Opinion of the Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE) on validation of test methods for phthalate migration - Opinion expressed at the 17th CSTEE plenary meeting, Brussels, 5 September 2000.
Background and Terms of
Reference
In the light of the
opinion of the CSTEE of 28th
September 1999 on "TNO, LGC and
US CPSC reports on Phthalate
migration test validation" and
the Addendum of 25th November
1999 to this opinion, a
programme for a full validation
of test methods for phthalate
migration has been developed
and that is presented to CSTEE.
The committee on the basis of
the Programme (CSTEE/2000/13
Add 1) for the validation of
methodologies to test migration
of plasticisers from toys and
childcare articles is to answer
the following questions:
- Is the approach of the
Programme suitable to provide
for a sound validation of
methods to measure
satisfactorily the release of
phthalates from toys and child
care articles as to distinguish
safe from unsafe products?
- Which comments can the
Committee offer to further
strengthen or complement the
programme?
Introduction
It is proposed in the
work programme that 3 of the in
vitro test methods, available
at present, will be validated:
- Horizontal shaking of
toy material with glass balls
in an artificial saliva at 37°
C (LGC mild horizontal shaking
method),
- Horizontal shaking of
toy material with relatively
heavy metal balls in an
artificial saliva at 60° C (LGC
stringent horizontal shaking
method)
- Dynamic extraction of
plasticiser from toy material
in an artificial saliva
employing a rotation method at
22° C (TNO rotation method)
The reasons given for
validating the 3 methods are:
"The stringent conditions may
allow to simulate the 'worse
case conditions' (referring to
DINP migration rate
9microgram/10 cm2/min, adopted
by CSTEE)1, whereas TNO method
and the mild version of LGC
method may be easily applied to
a wide range of testing and
other substances of various
target values".
The validation protocol
utilises to a great extent the
guidelines, which are described
in the CSTEE Opinion of 28th
September 1999 and in the
Addendum of 25th November 1999
to this Opinion. A major issue
identified, however, is the
problem of achieving the target
rate for the validation of test
method for DINP (diisononyl
phthalate) migration
(9microgram/10 cm2/min) from
toys and childcare articles
under in-vitro conditions. From
the data available so far, it
does not seem feasible to
obtain such a high DINP release
in in-vitro experiments
employing physiological like
conditions.
General Comments
The aim of the work
program is to validate 3 in
vitro test methods for the
migration of plasticiser(s)
from toys and childcare
articles using DINP as a model
plasticiser. It is expected
that the method(s) validated
through this exercise may be
applied for testing the
migration of other
plasticisers. The validation
protocol is mainly in
accordance with the recommended
guidelines, except for the
following (including the
unresolved issue mentioned
above):
1. The target migration
rate: CSTEE recommended the
need of a validated in vitro
test method1, which could
simulate the worst case DINP
migration (9 microgram/10
cm2/min) observed in the in
vivo study2. This value should
not be confused with the safety
of toys containing DINP. On the
basis of TDI, a toy/childcare
article will be safe when the
DINP migration rate from the
product is < 6.7
microgram/10 cm2/min (child<
3 years, weight 8 kg,
sucking/chewing period 180 min;
TDI 1200 microgram)1. Thus, to
distinguish between safe and
unsafe toys/childcare articles,
the target rate for the method
validation should be 6.7
microgram/10 cm2/min. But, the
validated method should also be
able to demonstrate that the
worst case migration, observed
in the in vivo study2, can be
simulated in in vitro
experiments. One of the several
toys/reference PVC discs used
in the in vivo studies2-4 can
be used for this demonstration,
as 15 % of the test population
in these studies was able to
achieve DINP migration >6.7
microgram/10 cm2/min.
2. The concern about
feasibility of achieving rather
high migration rate under
physiological like
conditions: The data
available to CSTEE indicated
that the target DINP migration rate
(9microgram/10 cm2/min) may not
be achieved under physiological
like conditions. Therefore,
CSTEE recommended in the
Addendum of 25th November 1999
that conditions 'as close as
possible' to in vivo conditions
should be used for the method
development and validation. The
only test method, available so
far, for relatively highly
release of DINP migration in
vitro employs rather aggressive
and non-physiological
experimental conditions - the
LGC stringent method5. It is
possible that microscopic
particles may release from the
test material under the
experimental conditions such as
those employed in the LGC
stringent method. In such a
case, it must be ascertained
that the measured DINP release
is not due to the presence of
microscopic particles in the
extraction medium. This will
require a suitable method for
testing of integrity of the
test material.
3. LGC mild horizontal
shaking method and TNO rotation
method: These methods have
not been able to simulate the
rather high phthalate migration
observed in the in vivo
studies. The reasons for
validating these methods is to
get an insight on dispersion of
data and sources of variation
in the results reported
recently5,6. Furthermore, it is
expected that these methods,
when validated using DINP as a
model plasticiser, can be used
as a general method(s) for
testing plasticiser migration
from toys and childcare
articles. However, no reason
has been provided to believe
that these methods may be
better for the analysis of
migration of other
plasticiser(s) than DINP.
It is proposed in the validation programme that the migration rate of a plasticiser from a toy/childcare article, determined by the validated method, may be corrected using a 'correction factor' to obtain maximum possible migration in vivo; and/or migration data may be statistically extrapolated to evaluate the safety of the product. It should be noted that correction factor for a plasticiser migration can only be derived when the migration rate of the plasticiser is available from a validated in vivo study. At present, correction factors can possibly be derived for DINP migration. As regards the statistical extrapolation of the migration data, an approach (using the DINP migration data from a TNO study6 and data from a child observation study7) for safety of toys has been proposed by NL8. However, the global acceptance of such an approach for safety assessment may require validation of the proposed method as well as an extended 'child observation study' with respect to children's mouthing behaviour may be required.
4. The CSTEE recommends
that the validation exercise
includes at least 15
laboratories (according to
international guidelines), but
it can be accepted if there are
acceptable results from 8
laboratories available for the
evaluation of the method
performance.
Specific Comments
Page 3, SOP, Expert
group discussion, line 3: The
migration limits have been
defined on the basis of the
risk assessment, and standard
deviation <20 % (of the mean
of the repeated observations)
has been recommended. The
performance of the validated
method for the determination of
DINP migration should take that
into account, but not vice
versa.
Page 3, Target rate,
Expert group discussion, para
2: See General Comments 2 &
3.
Page 5, Saliva simulant, Expert group discussion: According to the TNO report V2530, the proposed saliva simulant composition is based on the literature survey together with the prior consultation with an expert on oral biochemistry. The expert suggested the use of either amylase or albumin in the saliva simulant, but the saliva simulant described in the validation protocol does not contain any of these proteins. It should be noted that the CSTEE also recommended the use of some organic content in the saliva simulant. In the beginning of method development, TNO employed 0.16% mucin in the simulant but at a later stage the mucin was removed from the simulant. The apparent reason for using the mucin was that this protein has earlier been used by others as a substitute of natural proteins in saliva simulant. Furthermore, it appears that the determination of DINP migration rates should have been performed employing simulant containing mucin, amylase or albumin in preliminary experiments (TNO report V2530), but no results are available. The reason for removing mucin from the saliva simulant was that significantly lower (approximately 5x)2 DINP migration rate was observed in the presence of mucin. A possible explanation for this phenomenon may be that the DINP formed micelles/emulsion with the protein (mucin) present in the simulant. During extraction of the protein-simulant, the DINP may be precipitated together with the protein. This effect has possibly been observed in the determination of DINP in natural saliva - DCG in-vivo study2. In this study, recovery of DINP in test person's own saliva was determined and the results were corrected for the recovery percent. However, details of processing the saliva for DINP determination and the DINP recovery percents are not described in the report. (For a correct estimate recovery percent >50 may be accepted, provided that is reproducible). Therefore, it is not possible to comment on the results obtained in the in-vivo study.
One of the arguments to
validate TNO rotation method
and LGC mild shaking method is
that the DINP migration
determined by these methods may
be extrapolated, using a
correction factor, to estimate
the 'real' DINP migration
(in-vivo). To establish such a
'correction factor', it may
also be necessary to establish
the correction/correlation
factor for DINP migration
determined in the presence and
absence of natural saliva,
because the proposed saliva
simulant is devoid of proteins
present in natural saliva.
Page 6, # 3 from top
(check of sample integrity):
Check of sample integrity must
be performed when LGC stringent
method is used. In the first
Opinion on phthalate migration
from toys9, CSTEE already
mentioned that 'further
knowledge about possible
exposure to released particles
and direct transfer to the oral
cavity and ingestion is
needed'. However, particle
release from test materials has
not been checked in any of the
in vivo studies2-4.
Furthermore, the influence of
any particle release in
determination of DINP migration
rate has not been considered in
these studies. The CSTEE will
include this aspect in risk
assessment when data (from in
vivo studies) on the
possibility of released
particles is available.
Page 6, Analytical
determination, Expert group
discussion, para 3: Results of
all determinations must be
presented together with the
statistical analysis.
Page 7, Expert group
discussion, para 7: As
mentioned in General Comments
3, an evaluation of the
suitability of the validated in
vitro method (for DINP
migration) for other substances
will require migration data
from validated in vivo studies
employing test materials
containing the respective
substances.
Page 10, Homogeneity
testing, para 3: The
homogeneity testing should be
performed for the content(s) of
phthalate(s) concerned. CSTEE
is not aware of an accredited
method/ standard method for the
determination of phthalate
content in toy samples.
Page 11, Disks and toy
sources, para 4: It is a good
idea that 'JRC can act as
receiving office and interface
needed' (for future
distribution of reference
material as well).
Page 13, Item action 1,
HPLC method: It should not be
necessary to make use of a HPLC
method, because quantitative
analysis of phthalates can be
performed by the same GC-MS
method, which has to be used
for the qualitative analysis.
Page 14, Timeline:
Considering that some less
experienced laboratories may
participate in the validation
exercise, the timeline
described seems to be very
tight.
Conclusions
The programme for the validation methodologies aims to achieve a suitable method for testing DINP migration from toys and childcare articles. Furthermore, it is expected that the validated method will form a basis for the development of suitable methods for testing the migration of other plasticisers/additives from toys and childcare articles. The validation protocol is, to a great extent, in accordance with the guidelines recommended by CSTEE. The CSTEE's comments to strengthen/complement the validation programme are described above. The important suggestion for further improving the validation programme is that the validation exercise should focus on DINP migration employing the test method(s), which may possibly simulate the relatively high DINP migration (9 microgram/10 cm2/min) observed in an in vivo study. When absolutely necessary, non-physiological/ aggressive conditions may be used for simulating DINP migration in vitro, but the integrity of the test material must be ascertained. A correction/correlation factor between the DINP migration determined by the present protocol and in natural saliva has to be established. Furthermore, it must be demonstrated that a correct distinction can be made between safe and unsafe products, when the validated method is used for migration testing. An acceptance of NL approach8 for statistical extrapolation of DINP migration data (from in vitro studies) for distinguishing between safe and unsafe products requires validation of this approach. The 'correction factor' (for converting the observed in vitro migration to maximum possible migration in vivo), to evaluate the safety of a product may possibly be derived for DINP, but currently not for other substances. (In order to do this, in-vivo exposure studies with the specific substances under evaluation have to be conducted). For establishing a correction factor for DINP an additional in-vivo study will be required, because limited data is available on particle release during chewing/sucking of a toy. Furthermore, data on toxicokinetics of phthalates are also required. However, these recommendations should not postpone the proposed validation programme of in-vitro methods of migration testing.
1. References
1. Opinion of Phthalate
migration from soft PVC toys
and child-care article - data
made available since the 16th
of June 1998, opinion expressed
at the 6th CSTEE plenary
meeting, Brussels, 26/27
November 1998.
2. Report from the Dutch
Consensus Group: Phthalate
release from soft PVC toys.
RIVM report 613320 002, RIVM,
NL. September 1998.
3. Steiner I., Kubesch
K. and Fiala F.: Migration of
DEHP and DINP from PVC
articles. Vienna University of
Technology and Austrian
Standards Institute. September
3, 1998.
4. The risk of chronic
toxicity associated with
exposure to diisononyl
phthalate (DINP) in children's
products. US CPSC, December
1998.
5. Interlaboratory
validation of laboratory-based
agitation methods for the
determination of phthalate
plasticiser migration from PVC
toys and childcare articles.
LGC Technical Report No.
LGC/1999/DTI/004 , LGC, UK.
June 1999.
6. Validation of the
method "Determination of
diisononyl phthalate in saliva
simulant". TNO report V99.598,
TNO, NL. May 1999.
7. Groot M.E.,
LekkerkerK M.C. and
Dteenbekkers L.P.A.: Mouthing
behaviour of young children -
An observational study.
Wageningen Agricultural
University, Wageningen, NL.
September 1998.
8. CSTEE/97/1 - Add 174
9. Phthalate migration
from soft PVC toys and
child-care articles - opinion
expressed at the CSTEE plenary
meeting, Brussels, 24 April
1998.