.12.1999, report prepared by TNO & RIVM
Berg, J.M and Shi, Y., The galvanisation of biology: a growing appreciation for the rolesof zinc (1996), Science, 271, 1081-1085
Walsh, C.T. Sandstead, H.H., Prasad, A.s., Newberne, P.M. and Fraker, P.,J., Zinc:
Health effect and research priorities for the 1990s, (1994), Environmental Health
Perspectives, 102, 5-46
Prasad A., S., Discovery of human zinc deficiency and studies in an experimental
human model, (1991), Am. J. Clin. Nutri., 53, 403-412.
Loeser: Akute orale toxizitat, Bayer AG data: Orientierende toxikol. Pruefungen, 25.4.77
Dufour, P., Etude de la toxicite aiguë par voie cutanee chez le rat du produit oxyde dezinc lot 91.8207, EVIC-TOX, ref.: L 081/S477, EVIC-CEBA, laboratoire derecherche et d'experimentation, November 12, 1991.
Scott, D. A. and Fisher, A. M., Studies on the pancreas and liver of normal and of zincfed cats (1938), 253-260.
Hsu, F. S., Krook, L., Pond, W. G. and Duncan, J. R., Interactions of dietary calcium with
toxic levels of lead and zinc in pigs (1975), J. Nutr., 105, 112-118.
Straube, E. F., Schuster, N. H. and Sinclair, A. J., Zinc oxide in the ferret, (1980), J.
Comp. Path., 90, 355-361.
Ellis, T. M., Masters, H. G. and Mayberry, C., Examination of the susceptibility of different
breeds of sheep to zinc intoxication (1984), Australian Veterinary Journal, 61, 296-298.
Shankar, S., Ramnathan Sundaresan, P. and Mohla, S., Effect of chronic administration ofexcess dietary vitamin A and zinc on lipid metabolism in rats (1986), Intern. J. Vit.
Nutr. Res., 56, 329-337.
Maita, K., Hirano, M., Mitsumori, K., Takahashi, K. and Shirasu, Y., Subacute toxicity
studies with zinc sulfate in mice and rats (1981), J. Pesticide Sci., 6, 327-336.
Samman, S. and Roberts, D. C. K., The effect of zinc supplements on plasma zinc andcopper levels and the reported symptoms in healthy volunteers (1987), The medical
journal of Australia, 146, 246-249
Hooper, P. L., Visconti, L., Garry, P. J. and Johnson, G. E., Zinc lowers highdensity lipoprotein-cholesterol levels (1980), JAMA, 244, 1960-1961.
Chandra, R. K., Excessive intake of zinc impairs immune responses (1984), JAMA, 252,
1443-1446.
Hallbook, T. and Lanner, E., Serum-zinc and healing of venous leg ulcers (1972), The
Lancet, 780-782.
Greaves, M. W. and Skillen, A. W., Effects of long-continued ingestion of zinc sulphate inpatients with venous leg ulceration (1970),The Lancet, 879-891.
Dufour, P., Evaluation de l'irritation et/ou de la corrosion cutanees chez le lapin du produit
oxyde de zinc lot 91.8207, EVIC-TOX, ref.: L 082/5477, EVIC-CEBA, laboratoire derecherche et d'experimentation, November 12, 1991.
Lansdown, A. B. G., Interspecies variations in response to topical application of selected
zinc compounds (1991), Fd. Chem. Toxic., 29, 57-64.
Notox B. V. , Acute eye irritation/corrosion study with zinc oxide in the rabbit. Project
254352, (1999a), cited in reference (4).

28

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.

41.

42.

Atsunobo M., Eye irritation of ultra-fine oxide (6% w/w aqueous suspension) in rabbit, Life
Science Laboratory, October 19, 1994.
Notox B. V. , Assessment of contact hypersensitivity to zinc oxide in the albino guinea pig
maximisation test. Project 261214, (1999b1, b2), cited in reference (4).
Notox B. V., Assessment of contact hypersensitivity to zincweil3 pharma in the albino
guinea pig maximisation test. Project 263429, (1999i), cited in reference (4).
Cantor, S., 50 humans subject repeat insult patch test skin irritation/sensitisation
evaluation, ref n° WPCL 94-141Z/RIPT 00160.SSI, AMA laboratories, May 2, 1994.
Teratologic evaluation of FDA 71-49 (zinc sulfate), Food and Drug research labs., Inc.
(1973), PB-221 805 cited in reference 4.
Teratologic evaluation of compound. FDA 71-49. zinc sulfate in rabbits, Food and Drugresearch labs., Inc. (1974), PB-267 191 cited in reference 4.
Schlicker S.A., Cox D.H., Maternal dietary zinc and development and zinc, iron, andcopper content of the rats foetus (1968), J. Nutr., 95, 287-294.
Derry, J. E., McLean, W. M. and Freeman, J. B., A study of the percutaneous absorption
from topically applied zinc oxide ointment (1983), journal of Parenteral and Enteral
Nutrition, 7, 131-135.
Morgan, M. E. I., Hughes, M. A., McMillan, E. M., King, I. And Mackie, R. M., Plasma
zinc in psoriatic in-patients treated with local zinc applications (1980), British Journal of
Dermatology, 102, 579-583.
Gonül, B., Sdylemezoglu, T., Bab6l, A. and Celebi, N., Effects of epidermal growth factor
dosage forms onmice full-thickness skin wound zinc levels and relation to wound strength(1998), J. Pharm. Pharmacol., 50, 641-644.
Pirot, F., Millet, J., Kalia, Y. N. and Humbert, Ph., In vitro study of percutaneousabsorption, cutaneous bioavailability and bioequivalence of zinc and copper from five
topical formulations (1996), Skin Pharmacol., 9, 259-269.
Labor L+S AG, Final report, Cutaneous permeation of zinc oxide and zinc sulphate throughpig skin in vitro, Study Nr. 02073979/02073989. (1999) cited in refence 4.
Leonard, A., Gerber, G. B. and Leonard F., Mutagenicity, carcinogenicity and teratogenicity
of zinc (1986), Mutation Research, Elsevier, 168, 343-353.
United States Environmental Protection Agency, Zinc and compounds (1991), 1-24.
Henry, J. B., Clinical diagnosis and management by Laboratory methods, 7`h edition (1984),
pp. 1437.
Fong, L. Y. Y., Li, J.-X., Farber, J. L. and Magee, P. N., Cell proliferation and esophageal
carcinogenesis in the zinc-deficient rat (1996), 17, 1841-1848.
Mitchnick, M. A., Fairhurst, D. and Pinnel, S. R., Microfine zinc oxide (Z-Cote) asa photostable UVA/UVB sunblock agent, J. Am. Acad. Dermatol. (99), 40, 85-90.
Spielmann, H. Balls, M., Dupuis, J., Pape, W., De Silva, 0., Holzh6tter, H.-G., Gerberick,F., Liebsch, M., Lovell, W. and Pfannenbecker, U., A study on UV filter chemicals fromannex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU
phototoxicity test (1998),ATLA, 26, 679-708.
Spielmann, H. Balls, M., Dupuis, J., Pape, Pechovitch, G., W., De Silva, 0., Holzh6tter,
H.G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W., Maurer, T.,
Pfannenbecker, U., Potthast, J. M., Csato, M., Sladowski, D., Steiling, W. and Brantom, P.,
The international EU/COLIPA in vitro phototoxicity validation study: results of phase II(Blind trial). Partl: The 3T3 NRU phototoxicity test (1998), Toxicology in Vitro(Pergamon) 12, 305-327.
Beiersdorf AG test report, Diembeck, W., Testing of Zinc Oxide (H&R, PN 104702) with the
3T3 Neutral Red Uptake Phototoxicity Test (2001), report N° 0663-2001.
29

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.

Fink, E., Repeat photopatch test for photoxicity and photoallergy of HR 96/104702 and HR
96/104702 VeK in healthy adult male and female volunteers, part 1, project n)
9066/lib, GTLF, August 1, 1997.
Cantor, S., Lukin, A., Reidy, M. T. and Winne, D. R., Evaluation of phototoxicity potential
by UV-A irradiation on 10 human subjects (1994), AMA Laboratories, WPCL 94147Z/ PHT0016OSSI .10.
Cantor, S., Lukin, A., Reidy, M. T. and Winne, D. R., Photoallergy maximization test on
25 human subjects (1994), AMA Laboratories, WPCL 94-147Z/PHT00160SSI.25.
The SCC Notes of Guidance for testing of cosmetic ingredients for their safety evaluation(third revision, SCCNFP/01 19/99) (1999), Annex 3, Guidelines for the in vitro assessmentof the phototoxic potential of UV filters, 52-58.
Loprieno, N., In vitro assay systems for testing photomutagenic chemicals (1991),
Mutagenesis, 6, 331-333.
Loprieno, N., Guidelines for safety evaluation of cosmetics ingredients in the EC countries(1992), I'd Chem. Toxic., 30, 809-815.
RCC-CCR project, Czich, A. In vitro test on induction of chromosome aberrations in V79
cells with HR 99/ 104702 (a), 00/T00017 (b), 00/ 106407 (c), after simultaneous irradiation
with artificial
Ballantyne, M., HR 96/104702: reverse mutation in three histidine-requiring strains ofsalmonella typhimurium and a tryptophan-requiring strain of Escherichia coli, in thepresence of ultraviolet light (1998), Final report, 1-58.
Guidance document elaborated by the COLIPA task force "Photomutagenicity" (1995).
Riley S., HR 96/ 104702 : induction of chromosome aberrations in cultured Chinese
hamster ovary (CHO) cells in the presence of ultra violet light (1998), CovanceLaboratories Ltd., Harrogate, UK, final report n°. 1379/4-1052.
Dean, S. W., Lane, M., Dunmore, R. H., Ruddock, S. P., Martin, C. N., Kirkland, D. J.
and Loprieno, N., Development of assays for the detection of photomutagenicity ofchemicals during exposure to UV light-I. Assay developm (91), Mutagen.6,335-341.
Bayer report, Brendler-Schwaab, S., HR 00/ 106407: Photo-comet assay in vitro, (2001),
unpublished report n° T 8069508.
Yadrick, M. K., Kenney, M. A. and Winterfeldt, E. A., Iron, copper, and zincstatus: response to a supplementation with zinc or zinc and iron in adult females (1989),
Am. J. Clin. Nutr., 49, 145-150.
Brusick, D., Genotoxic effects incultured mammalian cells produced by low pH treatment
conditions and increased ion concentrations (1986), 8, 879-886.
Scott, D., Galloway, S. M., Marshall, R. R., Ishidate, M., Brusick, D. Ashby J. andMyhr, B. C., Genotoxicity under extreme culture conditions, report from ICPEMC task
group 9 (1991), Mutation Research, Elsevier, 257, 147-204.
Kirkland, D. J., Chromosomal aberration tests in vitro: problems with protocol design andinterpretation of results (1992), Mutagenesis, 7, 95-106.
Galloway, S. M., Aardema, M. J., Ishidate, M., Ivett, J. L., Kirkland D. J., Morita, T.,
Mosesso, P., Sofuni, T., Report from working group on in vitro tests for chromosomal
aberrations (1994), Mutation Research, Elsevier, 312, 241-261.
Kirkland, D. J. and Muller, L., Interpretation of the biological relevance ofgenetoxicity test results: the importance of thresholds (2000), Mutation Research,
Elsevier, 464, 137-147.
Ishidate, M., Harnois, M. C. and Sofuni, T., A comparative analysis of data on theclastogenicity of 951 chemical substances tested in mammalian cell cultures (1988),
Mutation Research, Elsevier, 195, 151-213.
30

SCCNFP/0649/03, final
Evaluation and opinion on : Zinc oxide____________________________________________________________________________________________

62.

Hilliard, C. A., Armstrong, M. J., Bradt, C. I., Hill, R. B., Greenwood, S. K. andGalloway, S. M., Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic
chemicals and metabolic poisons (1998), Environ.and Molecular Mutagenesis, 31, 316-326.
63. Blazak, W. F., Stewart, B. E., Galperin, I., Allen, K. L., Rudd, C. J., Mitchell, A. D. andCaspary, W. J., Chromosome analysis of trifluorothymidine-resistant LS 178Y mouse
lymphoma cell colonies (1986), Environmental Mutagenesis, 8, 229-240.
64. Speit, G., Autrup, H., Crebelli, R., Henderson, L., Kirsch-Volders, M., Madle, S., Parry,
J. M., Sarrif, A. M. and Vrijhof, H., Thresholds in genetic toxicology - conducting remarks(2000), Mutation Research, Elsevier, 464, 149-153.
65. Martindale, The extra Pharmacopoeia. 30th edition. 199331