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Endpoint:
health surveillance data
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2000-2002 (otherwise not specified).
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Levels and temporal trends of chlorinated pestcides, polychlorinated biphenyls and brominated flame retardants in individual human breast milk samples from Northern and Southern Norway.
Author:
Polder, A., Thomsen, C., Lindström, G., Løken & Skaare, J.U.
Year:
2008
Bibliographic source:
Chemosphere (2008) Vol. 73, pp.14-23.

Materials and methods

Study type:
biological exposure monitoring
Remarks:
World Health Organisation Survey
Test guideline
Qualifier:
according to
Guideline:
other: WHO designed study protocol
Deviations:
not specified
Principles of method if other than guideline:
As part of the 3rd WHO-coordinated exposure study, 29 breast milk samples were collected from primipara mothers living in Tromsø and Oslo. These samples were then analysed for levels of chlorinated pesticides, polychlorinated biphenyls and brominated flame retardants.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Type of population:
other: primipara mothers
Ethical approval:
confirmed and informed consent free of coercion received
Remarks:
The Norweigian Institute of Public Health was responsible for the study.
Details on study design:
SAMPLE COLLECTION
During 2000–2002, as part of the 3rd WHO-coordinated exposure study, a total of 29 breast milk samples were collected from primipara mothers living in Tromsø (N = 10) and Oslo (N = 19) (Fig. 1). Tromsø is a town located in Northern Norway (69°N, 19°E), 400 km north of the Arctic Circle. The city of Oslo is situated in Southern Norway (59°N, 10°E). The distance between the two locations is about 2000 km. The populations of Tromsø and Oslo counted 60.086 and 508.726 inhabitants in 2001, respectively (Tromsø Municipality, Plan og næring, oktober 2005; Annual statistics for Oslo 2001, nr. 101). In Oslo, two different sublocations were included in the study: Grünerløkka (Oslo GRÜ), which is a township close to the city centre and Søndre Nordstrand (Oslo SNO), situ­ated in the south-eastern part of the city. The SNO population is living in the vicinity of a municipal waste incinerator. The populations in these townships are multicultural. The Norwegian Institute of Public Health was responsible for the study. The collection of samples was organized through the local health centres. The local health personnel selected the mothers, chosen by given criteria. The mothers were informed about the objectives of the study and agreed with participation. They got instructions for collecting and storage of the milk samples at home. The samples were collected between 2 and 10 weeks after delivery, and kept frozen until analysis. Details of maternal age, body mass index (BMI), health status, occupation, dietary habits, place of birth, actual and previous places of living and the infant’s birth weight were described in questionnaires. All mothers were born in Norway, except for one mother from Tromsø, born in Singapore and two mothers from Oslo SNO, of which one was born in Sweden and one in USA. The ethnic background of the mothers was not registered. Some mothers reported that they had lived one or more years in countries as Sweden, France, Greece and U.S.A. In addition, the majority of mothers reported that they had moved within Norway once or several times before settling in Tromsø or Oslo.

CHEMICAL ANALYSIS
Concentrations of hexachlorobenzene (HCB); α-, β-, γ-hexachlorocyclohexane (HCHs); oxychlordane, cis-chlordane and transnonachlor (CHLs); 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p9-DDT) and its metabolites 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p9-DDD) and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p9-DDE) (DDTs); mirex; toxaphenes Parlar nos.: CHB-26, -40, -41, -44, -50 and -62; polychlorinated biphenyls (PCBs), IUPAC Nos.: PCB-28, -52, -74, -99, -101, -105, -114, 118, -123, -128, -138, -153, -156, -157, -167 -170, -180, -187, -189 and -194, polybrominated diphenylethers (PBDEs): PBDE-28, -47, -99, -100, -153, -154 and -209 and hexabromocyclododecane (total of α-, β- and γ-isomers of HBCD) were measured in individual breast milk samples at the Norwegian School of Veterinary Science in Oslo, Norway. Sum of indicator PCBs (PCBs6) consisted of non-dioxin-like (ndl) PCB-28, -52, -101, -138, -153, and -180. Sum-PCB12 consisted of PCBs6 and ndl PCB-74, -99, -128, -170, -187, and -194. Sum mono-ortho PCBs8 consisted of dioxin-like (dl) mono-ortho PCB-105, -114, -118, -123, -156, -156, -157, -167 and -189. Sum PCBs20 consisted of sum ndl and sum dl monoortho PCBs. The CHBs, PBDEs and HBCD were only analysed in the Tromsø samples. The analytical standards were provided by Cambridge Isotope Laboratories, Inc., Andover, MA, USA (OCPs PCBs, BFRs); Ultra Scientific, Rhode Island, USA (PCBs); Supelco, Bellefonte, PA, USA (OCPs); LGC Promochem, Wesel, Germany (CHBs).

EXTRACTION, SEPARATION AND DETECTION
The principles of the analytical method at the laboratory have been described by Brevik (1978). However, the method has been modified. The human milk samples (-10 g) were spiked with the internal standards PCB-29, -112 and -207, Detox-409 (2-endo,3-exo,6-exo,8,9,10,10-Heptachlorobornane), PBDE-77, -119, -181 and 13C-PBDE-209. Lipid extraction was done twice using cyclohexane and acetone (3:2) and an ultrasonic homogenizer (4710 Series, Cole Parmer Instruments Co., Chicago, IL, USA). Concentrated H2SO4 (purity 96%) (Scanpure, Chemscan AS, Elverum, Norway) was used for removal of lipids. The lipid content was measured gravimetrically. Other details of the analytical method have been described by Polder et al., 2008. For analyses of CHBs, the extracts were fractionated according to the method described by Føreid et al. (2000).

The separation and detection of OCs was performed by high resolution GC (Agilent 6890 Series gas chromatography system; Agilent Technologies, PA, USA) equipped with an auto sampler (Agilent 7683 Series; Agilent Technologies), a dual column system with specifications SPB-5 and SPB-1701, both 60 m, 0.25 mm i.d. and 0.25 µm film thickness (Supelco, Bellefonte, PA, USA) coupled to two 63Ni micro (µ) electron capture detectors (Agilent 6890 µ-ECD). Other details of the GC-ECD specifications have been described by Polder et al. (2008).

The separation and detection of CHBs, mono-ortho PCBs, PBDEs and HBCD were performed by GC (Agilent 6890 Series/5873 Network), equipped with a SPB-5 column (60 m, 0.25 mm i.d., 0.25 µm film thickness; Supelco) and connected to a low resolution MS (LRMS) quadrupol detector (Agilent Technologies, Avondale, PA, USA), operated in selected ion monitoring (SIM) mode with negative chemical ionization (NCI). Hydrogen 5.0 pure (Yara Industrial AS, Oslo) was used as carrier gas. An injection volume of 2 µl was used. The following temperature programs were used: CHBs: 90 °C–3 min–35 °C/min–200 °C–5 min–5 °C/min– 240 °C–8 min–5 °C/min–275 °C–15 min; mono-ortho PCBs: 90 °C–2 min–15 °C/min–220 °C–2 min–1.5 °C/min–235 °C–15 min–5 °C/ min–275 °C–5 min; PBDEs and HBCD: 90 °C–2 min–2.5 °C/min– 19 °C–1 min–5 °C/min–250–1 min–2.5 °C/min–320–10 min. The separation and detection of PBDE-209 was performed by GC¬LRMS (Agilent 6890 Series/5873 Network). The injection volume was 10 µl injected in a programmable temperature vaporization (PTV) injector (Agilent Technologies) connected to a DB-5-MS column (10 m, 0.25 mm I.D., 0.1 µm film thickness; J&W Scientific, Agilent Technologies). The following target ions were used: CHB-26: m/z 376.8; Detox-409: m/z 342.8; CHB-40, -41, -44: m/z 376.8; CHB-50: m/z 412.7; CHB-62: m/z 376.8; PCB-112, -123, -118, -114, -105: m/z 325.8; PCB-156, -157, -167: m/z 359.7; PCB¬189: m/z 395.7; PBDE-28, -47,-99, -100, -153, -154 and HBCD: m/z 79, 81; PBDE-209: m/z 486.5; 13C-PBDE-209: m/z 496.5. Further details have been described earlier (Andersen et al., 2006; Polder et al., 2008). Detection limits for individual compounds were determined as three times the noise level. The detection limits (ng g-1 wet weight (ww)) for determination on the GC-ECD ranged from 0.001 to 0.002 ng gg 1 ww for OCPs and PCBs. For the components detected on the GC–MS the detection limits were for mono-ortho PCBs: 0.0004 ng gg 1 ww; PBDEs: 0.003 ng gg-1 ww; PBDE-209: 0.0005 ng gg 1 ww and HBCD: 0.050 ng gg-1 ww, respectively. For all components, five- to eight-point linear calibration curves were used and calculations were done within the linear range for the component.

ANALYTICAL QUALITY
The laboratory is accredited by Norwegian Accreditation for determination of the above mentioned chemicals in biological material according to the requirements of the NS-EN ISO/IEC 17025 (TEST 137). The analytical quality of the determinations of OCPs, PCBs, CHBs, BDEs and HBCD was found satisfactory after participation in several relevant intercalibration tests: WHO, third round of coordinated exposure study on levels of PCBs, PCDD/PCDFs in breast milk (2000–2001); Quasimeme, Exercise 524, round 28, on PBDEs in human milk (2002) and AMAP ring-tests for PCBs and OCs in human serum (2001–2006).
Several procedural blanks (solvents) and one blind (low fat milk) were included in every series of 15 samples. For every batch of samples parallels of one or more samples were analysed. Deviation in response of the GC system was controlled by analysing a standard for every 10th sample. The between run repeatability of the method was tested by analysing the laboratory’s own reference sample of seal blubber (whale blubber for toxaphenes) which was analysed for each of the sample series. For recovery tests, low fat milk was spiked with OCPs, PCBs, and PBDEs. The relative mean recoveries were 90%, 86%, 107%, 98%, 99% and 116% for the OCPs, PCBs, mono-ortho PCBs, PBDEs, PBDE-209 and HBCD, respectively. The coefficient of variation for determinations in the laboratory’s reference samples ranged between 7% and 24% for OCs, 16% and 33% for CHBs, 16% and 41% for the PBDEs and HBCD and 7% for PBDE-209.

TEQS
In 2005, the WHO toxic equivalent factors (TEF) of PCDDs/ PCDFs, non-ortho- and mono-ortho PCBs for humans were re-evaluated. These changes influenced especially on the TEFs for the mono-ortho PCBs, which decreased substantially, compared to TEFs which were established by WHO in 1997. In the present study, we used the TEFs 1998 for calculations of the toxic equivalents (TEQs) when comprising with previous studies and with other countries. The re-evaluated TEFs2006 were used for the calculation of the estimated daily intake (EDI) of the dl mono PCBs8.

STATISTICS
The Shapiro-Wilk test was used to investigate if data were normal distributed. The results of this test showed that some data were normal distributed, but not all. Therefore non-parametric tests (Wilcoxon, 1-way Anova) were used for comparison of groups. Differences with p <0.05 were considered to be statistically significant. Two-tailed Spearman tests were used for investigation of bivariate correlations. The statistical analyses were performed using the statistical package JMP® Release 6.0.0 (SAS Institute Inc., Cary, NC, USA).

Results and discussion

Results:
Demographics
The mean age of the by criteria selected mothers (N = 29) was 29.3 years. This is 2.4 years more than the national mean age for primipara recorded in 2000 (26.9 years; N = 59806; (The Medical Birth Register of Norway). The reason for the higher age of the subjects in the present study could be that younger women did not fulfil the criteria of having lived at the same place during the last 5 years, or that elder women felt more confident to participate in the study. The means of maternal BMI, measured before pregnancy and before delivery (24 and 31 kg m2), were highest in Oslo SNO. The birth weights of all infants in the present study ranged from 2900 to 4890 g. The mean and median of infant birth weights were lowest in Oslo SNO (3338 and 3500 g) and were approximately 500 g lower than in Tromsø and Oslo GRÜ. The low infant birth weight in Oslo SNO was however not significantly different compared to Tromsø and Oslo GRÜ. The Norwegian average of birth weights was 3518 g in 2001 (The Medical Birth Register of Norway). Birth weights in Tromsø and Oslo GRÜ were thus somewhat higher than the national mean in 2001. In Tromsø, a weak positive relationship was found between maternal BMI and infant birth weight (r = 0.55). The dietary habits did not differ much between the studied locations. However, fish (fish species not specified) was consumed more often in Tromsø, and meat was consumed more often in Oslo. One mother from Tromsø was vegetarian.
A higher frequency of recent and earlier smokers was observed among the mothers from Oslo GRÜ.

HBCD
HBCD was only analysed in the samples from Tromsø. HBCD was only detected in one sample (0.13 ng g-1 lw).

Any other information on results incl. tables

Table 1.General demographic characteristics of mothers and infants, preseented as mean, standard deviation (SD), median (Med.), minimum (Min.) and maximum (Max.) values

Tromsø (N=10) Oslo GRÜ (N=10) Oslo SNO (N=9)
Mean SD Med. Min. Max. Mean SD Med. Min. Max. Mean SD Med. Min. Max.
Primipara mothers
Age (years)  29 5 28 20 37 29 4 29 24 37 30 3 30 26 34
Height (cm) 165 5 165 157 171 167 5 167 160 174 167 5 168 159 174
Weight before pregnancy (kg) 65 9 64 50 78 67 16 61 55 108 71 1 66 53 103
Weight before delivery (kg) 81 12 81 60 97* 84 15 79 69 118 85 16 87 65 117
BMI, kg m2 (before pregnancy) 24 3 24 19 28 24 6 22 19 41 26 5 24 20 36
BMI, kg m2 (before delivery) 27 10 28 23 36* 30 6 29 24 45 31 5 30 24 41
lipid% of breast milk  3.4 1 3.5 2.1 4.8 3.6 1 3.5 2.4 6 3.4 1 3.3 2.4 4.6
Infants
Birth weight (g)  3835 746 4003 2900 4890 3838 519 3863 3000 4740 3338 286 3500 2930 3640
Sex of infant (F, M)  4F 6M 4F 7M 4F 5M

Table 2. Mean standard deviation (SD), median, minimum (Min.) and maximum (Max.) of individual and sum concentrations (ngg-1 lipid weight) of HBCD in breast milk from primapara in Tromsø

Tromsø (N-10)
Nos Pos** Mean SD Median Min. Max.
HBCD 1/10 0.13 Na Na Na Na

*One missing value.

**Nos Pos - number of positive detected.

Applicant's summary and conclusion

Conclusions:
HBCD was only present in one sample from Tromsø (10 samples collected in total from Tromsø) to a level of 0.13 ng g-1 lw. Due to the presence of HBCD in the breast milk sample it is considered possible that the substance can be passed to the infant from the mother in breast milk.