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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
repeated dose toxicity: dermal, other
Remarks:
36 weeks, once per week (30 treatments)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies

Data source

Reference
Reference Type:
publication
Title:
Long-term exposure to commercially available sunscreens containing nanoparticles of TiO2 and ZnO revealed no biological impact in a hairless mouse model
Author:
Osmond-McLeod, M.J. et al.
Year:
2016
Bibliographic source:
Particle and Fibre Toxicology 13: 44.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A group of 10 female, albino, immune-competent, hairless SKH:QS mice were treated once per week for 36 weeks with a TiO2 containing sunscreen. The sunscreen was applied at 2 mg/cm² to the head, ears, back, sides and tail of each mouse and sunscreen were left to equlibrate for 20 min. Mice receiving no sunscreen application were sham treated. Mice were washed after 2 hrs. Once per month, the thickness of the dorsal skin on each mouse was measured 72 hrs after the last irradiation. After 36 weeks mice were sacrificed and skin neoplasms were counted and classified macroscopically. Major internal organs (brain, liver, spleen, kidneys, lung and heart) were histopathologically analyzed. Additionally, measurement of liver Ti by ICP-MS was conducted and liver tissue was chosen for an analysis of altered gene expression.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: nanoparticles in suncream
Details on test material:
- Particle size distribution (TEM): 21.5 ± 0.6 nm
- Shape of particles (TEM): spheroidal
- Composition of test material, percentage of components: 40 mg/mL TiO2, 70 mg/mL octylmethoxycinnamate,40 mg/mL butyl methoxydibenzoylmethane

This particular sunscreen was selected on the basis of its similarity to a sunscreen that had elsewhere been shown to contain nanoparticles with photocatalytic properties similar to Degussa P25, a commerciallyavailable product comprising both the anatase and rutile crystal phases of TiO2 nanoparticles (Barker & Branch, 2008)*; personal communication).

*Reference:
- Barker P, Branch A. The interaction of modern sunscreen formulations with surface coatings. Prog Org Coat. 2008;62: 313 – 20.

Test animals

Species:
mouse
Strain:
other: SKH:QS
Remarks:
hairless
Details on species / strain selection:
not specified
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CSIRO Animal House
- Age at study initiation: 8 weeks
- Housing: housed in groups of 10 in open-topped polycarbonate cages (bedding material was removed and replaced by lining of paper towel during treatment)
- Diet (ad libitum): Gordon´s rat and mouse pellets

ENVIRONMENTAL CONDITIONS
- Temperature: ca. 21°C
- Relative humidity: 55 - 65 %
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: head, ears, back, sides and tail

REMOVAL OF TEST SUBSTANCE
- Washing: pH balanced soapy water and soft cotton pads were used and mice were dried using fresh cotton pads
- Time after start of exposure: 2 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 mg/cm²
The total volume of sunscreen applied vaired slightly per mouse according to its size, but on average approx. 110 µL.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, Elizabethan collars

Mice were not observed to groom each other or themselves during treatment periods.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
36 weeks
Frequency of treatment:
once per week (total: 30 treatments)
Doses / concentrations
Dose / conc.:
2 other: mg/cm²
No. of animals per sex per dose:
10 animals/group
Control animals:
yes, concurrent no treatment
Details on study design:
not specified
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: no data
DETAILED CLINICAL OBSERVATIONS: no data
DERMAL IRRITATION: no data

BODY WEIGHT: Yes
- Time schedule for examinations: at study termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: no data
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: no data
WATER CONSUMPTION: no data:
OPHTHALMOSCOPIC EXAMINATION: no data
HAEMATOLOGY: no data
CLINICAL CHEMISTRY: no data
URINALYSIS: no data
NEUROBEHAVIOURAL EXAMINATION: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

After 36 weeks (30 treatments), under anaesthesia mice were weighed and then euthanized, and skin neoplasms were counted and classified macroscopically. The dorsal skin was dissected, mounted, and stored on ice in 1 X Histochoice tissue fixative for histopathology. Major internal organs (brain, liver, spleen, kidneys, lung and heart) were retrieved, weighed and sectioned. Sections were either stored in 4 % neutral buffered formalin at room temperature for histopathology, or snap frozen in liquid nitrogen and stored at -80 °C for other analyses.

Abnormalities identified in organs were classified, typically on their degree of severity, and the incidence within a treatment group was then compared to the incidence across all groups.
Other examinations:
MEASUREMENT OF LIVER Ti by ICP-MS
Total Ti concentrations in livers harvested from mice in control group and treatment group were determined in-house by ICP-MS.
Statistics:
Histological findings in treatment group was assessed for statistical significance compared to control group using a two-sided Fisher´s exact test. Differences between groups for skin thickness and/or organ concentrations of Ti were assessed for statistical significance by unpaired t-tests with Welch’s Correction (if only two groups), with significance set at p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
not specified
Dermal irritation:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
mortality was observed in the TiO2 group (1 animal)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
data not shown
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
data not shown
Gross pathological findings:
no effects observed
Description (incidence and severity):
no macroscopic outcomes were observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination of the prematurely deceased mouse in the TiO2 treated group showed peritonitis.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Levels of liver Ti
TiO2 treated animals showed significantly elevated levels of Ti in liver tissue (0.31 mg/g) compared to untreated mice. However, untreated mice had a Ti basal level of 0.19 mg/g tissue weight.
Details on results:
MORTALITY
- no treatment related mortality was observed. However, one animal of the TiO2-treated group died prematurely.

BODY WEIGHT AND WEIGHT CHANGES
- no changes in body weight could be observed (data not shown)

WATER CONSUMPTION
- water consumption remained unchanged (data not shown)

ORGAN WEIGHTS
- the test item had no effect on the organ weights of major internal organs (brain, liver, spleen, kidneys, lung and heart)

Macroscopic and histopathological outcomes
-TiO2 treated animals showed no tickening of the dorsal skin compared to the untreated animals.

Whole-genome analysis
-Whole genome gene-expression profiling showed no alterations in transcript levels of TiO2-treated animals.

Effect levels

Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
This study evaluated the impact of sunscreen containing TiO2 as well as organic substances as active ingredients. Hairless mice were treated once per week for 36 weeks. Mice were sacrificed after 30 treatments and selected organs were histopathologically examined. No TiO2- treatment related effects could be observed.
Elevated TiO2 levels in liver tissue of treated animals was the only observed treatment related effect and could probably be explained by the fact that all mice per group were treated at the same time in one cage and oral application of TiO2 by grooming or after treatment in their home cages could not be excluded.
This study is not in accordance with any dermal toxicity testing guideline and due to major methodological restriction this study cannot be used for hazard assessment. However, this study demonstrates that the use of TiO2 containing sunscreen is not linked to any adverse effect in a hairless mice model.