Calcium metaborate (Ca(BO2)2) and calcium tetraborate (CaB4O7), amorphous reaction products of boric acid with lime

Epidemiological data

Administrative data

Endpoint:

epidemiological data

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

other: Not relevant for occupational exposure study

Data source

Reference

Reference Type:

publication

Title:

Reproductive toxicity parameters and biological monitoring in occupationally and environmentally boron-exposed persons in Bandırma, Turkey.

Author:

Duydu Y, Başaran N, Ustündağ A, Aydın S, Undeğer U, Ataman OY, Aydos K, Düker Y, Ickstadt K, Waltrup BS, Golka K, Bolt HM.

Year:

2011

Bibliographic source:

Arch Toxicol. 2011 Jun;85(6):589-600. PMID:21424392

Materials and methods

Study type:

other: Worker reproductive toxicity study

Endpoint addressed:

basic toxicokinetics

toxicity to reproduction / fertility

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Assessment of environmental and occupational exposure to boron containing substances in a boric acid/borax production plant in Bandırma, Turkey.
- Sampling of Boron in food, water and workplace atmosphere (personal air monitoring or stationary ambient air monitoring) as well as in blood, urine, semen of participants.
- Determination of blood levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone and prostate-specific antigen (PSA) levels.
- Determination of sperm concentration and motility parameters and additionally sperm morphology s per the Kruger’s strict criteria.
- Assessment of individual boron exposures based on airborne occupational exposure measurements and intake calculations via food and drinking water.
- Groups:
Original groups:
exposed: 102 workers involved in the production of boron products (boric acid production workers (n=57), borax production workers (n=31), sodium perborate production unit workers (n=5), boric acid plus borax production workers (n=5), laboratory workers (n=2), a storage worker (n=1), a mechanic technician (n=1)).
controls: 102 sulfuric acid production workers, working at a different site.
Significant background exposure to boron via the diet prepared in the same cafeteria for both groups made a regrouping necessary which was based on the blood boron levels. See below for details.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid, borax and sodium perborate

IUPAC Name:

Boric acid, borax and sodium perborate

Details on test material:

- Name of test material (as cited in study report): Boric acid, borax and sodium perborate
- Molecular formula: boric acid: B(OH)3, borax: Na2B4O7*10H2O, sodium perborate: NaBO3
- Physical state: solids
- Analytical purity: different grades during the production process

Method

Type of population:

occupational

Ethical approval:

confirmed and informed consent free of coercion received

Details on study design:

HYPOTHESIS TESTED (if cohort or case control study):
The null hypothesis for each biologic fluid was that the mean of the exposed group is equal to the mean of the control group.

METHOD OF DATA COLLECTION
- Type: Questionnaire, Atmosphere measurement, boron level determination in of blood, semen and urine, determinetion of semen and sperm parameters.
- Details:
Questionnaire: demographic, exposure, reproductive and general health information, drinking and eating habits.
Atmosphere measurement: see under "Details on exposure"
Biological sampling: taken at the end of a work shift; no samples taken on the first working day of the week or shift period; workers were informed of the importance to avoid a possible contamination (sampling after showering and changing of clothes), for details see below under "Details on exposure"

STUDY PERIOD:
not described in detail
exposure periods (years):
Control 15.30 + 8.63
Low exposure 16.85 + 7.06
Medium 17.21 + 6.77
High 13.96 + 8.04

SETTING:
- 3 working shifts (24:00 — 08:00 h, 08:00 — 16:00 h, 16:00 — 24:00 h) for exposed and control workers, 8 hours per day / 6 days per week,
- exposed workers: 41of the midnight shift (24:00 — 08:00 h), 31 of the morning shift (08:00 — 16:00 h), 30of the night shift (16:00 — 24:00 h).
- control workers: working between 08:00 — 16:00 h.

STUDY POPULATION
- Total population (Total no. of persons in cohort from which the subjects were drawn):
exposed: 428 workers, 102 participated: boric acid production workers (n=57), borax production workers (n=31), sodium perborate production unit workers (n=5), boric acid plus borax production workers (n=5), laboratory workers (n=2), a storage worker (n=1), a mechanic technician (n=1)
controls: 432 workers, sulfuric acid production plant workers (n=28), steam power plant workers (n=17), demineralized water production (DWP) unit workers (n=2), energy suppliers (n=11), mechanical workshop workers (n=19), garage workers (n=14), steelyard workers (n=2), construction service workers (n=3), laboratory technicians (n=3), and office workers (n=3).
- Selection criteria:
original groups:
exposed: all married workers of the plants described above, wishing to participate, were enrolled.
controls: probably matched for age and years of employment (and possibly additional parameters), not described in detail
boron blood level based groups:
Exposure groups n (204) Re-classification (ng boron/g blood)
New control group 49 Low exposure group 72 >LOQ–100
Medium exposure group 44 >100–150
High-exposure group 39 >150
LOQ Limit of quantification, LOQ (for boron in blood): 48.5 ng/g
Significant background exposure to boron via the diet prepared in the same cafeteria for both groups made a regrouping necessary which was based on the blood boron levels. All participating workers were re-classifed both according to their calculated daily boron exposure levels and to the blood boron levels. For the re-classification of dose groups blood boron levels published in recent epidemiological studies were taken into account. Workers with a blood boron concentration below the LOQ were combined to form the new control group.

- Total number of subjects participating in study: 204
- Sex/age/race: males
original groups:
exposed: 42.62 ± 4.76 (range: 28 — 50) years, caucasian
controls: 41.75 ± 6.29 (range: 23 — 53) years, caucasian
new groups: see table 4
- Smoker/nonsmoker: not reported
- Total number of subjects at end of study: 204
- Matching criteria: not reported, probably age and years of employment (and possibly additional parameters)


COMPARISON POPULATION
- Type: Control group
- Details: Significant background exposure to boron via the diet prepared in the same cafeteria for both groups made a regrouping necessary which was based on the blood boron levels. The control group was defined as the group which had blood boron levels below the LOQ (level of quantification).

HEALTH EFFECTS STUDIED
- in the current publication only boron levels in blood, urine and semen were regarded

OTHER DESCRIPTIVE INFORMATION ABOUT STUDY:

Exposure assessment:

measured

Details on exposure:

TYPE OF EXPOSURE:
occupational: dusts of boron containing substances (see above)
environmentally: boron compounds dissolved in drinking water and contained in foodstuff

TYPE OF EXPOSURE MEASUREMENT:
- Personal sampling:
exposed group only, personal air sampler (SKC, AirCheck 2000), flow rate 2 L/min, sampling time 8 hours; low-ash PVC filters (SKC, 5 37 mm, preweighed) and SureSeal cassettes (SKC, 37 mm)
Analysis of dust collected in cassettes by gravimetric and instrumental methods (Selin B (2010) Boron Determination in Body Fluids by Inductively Coupled Plasma Optical Emission Spectrometry and Inductively Coupled Plasma Mass Spectrometry. Dissertation, Middle East Technical University, Ankara, Turkey. & OSHA (2009) Chemical Sampling Information, Boric Acid, www.osha.gov .)
- Area air sampling:
control group only: same devices and parameters were used as for the personal sampling but the devices were not carried by individuals, but used statically, to determine an average value for the control workers.
- Food sampling:
Water content of menus averaged 400 mL and was prepared with boron contaminated water of the central cantina (9.47 mg/L). Multiplication of the two values yielded the average daily boron exposure via food. Both groups received their menus from this source. Exposure through foodstuff is an addition of exposure from boron contaminated water and internal boron levels of food ingredients. Boron levels in food stuff were negligible compared to the boron levels from the tap water.
- Drinking water sampling:
at home: no measurable boron concentrations were found in the municipal tap water
at work place: - exposure group: water and tea consumption was assessed and an individual exposure was calculated based on the boron concentration of the boron contaminated water of the central cantina (9.47 mg/L).
- control group: no exposure as the water from the local source of their production site had no measurable boron level.

- Biomonitoring (urine): collected in polypropylene containers and kept at -20 °C until urinary boron and creatinine analysis (creatinine assay kit, Cayman Chem. Comp.); measurements in urine correlated to the creatinine contents of the samples.
- Biomonitoring (blood): collected by venipuncture in 3 separate blood collection tubes; 2 heparin containing tubes (5 mL each; BD vacutainer, plastic heparin tubes)one for blood boron level determination and one for Comet assay analysis, 1 5 mL tube containing clot activator (BD vacutainer, SST II Plus plastic tubes) for determination of FSH, LH, total testosterone, PSA (Immulite 2000 Immunoassay Analyzer).
- Biomonitoring (semen): at the infirmary in a special room (in accordance with the recommendations of the World Health Organization; WHO 2003). The workers were informed about the importance of an abstinence for at least 48 hours (but no longer than 7 days) prior to providing the semen samples.
Samples were collected in sterile polypropylene containers and allowed to liquefy (WHO 2003).
Additionally, three high-security sperm straws (Cryo BioSystem) per each semen sample were filled by means of an aspiration pump. The straws were sealed and immersed directly into liquid nitrogen for subsequent determination of DNA damage (Comet assay, in duplicate, one sample as backup).
Remaining semen sample was used for boron level determination (insufficient sample volumes in three samples each of the exposed and the control group)
Parameters tested:
Initial microscopic investigation, volume, viscosity and pH, sperm concentration, % motility, immotility, % normal morphology, motile sperm concentration, progressive motile sperm concentration, functional sperm concentration, average velocity, and sperm motility index), analyzed within 1 hour (including liquefaction), using a SQA-V Gold Sperm Quality Analyzer.


EXPOSURE LEVELS:
Calculated for individuals as summary of exposures via air (assuming 10 m³ inhaled volume per shift), food and water/tea consumption (assuming comparable absorption via the oral and the inhalation route)

EXPOSURE PERIOD:
exposed workers: 0.5 - 25.0 years employed, average = 16.02 ± 7.06
control workers: 0.17 - 26.00 years employed, average = 15.98 ± 8.22
new groups: see table 4

POSTEXPOSURE PERIOD:
not applicable

Statistical methods:

- Investigation of the global hypothesis (the means of the four groups are equal):
a Kruskal–Wallis test for all variables in Tables 4-9 except for the categorical variables pH and cytoplasmic droplets.
In case of P < 0.05 (i.e.: the hypothesis of equal group means was rejected) the two-sided Mann–Whitney U test was applied to find distinct differences between each of the pairs of groups + adjustment of the corresponding six P values with the Bonferroni-Holm method for each variable in order to account for the problem of multiple testing
- Nonparametric tests were deliberately used as some of the variables show outliers.
- Analysis of dependence of the groups and the categorical variables (pH and cytoplasmic droplets) with Fisher’s exact test.
- Software: R, a language and environment for statistical computing, version 2.11.1 (R Development Core Team 2010).

Results and discussion

Results:

EXPOSURE
- Number of measurements: not described in detail
- Average concentrations: see Tables 2 and 3
- Arithmetic mean: see Table 2 and 3

Discussion:
The high boron contamination of water sources for cafeteria and infirmary was not anticipated in the planning phase of the study. This "background" exposure lead to relatively high exposure of the "control" group:
Total average exposure of occupationally exposure exposed workers: 12.08 ± 6.18 mg boron/day)
Total average exposure of control workers: 5.83 ±1.71 mg boron/day
The average daily boron exposure (DBE, in mg B/d) calculated for the re-classified groups are:
Control 4.68 + 1.63
Low exposure 7.39 + 3.97
Medium 11.02 + 4.61
High 14.45 + 6.57
(see also Table 4)

FINDINGS

- Exposure to boron:
• restricted to the tap water in the infirmary and the cafeteria of the company (oral) and to the atmosphere in the boron production sites (inhalation) (see table 3 for details).
• mean levels of inhaled boron (mg/8 h) in medium and high-exposure group signiffantly higher than in the control group
- Mean ages of re-constituted exposure groups: not significantly different from the mean age of the new control group (see Table 4).
- Boron levels in biological fluids: (see Table 4)
• Mean calculated daily boron exposure levels (DBE): ignificantly higher in exposure groups than in the new control group (see Table 4).
• Mean urine boron levels: significantly higher in exposure groups than in the new control group (see Table 4).
• Calculated DBE levels: positively correlated with the blood boron concentrations of the workers (Pearson corr. coeff.: 0.635).
• Urine boron concentrations: positively correlated with the blood boron concentrations of the workers (Pearson corr. coeff: 0.633).
• Semen boron concentrations in exposed workers: exposure groups vs. new control group: signifficantly different; the dose response trend was not significant, variations within groups were great.
• Correlation between semen boron concentration and blood boron concentration: very low (Pearson corr. coeff.: 0.222).
• Boron concentrations in semen are a lot higher (p<0.001; t-test) than in blood and urine levels; concentrating factors semen boron concentration/blood boron concentration: low - ca. 20; medium - ca. 12; high - ca. 8.
- Hormone levels: (see Tables 5 + 6)
• no significant differences between grooups execpt for LH, mid dose vs. high dose
• very weak correlation between blood boron concentration and hormone levels (FSH: Pearson corr. coeff: 0.143; LH: Pearson corr. coeff: 0.164; total testosterone level: -0.053).
• No statistical significant difference in testosterone levels between shifts.
• No statistical significant difference in testosterone levels between new control group and exposure groups.
- Semen nd sperm parameters (including morphology): (see Tables 7 + 8 + 9)
• no significant difference in any parameter tested between the exposure groups and the new control group.
• correspondingly only a weak correlation between the percentages of the normal morphology and blood boron levels.
• Only weak correlation between inhaled boron (mg/8 h) and blood boron (0.279), inhaled boron–semen boron (0.185), and inhaled boron–urine boron (0.106) levels
- The PSA level was not statistically significantly different when groups are compared.

conclusions:
- The surprising contamination of the tap water of the cafeteria and the infirmary led to to significant exposure of the originally planned control group and made a re-assembly of dose groups necessary, based on the calculated daily boron exposure and the respective blood boron levels.
- Due to the background exposure via drinking water no clear realtion could be found between inhalation exposure and boron levels in biological fluids.
- Blood and urine boron levels increased steadily with rising DBE, while semen boron levels failed to follow a steady trend.
- Variation in semen boron levels was great.
- Boron is accumulated in semen and the concentration factor is highest at the lowest exposure.
- Adverse effects in hormone levels were absent when exposure groups are compared to the new control group.
- For any of the semen parameters a statistically significant differences was seen between new control group and exposure groups. Together with the affore mentioned fact this indicates that boron does not have an adverse effect on the male reproductive system at typical exposure conditions.

Confounding factors:

Age: no significant difference in the mean age between groups, see Table 4
Duration of employment: no significant difference in the mean years of employment between groups, see Table 4

Strengths and weaknesses:

Strengths:
- Measured inhalation exposures.
- Analysis of age and duration of employment as confounding factors

Weaknesses:
- Due to the contamination of the tap water of the infirmary and the cafeteria all workers are exposed.
- Exposure is a mixture of oral and inhalation exposure. Total daily boron exposure (DBE) is calculated as an addition of oral and inhalation exposure regardless of potential differences in toxicokinetic parameters for this routes.
- Due to the backgroud exposure four groups were assembled based on the calculated daily total boron exposure and the measured blood boron concentration. The new control group had blood boron levels below the LOQ of 48.5 ng/g. Nevertheless these workers have an average DBE of 4.7 mg boron/d.
- The correlation between blood boron levels and semen boron levels is very weak. Therefore the comparison of semen and sperm parameters between groups is of questionable explanatory power.
- Number of air measurements and study period not described.

Any other information on results incl. tables

- Table 2. Results of ambient air monitoring and calculated daily boron intake levels (inhaled and total)

Specific working areas of the workers		Inhalable dust, gravimetric (mg/m3)^1	Boron concentrations in workplace air (mg/rn3)	Boron inhaled at the workplace (mg/8h)	Calculated total daily boron intake (mg/day)
	n	Mean, SD (Range)	Mean. SD (Range)	Mean, SD (Range)	Mean, SD (Range)
Personal air monitoring Occup. exposed workers	102
Boric acid production	57	0.46 ± 0.94 (0.04 - 7.15)	0.27 ± 0.43 (ND - 2.69)	2.78 ± 4.31 (ND - 26.90)	11.22 ± 6.81 (2.56 - 35.62)
Borax production	31	0.99 ± 1.31 (0.16 - 4.95)	0.24 ± 0.46 (ND - 2.22)	2.35 ± 4.64 (ND - 22.16)	13.18 ± 5.57 (2.56 - 25.01)
Sodium perborate unit2	5	0.93 ± 0.59 (0.19 - 1.67)	0.09 ± 0.04 (0.03 - 0.14)	0.89 ± 0.41 (0.28 - 1.42)	13,60 ± 2.413 (9.63 - 16.10)
Boric acid - borax	5	0.55 ± 0.43 (0.15- 1.20)	0.38 ± 0.43 (1.09)	3.83 ± 4.27 (10.86)	14.48 - 4.21 (8.56- 19.57)
Miscellaneous'	4	0.25 ± 0.11 (0.09 - 0.35)	0.03 ± 0.05 (ND - 0.09)	0.26 ± 0.46 (ND -0.95)	10.54 ± 6.38 (3.51 - 17.05)
Mean		0.64 ± 1.04 (0.04 - 7.15)	0.25 ± 0.42 (ND - 2.69)	2.51 ± 4.23 (ND - 26.90)	12.08 ± 6.18 (2.56 - 35.62)
Ambient air monitoring Control workers	102
Sulfuric acid plant	28	0.14	ND	ND	3.79 (3.79 - 3.79)
Steam power plant	17	0.29	ND	ND	6.50 ± 1.00 (4.97 - 8.52)
Energy supply unit	11	0.09	ND	ND	6.59 ± 1.09 (4.97 - 8.52)
DWP unit	2	0.09	ND	ND	6.75 ± 0.84 (6.16 - 7.34)
Mechanical workshop	19	0.16	0.01	0.14*	6.73 ± 1.13 (5.11 - 8.66)
Garage + steelyard	16	0.16	0.02	0.20*	5.66 ± 2.32 (0.20 - 8.72)
Construction service	3	0.09	ND	ND	6.55 ± 0.68 (6.16 - 7.34)
Laboratory	3	0.09	ND	ND	8.52 - 0.00 (8.52- 8.52)
Office	3	0.07	ND	ND	5.37 ± 1.81 (3.79 - 7.34)
Mean		0.13 ± 0.07 (0.07 - 0.29)	-	-	5.83 ± 1.71 (0.2 - 8.72)

¹The dust was sampled using a 37mm total dust sampler, that the inhalable dust fraction was not actually measured. To get an estimate of the inhalable dust fraction (using IOM sampler) it is necessary to multiply values by factor of 2.5

Miscellaneous: laboratory technicians, storage worker, mechanic technician, The workers exposed to borax in sodium perborate unit (see "description of the cohort"), *Calculation based on ambient air measurement. ND: values below limit of quantification (LOQ).

- Table 3. Boron concentrations in water samples

Water samples		mg boron/L
Central cafeteria	3 x 250 mL	9.47 ± 0.18
Infirmary	3 x 250 mL	9.54 i 0.28
Tatlisu# fresh water source	3 x 250 mL	ND
Fresh water source of Bandirma	3 x 250 mL	ND
Tap water in Bandirma#	3 x 250 mL	ND

ND: not detected. # Hometowns of workers

- Table 4. Boron concentrations in biological fluids and calculated DBE levels of the workers

Parameters	Control (C)	Low exposure (L)	Medium exposure (M)	High exposure (H)	P value
Age	41.69 ± 6.52 (26–52)	42.32 ± 5.66 (23–50)	42.19 ± 4.65 (32–50)	42.67 ± 5.33 (28–53)	>0.05
Years employed	15.30 ± 8.63 (0.17–26)	16.85 ± 7.06 (0.17–25)	17.21 ± 6.77 (0.17–25)	13.96 ± 8.04 (0.17–23)	>0.05
Urine boron, mg/g creat.	2.59 ± 1.32 (0.78–5.62)	5.01 ± 2.07 (2.38–13.54)	7.03 ± 2.37 (2.57–13.43)	9.83 ± 5.13 (3.34–32.68)	<0.05 (all pairwise comparisons)
Blood boron, ng/g	<48.5 (LOQ)	72.94 ± 15.43 (48.46–99.91)	121.68 ± 15.62 (100.51–146.07)	223.89 ± 69.49 (152.82–454.02)	no test performed
Semen boron, ng/g	807.92 ± 1,625.58 (<LOQ–8,597)	1,422.07 ± 1,939.03 (<LOQ–8,615)	1,482.19 ± 1,410.71 (<LOQ–4,897)	1,875.68 ± 2,255.07 (<LOQ–9,522)	<0.05 (C-L; C-M; C-H)
Inhaled boron, mg/8 h	0.23 ± 0.79 (<LOQ–4.09)	1.15 ± 3.14 (<LOQ–22.16)	1.47 ± 2.69 (<LOQ–11.34)	2.58 ± 4.96 (<LOQ–26.9)	<0.05 (C-M; C-H; L-H)
DBE, mg B/day	4.68 ± 1.63 (0.2–7.54)	7.39 ± 3.97 (2.56–24.72)	11.02 ± 4.61 (2.56–20.88)	14.45 ± 6.57 (3.32–35.62)	<0.05 (all pairwise comparisons)

Mean + SD, range in brackets. LOQ Limit of quantification DBE calculated daily boron exposure levels of the workers (please refer to the electronic annex for the calculation method of DBE and the related LOQ values)

- Table 5: Hormone and total PSA levels in workers

Parameters	Control (C)	Low exposure (L)	Medium exposure (M)	High exposure (H)	P value
Total PSA ng/mL	1.18 ± 0.62	1.22 ± 0.70	1.30 ± 0.94	1.25 ± 0.65	>0.05
	(0.28–3.04)	(0.34–4.34)	(0.40–4.07)	(0.31–2.76)
FSH mIU/mL	5.97 ± 2.71	5.26 ± 2.39	4.97 ± 2.29	7.47 ± 6.36	<0.05(*)
	(2.06–17.40)	(1.63–17.40)	(1.81–14.10)	(1.81–40.20)
LH mIU/mL	4.15 ± 1.77	4.30 ± 2.00	3.67 ± 1.34	5.38 ± 3.20	<0.05 (M-H)
	(1.49–8.91)	(1.41–11.80)	(1.29–7.15)	(1.95–20.00)
Total testosterone ng/dL	351.78 ± 133.84	337.40 ± 118.64	347.30 ± 110.91	329.56 ± 106.04	>0.05
	(159–907)	(127–773)	(157–668)	(95.9–581)

Mean + SD, range in brackets. * The global null hypothesis that all group means are equal is rejected, but the pairwise Mann–Whitney U tests provide no P values below 0.05 after adjustment for multiple testing (Bonferroni-Holm). M-H: The global null hypothesis is rejected and the only significant pairwise difference is between medium and high-exposure group

- Table 6: The mean total testosterone levels of workers in different work shifts

	Shift time, n = 204
	00:00–08:00	16:00–24:00	08:00–16:00
n	41	30	133
Total testosterone, ng/dL	355 ± 118.25 (108–668)	325.53 ± 97.14 (95.9–512)	340.93 ± 122.49 (127–907)

- Table 7 Characteristics of the semen samples and sperm concentrations determined by SQA-V Gold Sperm Quality Analyzer

Semen parameters	Control n = 49/An. = 48	Low exposure n = 72/An. = 68	Medium exposure n = 44/An. = 43	High exposure n = 39/An. = 39	P value
Abstinence, day	3.98 ± 1.59	3.78 ± 1.17	3.95 ± 1.33	4 ± 1.32	>0.05
	(1–7)	(2–7)	(2–7)	(2–7)
Volume of ejaculate, mL, RV: ¸2	3.24 ± 1.24	3.13 ± 1.19	3.65 ± 1.93	3.13 ± 1.34	>0.05
	(1.5–7)	(1–7)	(1.5–10)	(1–6)	>0.05
pH	7.9 ± 0.29	8.04 ± 0.30	7.94 ± 0.35	7.92 ± 0.34	>0.05
	(7–9)	(7.5–8.5)	(7.5–8.5)	(7.5–8.5)
Sperm conc. M/mL, RV: ¸20	69.84 ± 40.76	64.94 ± 48.97	70.49 ± 61.26	65.34 ± 55.74	>0.05
	(5.2–166.3)	(5.2–231)	(5.9–277.2)	(5.9–292.3)
Motile sperm conc., M/mL	35.86 ± 28.33	35.15 ± 27.62	33.48 ± 26.14	35.92 ± 36.06	>0.05
	(1.4–161.2)	(1.40–133.5)	(0.3–135.3)	(0.3–207)
PMSC (grade a), M/mL	15.69 ± 14.3	16.28 ± 15.64	16.95 ± 14.68	17.48 ± 23.72	>0.05
	(0.1–55.9)	(0–52.8)	(0.3–51.1)	(0.3–136.4)
PMSC (grade b), M/mL	14.03 ± 13.95	12.51 ± 11.94	11.18 ± 11.03	12.6 ± 11.45	>0.05
	(0–89.8)	(0–67.6)	(0–66.4)	(0–53.9)
Functional sperm conc., M/mL	19.02 ± 20.75	18.7 ± 18.02	17.41 ± 15.33	19.9 ± 26.44	>0.05
	(0–120.8)	(0.1–82.8)	(0.1–69.37)	(0.1–152.9)
All sperm, M/ejac., RV: ¸40	223.59 ± 157.68	187.65 ± 140.49	233.55 ± 224.26	185.69 ± 139.01	>0.05
	(15.60–795.5)	(13–752.5)	(11.8–1,297)	(8.9–584.6)
Motile sperm, (a + b+c), M/ejac.	114.53 ± 93.33	102.1 ± 81.07	111.49 ± 72.24	104.85 ± 81.4	>0.05
	(4.2–503.5)	(3.5–409.5)	(3.2–298)	(2.6–414)	>0.05
PMS (grades a + b), M/ejac, RV: ¸20	92.84 ± 84.77	83 ± 72.91	89.86 ± 60.5	84.37 ± 73.14	>0.05
	(0.3–460)	(0–355.5)	(0.6–229)	(0.5–380.6)
Funtional sperm, M/ejac.	58.45 ± 63.92	53.86 ± 52.6	54.57 ± 37.32	55.19 ± 56.18	>0.05
	(0–342.5)	(0.4–249.6)	(0.2–122.4)	(0.2–305.8)

Mean ± SD, range in brackets. An. Analyzed samples, Conc. concentration, PMSC progressive motile sperm concentration, PMS progressive mo­tile sperm, M/ejac million/ejaculate, RV reference value

- Table 8: The motility parameters of the sperm cells determined by SQA-V Gold Sperm Quality Analyzer

Semen parameters	Control n = 49/An. = 48	Low exposure n = 72/An. = 68	Medium exposure n = 44/An. = 43	High exposure n = 39/An. = 39	P value
Motility %, (grades a + b+c)	50.6 ± 15.1 (6.1–96.9)	54.1 ± 14.1 (20.7–96.6)	52.41 ± 14.08 (21.4–74.1)	56.13 ± 15.57 (15.3–88.9)	>0.05
Rapid prog. mot. %, (grade a), RV: >= 25	20 ± 11.64 (0.4–42.3)	22.37 ± 14.74 (0–63.3)	22.65 ± 12.49 (3–48.4)	23.91 ± 13.86 (1.4–49.7)	>0.05
Slow prog. mot. %, (grade b)	18.8 ± 8.7 (0–54)	18.7 ± 7.92 (0–33.7)	17.67 ± 8.85 (0–37.5)	18.87 ± 8.85 (0–35.5)	>0.05
Non prog. mot. %, (grade c)	11.82 ± 5.1 (5.40–28.90)	12.95 ± 5.66 (5.3–27.3)	12.1 ± 5.7 (5.3–27.7)	13.35 ± 5.44 (5.6–3.1)	>0.05
Immotility %, (grade d)	49.38 ± 15.09 (3.1–93.1)	45.9 ± 14.1 (3.4–79.3)	47.59 ± 14.08 (25.9–78.6)	43.88 ± 15.57 (11.1–84.7)	>0.05
Sperm motility index, RV: >=80	163.06 ± 141.67 (1–663)	174.06 ± 141.61 (0–659)	173.53 ± 130.08 (0–475)	157.9 ± 146.68 (0–776)	>0.05
Velocity (APV), mic/sec, RV: >= 5	11 ± 3.3 (1–19)	11.01 ± 3.6 (1–19)	11.20 ± 3.56 (1–17)	10.86 ± 3.89 (2–22)	>0.05
Morphology (N.F.%), WHO criteria, RV: >= 30	35.48 ± 11.51 (14.6–63.7)	37 ± 10.68 (15.60–60.30)	36.57 ± 10.57 (16.5–57.8)	37.96 ± 11.46 (16.4–60.5)	>0.05

Mean ± SD, range in brackets. An. Analyzed samples, Prog. progressive, Mot. motility, N.F. normal forms, APV average path velocity, mic. mi­cron, sec. second, RV reference value

- Table 9: Sperm morphology parameters as per the Kruger’s criteria

Parameters	Control n = 49/An = 47	Low exposure n = 72/An = 65	Medium exposure n = 44/An = 42	High exposure n = 39/An = 37	P value
Normal morph. (%) RV: >14%	13.87 ± 8.05 (0–30)	16.74 ± 9.98 (1–36)	15.49 ± 7.94 (2–34)	17.95 ± 9.60 (2–40)	>0.05
Head defects (%)	56.55 ± 11.49 (32–90)	56.11 ± 10.17 (34–76)	57.10 ± 10.16 (31–80)	56.43 ± 9.26 (33–80)	>0.05
Neck/mid-piece defects (%)	12.23 ± 7.07 (2–36)	12.52 ± 6.87 (0–34)	14.48 ± 6.43 (2–29)	13.57 ± 6.65 (4–31)	>0.05
Tail defects (%)	9.09 ± 8.30 (0–34)	7.18 ± 5.96 (0–26)	7.79 ± 6.07 (0–24)	7.68 ± 5.95 (0–26)	>0.05
Cytoplasmic droplets (%)	0.45 ± 0.93 (0–4)	1.09 ± 1.97 (0–10)	0.79 ± 1.41 (0–6)	0.86 ± 1.51 (0–4)	>0.05

Mean ± SD, range in brackets. RV Reference value as per the Kruger’s strict criteria, An. Analyzed samples

Applicant's summary and conclusion

Conclusions:

In a worker reproductive toxicity study with 102 exposed workers and 102 control workers, the correlation between combined environmental and occupational exposure to boron and boron levels in blood, urine and semen was analyzed. A high environmental oral exposure via contaminated well water of the central cantina was found for both groups leading to a re-classification of all 204 workers into 4 groups based on their calculated total daily boron exposure (DBE) and their blood boron concentrations. The new control group had blood boron levels below the LOQ of 48.5 ng/g, additionally a low, medium and hig exposure group were derived. Average age and average period of employment compared vafourably between the groups. While blood and urine levels correlate reasonably well with the calculated exposure, the correlation between blood boron levels and semen boron levels is very weak. Boron is accumulated in semen and the concentration factor is highest at the lowest exposure.
Adverse effects in hormone levels were absent when exposure groups are compared to the new control group. For any of the semen parameters a statistically significant differences was seen between new control group and exposure groups. These facts indicates that boron does not have an adverse effect on the male reproductive system at typical exposure conditions.

Executive summary:

In a worker reproductive toxicity study with 102 exposed workers and 102 control workers, the correlation between combined environmental and occupational exposure to boron and boron levels in biological fluids (blood, urine semen) or hormone levels (FSH, LH, total testosterone) was analyzed in a boric acid/borax production plant in Bandinna, Turkey.

From a group of 428 occupationally exposed workers (borax and boric acid), 102 participated in the study (boric acid production workers (n=57), borax production workers (n=31), sodium perborate production unit workers (n=5), boric acid plus borax production workers (n=5), laboratory workers (n=2), a storage worker (n=1), a mechanic technician (n=1)). Of 432 workers from a nearby sulfuric acid production plant 102 were selected as control group (sulfuric acid production plant workers (n=28), steam power plant workers (n=17), demineralized water production (DWP) unit workers (n=2), energy suppliers (n=11), mechanical workshop workers (n=19), garage workers (n=14), steelyard workers (n=2), construction service workers (n=3), laboratory technicians (n=3), and office workers (n=3)).

Both groups were served meals from the same central cantina. This lead to significant boron exposure exposure via the contaminated tap water (9.47 ± 0.18 mg boron/L). Occupational exposure was assessed by personal (exposed group) or static (control group) atmospheric measurement with personal air sampler (SKC, AirCheck 2000). Total individual exposure was calculated as a sum of occupational exposure (via inhalation), exposure through food (assumption of 400 mL contaminated tap water/meal) and exposure via beverages prepared from the contaminated tap water. Noteworthy exposure from municipal water sources at the homes of the workers or foodstuff was excluded be measurements. A questionnaire was used to assess demographic, exposure, reproductive and

general health information. Post-shift urine and blood samples (venipuncture ) were collected and analysed for boron content, hormone levels (FSH, LH, total testosterone) and prostate specific antigen concentration (PSA). Semen and sperm samples were collected in accordance with the recommendations of the World Health Organization (WHO (2003) Laboratory manual for the examination and processing of human semen and sperm, 4th edn. WHO Press, Geneva) and analysed for volume, sperm concentration and motility and morphological parameters per the Kruger’s strict criteria (Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, Van Zyl JA, Smith K (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 46:1118–1123; Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S (1988) Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 49:112–117).

Results:

The high boron contamination of water sources for cafeteria and infirmary was not anticipated in the planning phase of the study. This "background" exposure lead to relatively high exposure of the "control" group:

Total average exposure of occupationally exposure exposed workers: 12.08 ± 6.18 mg boron/day)

Total average exposure of control workers: 5.83 ±1.71 mg boron/day

The new exposure groups were assembled as follows: new control group (49 participants, blood boron level <LOQ (48.5 ng/g blood)), low exposure group (72 participants, blood boron level > LOQ - 100 ng/g blood), medium exposure group (44 participants, blood boron level > 100 - 150 ng/g blood), high-exposure group (39 participants, blood boron level > 150 ng/g blood).

Mean ages of re-constituted exposure groups were not significantly different from the mean age of the new control group. The average daily boron exposure (DBE, in mg B/d) calculated for the re-classified groups are: control: 4.68 + 1.63, low exposure: 7.39 + 3.97, medium: 11.02 + 4.61, high 14.45 + 6.57. Mean levels of inhaled boron (mg/8 h) in medium and high-exposure group were signiffantly higher than in the control group (0.23 ± 0.79 (control) vs. 1.47 ± 2.69 (medium) or 2.58 ± 4.96 (high)). The mean calculated daily boron exposure levels (DBE, in mg B/d) was significantly higher in exposure groups than in the new control group (4.68 ± 1.63 (control) vs. 7.39 ± 3.97 (low), 11.02 ± 4.61 (medium) and 14.45 ± 6.57 (high)). Mean urine boron levels (mg B/g creatine) were significantly higher in exposure groups than in the new control group (2.59 ± 1.32 (control) vs. 5.01 ± 2.07 (low), 7.03 ± 2.37 (medium) and 9.83 ± 5.13 (high)). Blood boron concentrations were positively correlated with the calculated DBE levels (Pearson corr. coeff.: 0.635) and the urine boron concentrations of the workers (Pearson corr. coeff: 0.633). The semen boron concentrations in exposed workers was significantly different between the exposure groups and the new control group, though the dose response trend was not significant. Variations of these levels within groups were great. Correspondingly the correlation between semen boron concentration and blood boron concentration was found to be very low (Pearson corr. coeff.: 0.222). Boron concentrations in semen are a lot higher (p<0.001; t-test) than in blood and urine levels. concentrating factors for semen boron concentration/blood boron concentration: low - ca. 20; medium - ca. 12; high - ca. 8. In hormone levels no significant differences were seen between groups execpt for LH when mid dose and high dose are compared which is not of biological significance.

The correlation between blood boron concentration and hormone levels are all very weak (FSH: Pearson corr. coeff: 0.143; LH: Pearson corr. coeff: 0.164; total testosterone level: -0.053). In addition no statistical significant difference in testosterone levels between shifts. In semen and sperm analysis including morphology no significant difference was detected between the exposure groups and the new control group in any tested parameter. Only weak a correlation was found between inhaled boron (mg/8 h) and blood boron (0.279), inhaled boron–semen boron (0.185), and inhaled boron–urine boron (0.106) levels. Finally the PSA level was not statistically significantly different when groups are compared.

Conclusions:

The surprising contamination of the tap water of the cafeteria and the infirmary led to to significant exposure of the originally planned control group and made a re-assembly of dose groups necessary. The re-assembly was based on the calculated daily boron exposure (DBE) and the respective blood boron levels. Due to the background exposure via drinking water no clear relation could be found between inhalation exposure and boron levels in biological fluids. Blood and urine boron levels increased steadily with rising DBE, while semen boron levels failed to follow a steady trend. Variation in semen boron levels was great. Boron is accumulated in semen and the concentration factor is highest at the lowest exposure. Adverse effects in hormone levels were absent when exposure groups are compared to the new "control group". For any of the semen parameters a statistically significant differences was seen between new control group and exposure groups. Together with the affore mentioned fact the authors of the publication come to the conclusion that this indicates that boron does not have an adverse effect on the male reproductive system at typical exposure conditions.

There are nevertheless several caveats in this report:

- All groups have significant exposures, including the control group (average daily boron exposure (DBE) of 4.7 mg boron/d). The controlgroup also showed a high fluctuation in boron semen levels. So no non-exposed comparison group is available.

- The correlation between blood boron levels and semen boron levels is very weak (0.222). This leaves in question whether blood boron levels are a good comparison partner for semen parameters. Therefore the comparison of semen and sperm parameters between the current groups is of arguable explanatory power.

- Total boron exposure in this study is calculated as a sum of oral and inhalation exposure without discussing possible differences in uptake and distribution of these two route.

- The number of samplings and the duration of the sampling period is not reported.

- The determination of the oral boron intake is based on the intake of contaminated water. It remains unclear how the water intake was graduated (glasses (assumed to contain 250 mL = 2.37 mg B/glass), half glasses, etc.). Also drinking of tea on the job was reported, but it remains unclear whether contamination of the beverages through ambient dust or dirty hands was accounted for. It is assumed that this might have led to a significant imprecision in the determination of daily boron uptake.

Because of these caveats a final conclusion on the effects of occupational exposure to boron on the male reproductive system in humans based on the data presented here is deemed arguable.

Despite these issues the study presents interesting and valuable data. The scientific value of the findings might benefit from the following additional analysis:

- Comparison of semen parameters with semen boron levels.

- Comparison of semen parameters with urine boron levels.

- Inclusion of a true negative group, or at least semen parameters for the average turkish population for comparison and evaluation of the effects seen in current study.