European Union Risk Assessment Report 

2-BUTOXYETHANOL 

CAS No: 111-76-2 

EINECS No: 203-905-0 

RISK ASSESSMENT 

GENERAL NOTE 

This document contains two different reports: 

- Volume 68 Part I Environment (Publication: EUR 22501 EN) – pages 2-97 

- Part II Human Health (Final approved version awaiting for publication) – pages 98-419 
OH 

H3C O

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 Existing Substances

 European Chemicals Bureau 

European Union 

Risk Assessment Report

CAS No: 111-76-2 EINECS No: 203-905-0

2-butoxyethanol (EGBE) 

 European

Part I - environment 

 Union Risk Assessment Report 

 2-butox

yethanol (EGBE) 203-905-0 

111-76-2 

EC:

CAS: 

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EUR 22501 EN 
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EUR 22501 EN 

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© European Communities, 2006 

Reproduction is authorised provided the source is acknowledged. 

Printed in Italy
European Union Risk Assessment Report 

2-BUTOXYETHANOL (EGBE) 

Part I - Environment 

CAS No: 111-76-2 

EINECS No: 203-905-0 

RISK ASSESSMENT 
LEGAL NOTICE 

Neither the European Commission nor any person 

acting on behalf of the Commission is responsible for the use which might 

be made of the following information 

 

A great deal of additional information on the European Union 

is available on the Internet. 

It can be accessed through the Europa Server 

(http://europa.eu.int). 

 

Cataloguing data can be found at the end of this publication 

Luxembourg: Office for Official Publications of the European Communities, 2006 

 

© European Communities, 2006 

Reproduction is authorised provided the source is acknowledged. 

Printed in Italy 
2-BUTOXYETHANOL (EGBE) 

Part I – Environment 

CAS No: 111-76-2 

EINECS No: 203-905-0 

RISK ASSESSMENT 

Final Report, 2006 

France 

The environmental part of the risk assessment of 2-butoxyethanol (EGBE) has been prepared by 

Ministry of the Environment (MEDD) on behalf of the European Union. 

The scientific work on this report has been prepared by: 

Institut National de l’Environnement Industriel et des Risques (INERIS) 

Direction des Risques Chroniques 

Unité Evaluation des Risques Ecotoxicologiques 

Parc Technologique ALATA 

BP n°2 

60550 Verneuil-en-Halatte 

France 
Date of Last Literature Search: 2004 

Review of report by MS Technical Experts finalised: 2005 

Final report: 2006 



0 OVERALL RESULTS OF THE RISK ASSESSMENT 

CAS No: 111-76-2 

EINECS No: 203-905-0 

IUPAC Name: 2-butoxytehanol 

Synonyms: EGBE (this synonym will be used in the present study to refer to the 

chemical Ethylene Glycol Butyl Ether). Other synonyms: 2-BE; 

butoxyethanol; 2-butoxy-1-ethanol; n-butoxyethanol; butyl ethoxol; 

3-oxa-1-heptanol; o-butyl ethylene glycol; butyl glycol; butyl 

monoether glycol; ethylene glycol butyl ether; EGBE; ethylene glycol 

n-butyl ether; ethylene glycol monobutyl ether; glycol butyl ether 

Commercial trade names: Dowanol EB; Butyl Cellosolve; Butyl 

Icinol; Butyl Oxitol; Eastman EB Solvent 

Environment 

Conclusions to the risk assessment for the aquatic compartment

Conclusion (ii) There is at present no need for further information and/or testing and for risk 

reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for the terrestrial compartment

Conclusion (ii) There is at present no need for further information and/or testing and for risk 

reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for the atmospheric compartment

Conclusion (ii) There is at present no need for further information and/or testing and for risk 

reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for secondary poisoning

Conclusion (ii) There is at present no need for further information and/or testing and for risk 

reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Human Health 

(to be added later). 

VII

CONTENTS 

1 GENERAL SUBSTANCE INFORMATION................................................................................................ 5

1.1 IDENTIFICATION OF THE SUBSTANCE........................................................................................ 5

1.2 PURITY/IMPURITIES, ADDITIVES.................................................................................................. 5

1.3 PHYSICO-CHEMICAL PROPERTIES .............................................................................................. 6

1.3.1 Physical state ................................................................................................................................. 6

1.3.2 Melting point ................................................................................................................................. 6

1.3.3 Boiling point.................................................................................................................................. 7

1.3.4 Relative density ............................................................................................................................. 7

1.3.5 Vapour pressure ............................................................................................................................. 7

1.3.6 Surface tension .............................................................................................................................. 7

1.3.7 Water solubility ............................................................................................................................. 8

1.3.8 Partition coefficient n-octanol/water.............................................................................................. 8

1.3.9 Granulometry................................................................................................................................. 9

1.3.10 Flash point ..................................................................................................................................... 9

1.3.11 Autoflammability........................................................................................................................... 9

1.3.12 Flammability.................................................................................................................................. 9

1.3.13 Explosive properties ...................................................................................................................... 9

1.3.14 Oxidising properties....................................................................................................................... 9

1.3.15 Viscosity ........................................................................................................................................ 10

1.3.16 Henry’s constant............................................................................................................................ 10

1.4 CLASSIFICATION ................................................................................................................................ 11

1.4.1 Current classification..................................................................................................................... 11

1.4.2 Proposed classification (environmental part only)......................................................................... 11

2 GENERAL INFORMATION ON EXPOSURE........................................................................................... 12

2.1 PRODUCTION ....................................................................................................................................... 12

2.1.1 Production processes ..................................................................................................................... 12

2.1.2 Detailed production capacity ......................................................................................................... 12

2.2 USES ........................................................................................................................................................ 13

2.2.1 Paints and coatings ........................................................................................................................ 16

2.2.2 Detergents, cleaners....................................................................................................................... 17

2.2.3 Chemicals used in synthesis: intermediate for EGBEA production .............................................. 18

2.2.4 Printing inks................................................................................................................................... 18

2.2.5 Other uses...................................................................................................................................... 18

3 ENVIRONMENT............................................................................................................................................ 21

3.1 ENVIRONMENTAL EXPOSURE........................................................................................................ 21

3.1.1 Environmental fate......................................................................................................................... 21

3.1.1.1 Degradation in the environment ...................................................................................... 21

3.1.1.2 Distribution...................................................................................................................... 24

3.1.1.3 Accumulation................................................................................................................... 25

3.1.2 Environmental releases.................................................................................................................. 26

3.1.2.1 Release from production.................................................................................................. 26

3.1.2.2 Release from formulation, processing and private use .................................................... 29

3.1.2.2.1 Continental and regional releases ................................................................... 30

3.1.2.2.2 Local releases: EGBE used in leather finishing operations............................ 31

3.1.2.2.3 Local releases: EGBE used for metal cleaning operations (processing)......... 31

3.1.2.2.4 Local releases: EGBE used in oilfield chemicals (processing)....................... 32

3.1.2.2.5 Local releases: EGBE used in oil spill dispersants (marine compartment)..... 36

3.1.2.2.6 Local releases for other uses........................................................................... 37

1
3.1.3 Continental and regional predicted environmental concentrations................................................ 42

3.1.4 Local predicted environmental concentrations (PEClocal) .............................................................. 43

3.1.4.1 Aquatic compartment....................................................................................................... 43

3.1.4.1.1 PEClocal for production.................................................................................... 43

3.1.4.1.2 Calculation of PEClocal for formulation, processing and private use............... 43

3.1.4.1.3 Measured levels.............................................................................................. 45

3.1.4.1.4 Comparison between predicted and measured levels...................................... 46

3.1.4.2 Terrestrial compartment................................................................................................... 46

3.1.4.2.1 PEClocal for production.................................................................................... 46

3.1.4.2.2 Calculation of PEClocal for formulation, processing and private use............... 46

3.1.4.3 Atmosphere...................................................................................................................... 48

3.1.4.3.1 PEClocal for production.................................................................................... 48

3.1.4.3.2 Calculation of PEClocal for formulation, processing and private use............... 48

3.1.4.3.3 Measured levels.............................................................................................. 49

3.1.4.3.4 Comparison between predicted and measured levels...................................... 49

3.1.4.4 Secondary poisoning........................................................................................................ 50

3.2 EFFECTS ASSESSMENT: HAZARD IDENTIFICATION AND DOSE (CONCENTRATION) - 

RESPONSE (EFFECT ASSESSMENT)............................................................................................... 51

3.2.1 Aquatic compartment (incl. sediment)........................................................................................... 51

3.2.1.1 Fish .................................................................................................................................. 51

3.2.1.2 Aquatic invertebrates....................................................................................................... 53

3.2.1.3 Algae................................................................................................................................ 58

3.2.1.4 Micro-organisms.............................................................................................................. 59

3.2.1.5 Endocrine disruption........................................................................................................ 60

3.2.1.6 PNEC for the aquatic compartment................................................................................. 61

3.2.1.6.1 Calculation of the intermittent PNEC for freshwater...................................... 62

3.2.1.6.2 Calculation of the PNEC for the seawater compartment ................................ 62

3.2.1.6.3 Calculation of the intermittent PNEC for seawater......................................... 62

3.2.1.7 Calculation of a PNEC for the sediment compartment.................................................... 62

3.2.1.7.1 Calculation of the PNEC for the marine sediment compartment.................... 63

3.2.1.8 PNEC for micro-organisms in STP ................................................................................. 63

3.2.2 Terrestrial compartment................................................................................................................. 63

3.2.3 Atmosphere.................................................................................................................................... 64

3.2.4 Secondary poisoning...................................................................................................................... 64

3.3 RISK CHARACTERISATION ............................................................................................................. 65

3.3.1 Aquatic compartment (incl. sediment)........................................................................................... 65

3.3.2 Terrestrial compartment................................................................................................................. 67

3.3.3 Atmosphere.................................................................................................................................... 68

3.3.4 Secondary poisoning...................................................................................................................... 69

4 HUMAN HEALTH ......................................................................................................................................... 70

5 RESULTS......................................................................................................................................................... 71

5.1 ENVIRONMENT.................................................................................................................................... 71

5.2 HUMAN HEALTH................................................................................................................................. 71

6 REFERENCES................................................................................................................................................ 72

Euses Calculations can be viewed as part of the report at the website of the European Chemicals Bureau: 

http://ecb.jrc.it

2 
TABLES 

Table 1.1 Physico-chemical properties................................................................................................................ 6

Table 1.2 Peroxide levels in EGBE during storage under adverse (daylight) and recommended (dark) 

conditions. No antioxidants used. Results in mmol active oxygen/litre.............................................. 9

Table 1.3 Henry’s law constant values (at 25°C) in EPI suite (US EPA, 2001).................................................. 10

Table 1.4 Henry’s law constant values as measured in Kim et al., 2000............................................................. 11

Table 2.1 Overview of EGBE production and sales in Europe for years 2001 to 2003 (data provided by 

CEFIC)................................................................................................................................................. 12

Table 2.2 EGBE production sites in EU (larger than 1,000 tonnes/year)............................................................ 13

Table 2.3 Breakdown of EGBE uses in Europe .................................................................................................. 14

Table 2.4 EGBE end use information (OECD, 1996) ......................................................................................... 16

Table 2.5 Detailed view of paint uses.................................................................................................................. 17

Table 2.6 EGBE concentrations in cleaning products......................................................................................... 17

Table 3.1 Biodegradation test results for EGBE ................................................................................................. 22

Table 3.2 Estimated biodegradation rate constants for EGBE in WWTP, surface water, soil and sediment ...... 24

Table 3.3 Estimated solids / water partition coefficients..................................................................................... 25

Table 3.4 Calculated distribution of EGBE in the different compartments of the environment......................... 25

Table 3.5 Estimated distribution in a STP (SIMPLETREAT) ............................................................................ 25

Table 3.6 Aquatic emission data from production sites of EGBE in EUU............................................................. 28

Table 3.7 Atmospheric emissions of EGBE from European producers .............................................................. 29

Table 3.8 Emissions of EGBE to soil from European producers ........................................................................ 29

Table 3.9 Environmental exposure scenarios for formulation, processing and private uses of EGBE................ 29

Table 3.10 Total continental and regional EGBE emissions................................................................................. 30

Table 3.11 Default values to be used for hazard assessment of completion and workover chemicals 

(specified as ‘cleaning chemicals’, other chemicals’, ‘squeeze treatments’ and ‘hydrotest 

chemicals’) (Thatcher et al., 2004). ..................................................................................................... 33

Table 3.12 PECintermittent for the use of oilfield chemicals containing EGBE on offshore platforms (marine 

compartment)....................................................................................................................................... 34

Table 3.13 Examples of EGBE containing products registered in UK.................................................................. 35

Table 3.14 Dispersants used today, application methods and dosages (Bonn Agreement, 2001)......................... 36

Table 3.15 Local releases of EGBE from formulation of paints ........................................................................... 38

Table 3.16 Local releases of EGBE from formulation of paints (continue).......................................................... 38

Table 3.17 Local releases of EGBE from processing of paints............................................................................. 39

Table 3.18 Local releases of EGBE from processing and private use of paints.................................................... 39

Table 3.19 Local releases of EGBE from formulation, processing and private use of detergents......................... 40

Table 3.20 Local releases of EGBE from formulation and processing of other uses............................................ 40

Table 3.21 Local releases of EGBE from formulation and processing of other uses (continued)......................... 41

Table 3.22 Local releases of EGBE from formulation and processing of other uses (continued)......................... 41

Table 3.23 Local releases of EGBE from formulation and processing of other uses (continue)........................... 42

Table 3.24 Regional PECs in air, water and soil (calculations made by EUSES – SIMPLEBOX model) ........... 43

Table 3.25 Local PEC in water at production........................................................................................................ 43

Table 3.26 Local PECSTP and PECaqua for EGBE .................................................................................................. 44

Table 3.27 PEClocalsoil at production and in situ processing (according to EUSES)............................................ 46

Table 3.28 Local PECsoil for EGBE....................................................................................................................... 47

Table 3.29 Local PECair for EGBE........................................................................................................................ 48

Table 3.30 Short term fish toxicity data for EGBE ............................................................................................... 51

Table 3.31 Long term fish toxicity data for EGBE ............................................................................................... 53

Table 3.32 Short term invertebrate toxicity data for EGBE .................................................................................. 54

Table 3.33 Long term invertebrate toxicity data for EGBE .................................................................................. 56

Table 3.34 Pros and cons for the validation of the test performed with Brachionus calyciflorus......................... 56

Table 3.35 Algae toxicity data for EGBE.............................................................................................................. 58

Table 3.36 Micro-organisms toxicity data for EGBE............................................................................................ 60

Table 3.37 Toxicity tests retained for the derivation of PNECaqua......................................................................... 61

Table 3.38 QSAR ecotoxicity data for EGBE....................................................................................................... 61

Table 3.39 Toxicity tests retained for the derivation of the intermittent PNECaqua ............................................... 62

Table 3.40 Risk characterisation for micro-organisms in STP and aquatic organisms ......................................... 65

Table 3.41 Risk characterisation for terrestrial compartment................................................................................ 67 

3

1 GENERAL SUBSTANCE INFORMATION 

1.1 IDENTIFICATION OF THE SUBSTANCE 

CAS Number: 111-76-2 

EINECS Number: 203-905-0 

IUPAC Name: 2-butoxyethanol 

Molecular formula: C6H14O2

Structural formula: CH3-CH2-CH2-CH2-O-CH2-CH2-OH 

3CH O

OH

Molecular weight: 118.17 g.mol-1

Synonyms: EGBE (this synonym will be used in the present study to refer to the 

chemical Ethylene Glycol Butyl Ether). Other synonyms: 2-BE; 

butoxyethanol; 2-butoxy-1-ethanol; n-butoxyethanol; butyl ethoxol; 

3-oxa-1-heptanol; o-butyl ethylene glycol; butyl glycol; butyl 

monoether glycol; ethylene glycol butyl ether; EGBE; ethylene glycol 

n-butyl ether; ethylene glycol monobutyl ether; glycol butyl ether 

Commercial trade names: Dowanol EB; Butyl Cellosolve; Butyl 

Icinol; Butyl Oxitol; Eastman EB Solvent 

Annex I entry: 603-014-00-0 

1.2 PURITY/IMPURITIES, ADDITIVES 

Purity: the purities quoted in IUCLID were all ≥ 99% w/w 

Impurity: as the synthesis reaction produces other glycol ethers, small concentrations of 

them can be found. 

 2-butoxyethoxyethanol (CAS 112-34-5) ≤ 0.3% w/w 

 1,2-ethanediol (CAS 107-21-1) ≤ 0.3-0.5% w/w 

 1-butanol (CAS 71-36-3) ≤ 0.1-0.2% w/w 

 water < 0.1-0.2% w/w 

Additives: 0.008-0.012% w/w 2,6-bis(1,1-dimethylethyl)-4-methylphenol 

(CAS 128-37-0) added to prevent the formation of peroxides. 

5
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

1.3 PHYSICO-CHEMICAL PROPERTIES 

Note: When the reliability of values do not enable a clear choice between one and another, a 

median value is chosen or calculated taking into consideration all figures supplied by the 

industry and only once the values found in handbooks or reports and which differ. 

The physico-chemical properties are discussed below and summarised in Table 1.1. 

Table 1.1 Physico-chemical properties 

Property Value 

Physical state Liquid 

Melting point -74.8°C 

Boiling point 171°C 

Relative density 0.9 at 20°C 

Vapour pressure 1.41 hPa, calculated at 25°C (initial value: 1 hPa 

at 20°C) 

Surface tension 26.6 mN/m at 20°C 

Water solubility Highly miscible 

Partition coefficient 

n-octanol/water (log value) 

0.8 

Granulometry Not applicable 

Flash point 67°C 

Autoflammability 244.5°C 

Flammability Upper limit: 12.7% (volume) 

Lower limit: 1.1% (volume) 

Explosive properties Not explosive 

Oxidising properties No oxidising properties 

Viscosity 3.28 mPa.s at 20°C 

Henry’s constant 0.08 Pa.m3/mol at 25°C 

Conversion factors (101 kPa, 20°C) 1 ppm = 4.9 mg/m3

1 mg/m3 = 0.204 ppm 

1.3.1 Physical state 

EGBE is a neutral, colourless liquid with a very weak pleasant odour (Ullmann, 2000). An 

absolute perceptible limit in air of 0.1 ppm (50% recognition = 0.35 ppm and 100% recognition 

= 0.48 ppm) was referred to EGBE (Verschueren, 2001). 

1.3.2 Melting point 

Several values are reported in literature. The value of -77°C appears in several reports and 

studies (ECETOC, 1994; OECD, 1996; Dow Chemical Co, 2001) and handbooks show values 

6 
CHAPTER 1. GENERAL SUBSTANCE INFORMATION

ranging between -75 and -70°C (-74.8°C Lewis, 1999; < -70°C Ullmann, 2000; -70°C Howard, 

1989; -75°C Eastman, 2001; -70.4°C BASF, 2002; -70°C Verschueren, 2001). 

The median of all different values found is retained for this study: -74.8°C. 

1.3.3 Boiling point 

Most of the values found in literature are in the following range: 168-172°C (BASF, 2002; 

Ullmann, 2000). A boiling point of 170.8°C for EGBE can be found (ECETOC, 1994) which is 

close to those appearing in handbooks (170°C in Kirk-Ohtmer, 1983 and Verschueren, 2001). 

Two other handbooks show values between 171 and 172°C (Lewis, 1999 ; Howard, 1989). 

Boiling point of 171°C was found in technical data sheets or product information reports (BP, 

2002; Dow Chemical Co, 2001). 

The median of all the different values above is 171°C. This value will be used for the risk 

evaluation. 

1.3.4 Relative density 

Several relative density values are available too. Most of them are about 0.9 (Verschueren, 2001 

- value measured at 20°C; Merck, 1996). Technical data sheets or product information reports 

give values of 0.9011 and 0.902, at 20°C (BP, 2002; Dow Chemical Co, 2001). 

A rounded value of 0.9 will be chosen for this study. 

1.3.5 Vapour pressure 

Vapour pressures ranging from 0.79 to 1.3 hPa, at 20°C have been reported. Data come from 

technical data sheets: 0.8 hPa (Shell Chemicals, 2001), 0.89 hPa (Dow Chemical Co, 2001), 

1.3 hPa (BP, 2002). The same range appears in Sax, 1986 and other chemical handbooks give 

almost the same values: 0.89 hPa (Verschueren, 2001), 1 hPa (Ullmann, 2000). 

The rounded value of the median gives a vapour pressure of 1 hPa, at 20°C. It will be used in the 

risk assessment (based on this value, a vapour pressure of 1.41 hPa has been recalculated at 

25°C, by EUSES, EC, 2004). 

1.3.6 Surface tension 

Several values of surface tension appear in technical data sheets for commercial EGBE: Dow 

Chemical Co, 2001 reports a value of 27.4 mN/m at 25°C, Eastman, 2001 gives a value of 

26.6 mN/m at 20°C and Shell Chemicals, 2001 another of 28.9 mN/m. A value of 27.4 mN/m at 

25°C is also reported in a handbook (Kirk-Ohtmer, 1983). 

The value reported in Eastman, 2001 will be retained for the study: 26.6 mN/m, at 20°C, the 

temperature recommended in the OECD guideline No 115. 

Surface active properties can be assumed for glycol ethers, and especially for ethylene glycol 

butyl ether, because of its quite long carbon chain. The values reported in the literature for 

EGBE tend to indicate that this substance is a surface-active reagent even if no indication has 

been found about the concentration of the substance during the tests listed above. Indeed, OECD 

7
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

guideline n°115 suggests that surface tension measurements should be performed using a 

concentration of 1 g/L for soluble substances. 

The fact that EGBE shows surface-active properties could thus lead to the disturbance of 

analytical method employed to measure some physico-chemical characteristics. 

However, there is a difference between the surface activity of traditional surfactants and 

substances that can reduce the surface activity of solutions like EGBE. What is observed with the 

glycol ethers during the surface tension measurements is the typical non-ideal behaviour of a 

mixture of a water miscible solvent such as methanol and ethanol. The reason for the observed 

relationship between surface tension and concentration is the disruption of the hydrogen bonding 

of the water causing non-linear behaviour of the surface tension against the concentration. In this 

case the substance is not migrating to the surface; it is not acting in the traditional surface-active 

manner. Therefore it would not affect the measurements of the physical chemical properties. One 

should also notice that glycol ethers do not form micelles. They are fully miscible with water and 

form clear solutions. 

Furthermore, considering the other properties of this substance (EGBE is highly miscible in 

water, hydrosphere is the preferential target of EGBE in the environment: > 90%, see 

Section 3.1.1.2), surface active properties of EGBE will not be considered in this assessment. 

1.3.7 Water solubility 

EGBE is often reported as miscible with water in several data sheets and product information 

reports (Hoechst, 1992; Hoechst, 1993; BP, 2002). A solubility of about 50,000 mg/L is reported 

by Eckel et al., 1996, 63,500 mg/L by ASTER, 1996. A value of 1.106 mg/L has been measured 

(Riddick et al., 1985 cited in HSDB, 2000). 

EGBE can be considered as highly miscible in water and the upper value of the range advised by 

EUSES (EC, 2004), 1.105 mg/L, is retained for modelling purposes. 

1.3.8 Partition coefficient n-octanol/water 

Several QSAR calculations of partition coefficient n-octanol/water for EGBE have been 

performed. They are presented hereafter using an ascending order of logarithmic values: 

0.57 (calculated with KOWWIN v.1.66. - US EPA and Syracuse Research Corporation, 2001); 

0.81 (Verschueren, 2001), 0.84 (ASTER, 1996). 

Partition coefficient measurements have been performed (Tanii and Hashimoto, 1982): 0.77 is 

the mean value of three determinations. Measurements have been carried out with gas 

chromatography. 

Other measured log Kow can be found in literature: 0.74 (Verschueren, 2001), 0.81 (Staples et al., 

1998), 0.83 (Korenman and Dobromyslova, 1975; Hansch and Leo, 1985, cited in Prager, 1995; 

Staples et al., 1998). 

The rounded value of the median obtained with all measured data gives a log Kow of 0.8 that is 

retained for this study. 

8 
CHAPTER 1. GENERAL SUBSTANCE INFORMATION

1.3.9 Granulometry 

Not applicable: the substance is a liquid. 

1.3.10 Flash point 

Values reported in literature range from 60 (INRS, 1996 and Merck, 1996) to 68°C (BP, 2002). 

Both values are measured with closed cup method. Two other values were obtained using this 

method: 67°C (Shell Chemicals, 2001; BASF, 2002) and 61°C (Sax, 1986), while methods 

leading to values of 65°Dow Chemical Co, 2001) and 67°C (Ullmann, 2000) are not mentioned. 

The median of the values measured using a closed cup is retained: 67°C. 

1.3.11 Autoflammability 

Two temperatures of autoflammability have been found in technical data sheets: 244°Dow 

Chemical Co, 2001) and 245°C (BP, 2002). The median value of 244.5°C is retained. 

1.3.12 Flammability 

Limits of flammability are reported by BP, 2002 for EGBE. The upper limit is 12.7% (volume) 

and the lower one is 1.1% (volume). 

1.3.13 Explosive properties 

Not explosive. 

1.3.14 Oxidising properties 

There are some references which suggest that glycol ethers can be prone to the formation of 

peroxides on storage. However data from one of the producers, shown below, indicates that 

peroxide production levels for EGBE remain low, even during prolonged storage under adverse 

conditions, as shown in Table 1.2. 

Table 1.2 Peroxide levels in EGBE during storage under adverse 

(daylight) and recommended (dark) conditions. No 

antioxidants used. Results in mmol active oxygen/litre 

In daylight In the dark 

Time 0 0.018 0.018 

3 months 0.040 - 

6 months 0.052 0.019 

18 months 0.95 - 

The National Fire Protection Association’s code for the reactivity of EGBE is 0 indicating 

minimal hazard. In consequence, there is no requirement for classification R19. 

9
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

1.3.15 Viscosity 

The range of viscosity values found in technical data sheets for EGBE goes from 2.9 mPa.s (at 

25°C, Dow Chemical Co, 2001) to 3.3 mPa.s (Shell Chemicals, 2001). A value of 3.28 mPa.s is 

also reported by BP, 2002, at 20°C. BP Chemicals, 1998 also reports a range of viscosity values 

for several concentrations of EGBE in water (between 20 and 100% volume): 2-6 mPa.s at 20°C. 

Several values have been found in handbooks: 3.26 mPa.s, at 20°C (Ullmann, 2000), 2.83 mPa.s 

at 25°C (Prager, 1995) and a catalogue of commercial substances gives a viscosity of 5.83 mPa.s, 

at 20°C (Merck, 1996). 

The most common values are rounded and a viscosity of 3.3 mPa.s (at 20°C) is retained for this 

study. 

1.3.16 Henry’s constant 

Different QSAR estimation methods (calculated with HENRYWIN v.3.10. - US EPA and 

Syracuse Research Corporation, 2001) give values of Henry’s law constant presented in Table

1.3. 

Table 1.3 Henry’s law constant values (at 25°C) in EPI suite (US EPA, 2001) 

Method Value (Pa.m3/mole) 

Bond method 0.009 9 

Group method 0.002 1 

Experimental value quoted in EPI suite database 0.162 1 

Calculated with VP.M / WSol ratio using EPI suite estimated values (QSAR) 0.116 1 

Henry’s law constant values have also been calculated using another program (MPBPVP), at 

25°C. Depending on the method used, values of 0.01 and 0.002 Pa.m3/mol are reported (SRC, 

1988, cited in Envirofate). 

Another study shows a calculated Henry’s law constant of 0.551 Pa.m3

/mol at 25°C ASTER, 

1996). 

Henry’s law constant can also be estimated from the ratio of the vapour pressure to the water 

solubility using selected values from this study: 141 Pa for vapour pressure and 1.106

 mg/L for 

water solubility. Calculation gives a Henry’s law constant of 0.017 Pa.m3

/mol. 

Experimental measurements of Henry’s law constant have also been Kim et al., 2000). Two 

different methods were used to determine Henry’s law constant for the equilibrium partitioning 

of EGBE contained in an aqueous solution and the air. Results of measurements are presented in 

Table 1.4. 

10 
CHAPTER 1. GENERAL SUBSTANCE INFORMATION

Table 1.4 Henry’s law constant values as measured in Kim et al., 2000

Bag method for equilibrium partitioning Batch stripping method 

T = 20°C ; H = 0.041 Pa.m3/mol T = 22°C ; H = 0.063 Pa.m3/mol 

T = 25°C ; H = 0.081 Pa.m3/mol T = 23°C ; H = 0.068 Pa.m3/mol 

T = 25°C ; H = 0.082 Pa.m3 T = 30°C ; H = 0.101 Pa.m /mol 3/mol 

T = 30°C ; H = 0.135 Pa.m3/mol 

In the previous study, there is a good correlation between both method used, bag method for 

equilibrium partitioning and batch stripping method, and particularly at 25°C. Moreover, direct 

measurement of the Henry’s law constant is recommended for water miscible compounds (TGD 

- EC, 2003). So a Henry’s law constant of 0.08 Pa.m3

/mol will be used. 

1.4 CLASSIFICATION 

1.4.1 Current classification

There is no classification for the environment. 

1.4.2 Proposed classification (environmental part only) 

According to the data presented and the criteria of Directive 67/548/EEC EGBE is not classified 

as dangerous for the environment. 

11
2 GENERAL INFORMATION ON EXPOSURE 

2.1 PRODUCTION 

The chemical 2-butoxyethanol (here referred to as EGBE) belongs to the group of glycol ethers, 

which are mainly used as solvents. During 2003, the production of EGBE in the European Union 

was approximately 161,000 tonnes (personal communication from CEFIC). According to 

information provided by industry and compiled by SICOS, 1999, the total EU production of all 

glycol ethers was 350,000 tonnes in 1998. 

2.1.1 Production processes 

EGBE is manufactured by adding n-butyl alcohol and ethylene oxide. The standard ethoxylation 

reaction for glycol ethers is as follows: 

Equation 2.1 EGBE synthesis reaction 

O + H3C OH → H3C O

OH

ethylene oxide butanol EGBE

Ethylene oxide and butanol react together with a typical industrial catalyst. This catalysed 

reaction produces a range of butyl glycol ethers (monoglycol, diglycol and triglycol ethers) 

which are separated by fractionation using, for example, vacuum distillation (personal 

communication from OSPA). 

2.1.2 Detailed production capacity 

The production and sales data for years 2001 to 2003 are given in Table 2.1. 

Table 2.1 Overview of EGBE production and sales in Europe for years 2001 to 2003 (data provided by CEFIC) 

(In kilo tonnes) 2001 2002 2003 Figures retained 

Production 135 158.5 161 155.1** 

Imports 9 6 6.5 6*** 

Exports 49 59 68 64*** 

Net into stock * 7.5 3 - 

Captive use (EGBEA 

production) 9 8 10 9 

Table 2.1 continued overleaf 

12 
CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.1 continued Overview of EGBE production and sales in Europe for years 2001 to 2003 (data provided by CEFIC) 

(In kilo tonnes) 2001 2002 2003 Figures retained 

Sales in EU 86 90 86.5 88.1 

Total use in EU 95 97.5 96.5 97.1¤

* Included in exports figure 

** The production volume has been obtained using the following calculation: (Exports + Captive use + Sales) – (imports) 

*** Mean of 2002 and 2003 figures. This figure has been retained in order to take into account the evolution of import and 

 export volumes the last two years 

¤ See Table 2.3 for a breakdown of total use in EU 

The figures presented above show that there is a trend for an increase in production year by year: 

135, 158.5 and 161 kt for years 2001, 2002 and 2003 respectively. However this is almost 

entirely due to increased demand for exports: 49, 59 and 68 kt each year between 2001 and 2003. 

The overall demand within the EU remains flat. 

A production volume of 155,000 tonnes/year will be retained for the risk assessment. 

The production in the European Union is located at five different sites (see Table 2.2). 

Table 2.2 EGBE production sites in EU (larger than 1,000 tonnes/year) 

Company Location 

BASF AG Ludwigshaven, Germany 

BP* Lavera, S. France 

Dow Chemical Company** UCL, Wilton International, Middlesbrough, United Kingdom 

Sasol Gmbh*** Marl, Germany 

Shell Nederland BV Hoogvliet Rt, the Netherlands 

* EGBE business of Hoescht renamed as Clariant in 1998 and then sold to BP (2001) 

** EGBE business of ICI sold to Union Carbide in 1995 and then to Dow in 2001 / EGBE business 

of Enichem sold to Dow in 2001 

*** Huels sold to Condea in 1998 and then EGBE business sold to Sasol in 2001 

From the Table 2.2, it appears that some production sites are located in the same area. 

Consequently the locations of both German sites and the Dutch one have been checked so as to 

establish whether they could pertain to the same region (TGD definition EC, 2003). Distances 

between Marl and Ludwigshaven on one hand and Marl and Hoogvliet on the other hand are 259 

and 188 km, respectively. So, in the regional assessment, none of these sites will be considered 

in a same region. 

Ten EGBE importers are also reported. Most of them are paint or detergent manufacturers and 

some are distributors. They import less than 1,000 tonnes/year except one which has reported a 

higher tonnage (but detailed figures are not available). 

2.2 USES 

EGBE has a wide range of uses as a solvent in paints and surface coatings, detergents and 

surface cleaners, inks or dyes. A breakdown of the uses of EGBE in Western Europe has been 

established based on the data collected for years 2001 to 2003 by CEFIC (see Table 2.3). 

13
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Values presented in this table are based both on volumes shown in Table 2.1 and on the EGBE 

volumes declared by uses reported in 2001, 2002 and 2003. The analysis of this set of data has 

led to a choice which is meant to represent a reasonable worst case. The final data choice is 

based mainly on averages but some expert judgement has also been applied to adjust for market 

knowledge and the fact that supply via distributors adds some uncertainty to the numbers. This 

uncertainty explains the significant fluctuation in the annual tonnage figures for the smaller uses. 

Typically, 25-40% of volume goes via distributors. To reflect these uncertainties, the figures are 

quoted as rounded numbers. 2002 and 2003 data should be given more weight as some errors 

have possibly been made during assessment of the 2001 data in allocating users to the 

appropriate end use categories. 

Table 2.3 Breakdown of EGBE uses in Europe 

Retained proposal

End use Stage of the 

life cycle 

Industry 

category 

Use 

category 2001¤ 2002 2003 Quantity 

used 

(Tonnes) 

Percentage 

of total use 

Paints and 

coatings 

(including 

estimation for 

indirect sales via 

distributors) 

Formulation 

Processing 

Private use* 

14: paints, 

lacquers and 

varnishes 

industry 

48: 

solvents 51,275 63,000 56,700 57,000 58.70 

Detergents, 

cleaners 

Formulation 

Private / 

public use 

5: personal/ 

domestic 

6: public 

domain 

9: cleaning, 

washing 

agents 

1,4921 7,000 10,000 11,000 11.33 

Chemicals used 

in synthesis Processing 

3: chemicals 

used in 

synthesis 

33: organic 

intermediat

es 

6,916 13,600 11,000 10,000 10.30 

Captive use 

(EGBEA 

production) 

Processing 

3: chemicals 

used in 

synthesis 

33: organic 

intermediat

es 

9,000 8,000 10,000 9,000 9.27 

Printing inks 

Formulation 

Processing 

12: pulp, 

paper and 

board industry 

48: 

solvents 5,340 1,100 2,500 2,500 2.57 

Oilfield 

chemicals 

Formulation 

Processing 

6: public 

domain 

48: 

solvents 0 3,000 2,000 2,500 2.57 

Metal cleaning 

Formulation 

Processing 

8 : metal 

extraction, 

refining and 

processing 

industry 

9: cleaning, 

washing 

agents 

0 50 2,000 2,000 2.06 

Electronic 

industry Processing 

4: electrical / 

electronic 

industry 

48: 

solvents 1,590 0 30 600 0.62 

Table 2.3 continued overleaf 

14 
CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.3 continued Breakdown of EGBE uses in Europe 

Retained proposal

End use Stage of the 

life cycle 

Industry 

category 

Use 

category 2001¤ 2002 2003 Quantity 

used 

(Tonnes) 

Percentage 

of total use 

Leather 

treatment 

operations 

Processing 

7: leather 

processing 

industry 

48: 

solvents 1,349 0 160 500 0.51 

Pharmaceuticals Processing 

3: chemicals 

used in 

synthesis 

48: 

solvents 

(extraction 

agents, 

reaction 

media) 

2,073 400 400 500 0.51 

Adhesives 

Formulation 

Processing 

6: public 

domain 

48: 

solvents 223 500 500 500 0.51 

Cosmetics / 

Personal care 

Formulation 

Private use 

5: personal / 

domestic 

6: public 

domain 

15: 

cosmetics 102 0 700 500 0.51 

Fire foams 

Formulation 

Processing 

6: public 

domain 

25: 

foaming 

agents 

65 200 330 300 0.31 

Agricultural 

products 

Formulation 

Processing 

1: agricultural 

industry 48: solvent 207 0 140 200 0.21 

Paper industry 0 0 20 0 0.00 

Textile manufacture 0 50 20 0 0.00 

Miscellaneous (unknown) 917 0 0 0 0.00 

Rubber, oil industry 674 0 0 0 0.00 

Oil spill dispersants 191 0 0 0 0.00 

Construction chemicals 153 0 0 0 0.00 

Total 94,996 96,900 96,500 97,100 ~ 100 

* For personal decorative paints only 

¤ Original 2001 data provided by CEFIC have been modified so as to integrate sales via distributors and volume of EGBE used for 

EGBEA production on site (captive use) 

Four major uses can be identified from sale figures. The use of EGBE in paint and lacquer 

industry represents ~ 58% of the total volume used in EU. The two other main uses, intermediate 

for EGBEA synthesis (including captive use) and cleaning agent, represent respectively ~ 20% 

and ~ 11% of the total quantity of EGBE used. 

Information for other minor uses for EGBE is also available (light dimmed cells in Table 2.3). 

The sum of the other uses represents about 10% of the total use of EGBE. Due to the low 

toxicity of the substance, these uses might be evaluated in a second time, if they show any 

concern. Moreover, some uses have been reported in the past that seem to no longer exist or 

errors could have occurred when allocating volumes to end-uses (dimmed lines in Table 2.3). 

For some of these uses, the percentage of total use has been set at 0 since no information has 

confirmed that EGBE was still used in this sector. For some other uses (e. g. oil spill dispersant), 

15
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

figures reported does not seem to indicate a real annual use of the substance since stockpiles 

could be made during several years without using the product (see Section 3.1.2.2.5). 

Another information source (OECD, 1996), presented in Table 2.4, shows supplies of EGBE to 

EU market. It gives a more detailed view on the major uses for EGBE. 

Table 2.4 EGBE end use information (OECD, 1996) 

End use kt/year (% of total use) 

Anticorrosion coatings 2.6 (2.9%) 

Can coatings 9 (10.0%) 

Coil coatings 6.5 (7.2%) 

Decorative trade coatings* 15.5 (17.2%) 

General industrial coatings* 16.8 (18.7%) 

Automotive OEM coatings 1.3 (1.4%) 

Automotive OEM coatings* 6.5 (7.2%) 

Wood coatings* 1.3 (1.4%) 

Detergents / cleaners 3 (3.3%) 

Inks (screen) 5 (5.6%) 

Feedstock for EGBEA production 11 (12.2%) 

Industrial use 

Subtotal 78.5 (87.3%) 

Decorative retail coatings* 10.4 (11.6%) 

Detergents / cleaners 1 (1.1%) 

Consumers uses 

Subtotal 11.4 (12.7%) 

Total 89.9 (100%) 

* Water based 

In this study, the detailed percentages for paint and detergent uses presented in Table 2.4 will be 

applied to the volumes retained in Table 2.3. These figures will be presented with further 

explanations given in the following paragraphs concerning each reported use. 

2.2.1 Paints and coatings 

Glycol ethers and their acetates are effective in many surface coating technologies including, for 

example, high and low solid coatings, waterborne emulsions, water-reducible coatings. As ‘true 

solvents’ glycol ethers are able to dissolve many resins in all proportions, and are particularly 

useful in promoting flow properties. They also encourage good gloss and film properties by 

maintaining the resin(s) in solution throughout the film formation process (Shell Chemicals). 

Detailed percentages for different paint types were calculated from the figures in Table 2.3 and 

end use information for EGBE (OECD, 1996). They are presented in Table 2.5. 

16 
CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Table 2.5 Detailed view of paint uses 

End use % of total use Typical maximum % of EGBE 

in formulated products 

General industrial coatings* 14.11 3 

Decorative trade coatings* 13.02 1.5 (3**) 

Decorative retail coatings (consumers uses)* 8.73 1.5 (3**) 

Can coatings 7.56 7 

Coil coatings 5.46 7 

Automotive OEM coatings* 5.46 8 

Anticorrosion coatings 2.18 1 

Automotive OEM coatings 1.09 2 

Wood coatings* 1.09 2 

Total 58.70 

* Water based 

** Personal communication, OSPA (value used for the assessment) 

2.2.2 Detergents, cleaners 

Glycol ethers can be used in all types of cleaners. They reduce surface tension and are excellent 

coupling agents. Working in conjunction with a surfactant, they facilitate wetting of the surface, 

penetration of the soil, and help to suspend the soil in water until it is wiped or rinsed away. 

The analysis of uses of 434 cleaning products identified during risk assessment of EGBE in 

cleaning products (NICNAS, 1996) revealed the range of EGBE concentrations below. 

Table 2.6 EGBE concentrations in cleaning products 

EGBE conc. % of total products Examples of cleaning products 

< 10% 68 

10-30% 14 

30-60% 1 

10-60% 2 

> 60% 1 

unknown 14 

Surface cleaner (multi-purpose cleaners) 

Floor stripper 

Glass / window cleaner ; Oven cleaner 

Carpet cleaner 

Laundry detergent 

Rust remover 

In this study, most of the cleaning products have an EGBE concentration < 10%. However, 

extreme values go from < 1% to 94%. The fraction of chemical in formulated detergent for both 

industrial and consumer uses (0.1) quoted in the OECD, 1996 will be retained for this study. In 

this reference EGBE end use volumes show that 3⁄4 (~ 8.50% of total use) of EGBE used as a 

detergent takes place in the industrial domain and that only 1⁄4 (~ 2.83% of total use) remains for 

consumers uses. 

17
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

2.2.3 Chemicals used in synthesis: intermediate for EGBEA production 

EGBE is used in the synthesis of acetate esters as well as phthalate and stearate plasticisers. 

Although the major use of EGBE as an intermediate is in 2-butoxyethanol acetate synthesis, it is 

also employed for the synthesis of di(-2butoxyethyl) phthalate (ATSDR, 1998). 

2.2.4 Printing inks 

Combination of glycol ethers and other solvents can be used to achieve the right evaporation 

rates, levelling properties, water miscibility and solubility for ink formulations. They can also be 

used to adjust surface tension and viscosity, promoting deep and even penetration of inks in soft 

substrates (Shell Chemicals). The typical maximum percentage of EGBE in formulated printing 

inks is 20% (OECD, 1996). Specific information shows a decline in this particular use due to the 

switch to radiation cured screen printing inks. 

Rastogi, 1991 analysed a series of printer’s inks in order to characterise their levels of organic 

solvents. EGBE was found in 2/29 samples and identified, for example, in printer’s inks for 

screen printing on paper and paper boards. The EGBE concentrations range in such formulated 

products was 0.1-0.4%. 

The value quoted in OECD, 1996, 20%, will be retained as the typical percentage of EGBE in 

printing inks. 

2.2.5 Other uses 

Oilfield chemicals (2.57%): oilfield chemicals include brines and brine components used as 

completion, workover4 and drill-in base fluids. Other end-use and intermediate applications 

include antifoams, drilling lubricants, emulsifiers, corrosion inhibitors, surfactants, oilfield 

biocides and scale inhibitors. They can be used in offshore platforms. Based on a search of 

patents on such products (personal communication, OSPA), typical concentrations are in the 

range 5-40%. However, one product used for the cleaning of oilfield equipment has been 

reported with concentrations of EGBE between 60 and 100%. This latter product is a 

concentrated one intended for dilution before use. 

Metal cleaning (2.06%): metal cleaning/degreasing is used in industries where the production 

process includes fabricating and/or assembling metal parts: mainly the automotive, aviation, 

appliance and railroad industries. During the various steps of the production process, metal parts 

must be cleaned of oils, fluxes and grease. 

Electronic industry (0.62%): among the uses of glycol ethers in electronic industry, those in 

copper clad laminating, laminates or photoresistors are reported (Shell Chemicals). 

Pharmaceuticals (0.51%): glycol ethers are reported to be process chemicals in the synthesis of 

pharmaceuticals. Usually they provide the medium where reaction takes place but they are also 

used in separation steps in order to obtain the desired drug or to maximise its purity. 

Leather treatment operations (0.51%): use as a solvent in leather protection system confirmed by 

appropriate EU trade association (OSPA enquiries). Nevertheless, EGBE is being substituted by 

other solvents (personal communication from the European Leather Industry). 

 

4 Major maintenance or remedial treatments. 

18 
CHAPTER 2. GENERAL INFORMATION ON EXPOSURE

Glycol ethers are used in aqueous degreasing systems for sheep and goatskin leather at 

concentrations of 1-5% (worst case). Only EGBE is reportedly used in this part of the process. 

Usage rates can therefore be calculated as up to 3 g/kg of dry finished leather (worst case based 

on 5% EGBE in formulation). Based on the total production information in the TGD of 314 Mm2

leather per year, 25% of which is sheep/goatskin, and assuming a leather weight of 2 kg/m2

would suggest total GE use in this application of around 500 tonnes/year. 

Glycol ethers are also used in leather finishing. EGBE is quoted as being used in this application, 

which is effectively a “coating” operation where a preparation is applied by air atomised 

spraying in a spray booth. Actual usage rates are quoted as up to 0.4 g/kg of dry finished leather, 

which, following the above assumptions, would lead to total GE consumption of up to 

250 tonnes/year. There will in addition be wastage from over spray and emissions to water from 

the over spray control systems. However, since this is a coating operation, it is already covered 

by the existing painting scenarios. Consequently, there will not be a separate risk characterisation 

for this use. 

Adhesives (0.51%): in the industry of adhesives and sealants, glycol ethers are employed as 

solvents and enter formulated products. They are employed both in domestic and industrial 

adhesives. EGBE is most likely used in water-based adhesives. A percentage of 10% of EGBE in 

formulation will be adopted for the assessment. This figure is similar to the highest figures used 

for water-based coatings (personal communication, OSPA). 

Cosmetics/Personal care (0.51%): the solvent and coupling properties of glycol ethers are 

utilised in cosmetic formulations. They can be used to stabilise emulsions used in lotions, 

preventing the active ingredient particles from dropping out of suspension. This helps to 

maintain the aesthetic appearance of the product and can increase shelf-life. That usage is now 

believed to be superseded by DEGBE (OSPA enquiries). Actually, EGBE is used in few 

cosmetic formulations and is only permitted in hair dyes up to a concentration of 10%. 

Fire foams (0.31%): EGBE can be used in synthetic foam formulations. No firm information was 

available but an estimation of the market size would be 1-3 kt/year. Some of this would be 

exported however (OSPA enquiries). Most synthetic foams use glycol ether solvents as a foam 

booster and freezing point depressant. Butyl glycol ether use is now more and more superseded 

by other glycol ether. The solvent composition of fire foams is typically 16% as the concentrate 

but 0.5% in use (personal communication, OSPA). 

Agricultural products (0.21%): the use of EGBE as a solvent or a co-solvent in pesticide 

formulations represents a minor application for this chemical. EGBE is used as a coupling 

solvent to help disperse the active component in water when the concentrate is diluted for use. It 

may be used at a significant level in the concentrate but these are usually diluted manyfold 

before use. For a conservative worst case “in use” figure, 1% will be retained (this would 

correspond to 1 L diluted in 100 L which represents a low dilution factor compared to typical use 

of pesticides for field application). 

Construction chemicals: EGBE is an additive for construction materials. It is used as a 

water-reducing agent for example. 

Cutting oils: declining use in soluble oils. Glycol ethers used ‘very occasionally’ at use levels of 

5-7%. Total EU sales in 1996: 110 kt/year for such oils. As a worst-case, if 5% of oils use glycol 

ethers and all assumed to be EGBE, total market would be a maximum of 400 tonnes/year 

(~ 0.41% of total use) (OSPA enquiries). 

19
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Oil spill dispersants: this use was reported to be declining. It is now only found in three of the 

44 dispersants approved for use in UK waters. Total stocks of dispersants in EU are estimated at 

5 kt and the estimated use in EU would be < 300 tonnes/year (OSPA enquiries). 

Photographic solutions: EGBE seems to be no longer used in photographic solutions. 

Rubber, oil industry: EGBE is reported as used in polymer manufacturing. 

20 
3 ENVIRONMENT 

3.1 ENVIRONMENTAL EXPOSURE 

The level of exposure of the environment to a chemical depends on the quantities and 

compartments of release and subsequent degradation, distribution and accumulation in the 

environment. This section discusses the behaviour of EGBE and its releases into the 

environment. 

3.1.1 Environmental fate 

3.1.1.1 Degradation in the environment 

Hydrolysis

No experimental data are available on hydrolysis. However, alcohols and ethers are generally 

resistant to hydrolysis (Lyman et al., 1990). 

Photodegradation

If EGBE is present in ambient air it is expected to exist almost entirely in the vapour phase, 

based on a vapour pressure of 1 hPa at 20°C, where vapour phase reactions with 

photochemically produced hydroxyl radicals may be important. A QSAR method is applied for a 

first estimation of primary transformation rates (SRC, 1988, cited in Environment Canada and 

Health Canada, 2000): the overall OH rate constant for EGBE has been calculated by the AOP 

program v1.90 and is estimated to be 2.3.10-11 cm3/molecule.second at 25°C. The estimated 

value corresponds to an atmospheric half-life of about 16.7 hours at an atmospheric 

concentration of 5.105 hydroxyl radicals/cm3. Atkinson, 1987 has calculated an OH rate constant 

for EGBE of 1.96.10-11 cm3

/molecule.second. 

Estimated half-lives values for EGBE, based on photooxydation, are also mentioned in Howard

et al., 1991, cited in Environment Canada and Health Canada, 2000. They range between 3.3 and 

32.8 hours. 

Values retained in this study to represent the photodegradation ability of EGBE were taken from 

a study performed by Aschmann and Atkinson, 1998. EGBE rate constants were measured using 

a relative method, i.e. measures were made indirectly using a well-known compound, for three 

different sensitizers: OH, O3 and NO3. Test conditions were as follow: light spectrum > 300 nm, 

EGBE concentration was 4.71.10-3 mg/L. Measurements revealed that reactions between EGBE 

and O3 or NO3 are of minor or negligible importance as tropospheric loss processes. Indeed, rate 

constants with NO3 and O3 have been measured at 3.10-15 cm3/molecule.second and 

< 1.1.10-19 cm3/molecule.second respectively. The main loss process for EGBE in ambient air is 

the reaction with OH radicals. The corresponding measured rate constant was 

2.94.10-11 cm3/molecule.second. 

Using this last data, a pseudo first order rate constant for degradation in air is calculated using 

TGD (EC, 2003): Kdeg-air = 1.27 d-1. This rate constant gives a half-life value of ~ 13 hours for 

EGBE in air. 

21
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Degradation products of EGBE after its reaction with OH radicals have also been measured by 

Tuazon et al., 1998. An EGBE concentration of 4.7.10-3 mg/L was used for the experiment 

performed at 25°C. Several products have been identified: propionaldehyde (CAS 123-38-6), 

butyraldehyde (CAS 123-72-8), butyl formate (CAS 592-84-7) and 2-hydroxyethyl formate 

(CAS 628-35-3), 3-hydroxybutyl formate, 2-hydroxybutyl formate and an organic nitrate. 

Since EGBE does not absorb ultraviolet radiation within the solar spectrum (> 290 nm), direct 

photolysis in the atmosphere is not expected to occur (Silverstein and Bassler, 1963, cited in 

HSDB, 2000). 

Biodegradation

The available aerobic biodegradation test results for EGBE are summarised in Table 3.1. A 

number of tests were carried out according to (international) standard test guidelines. Although 

the current information on several technical aspects is incomplete for some biodegradation tests 

the total data set available is regarded as being sufficient to draw conclusions on the 

biodegradation potential of EGBE. 

Table 3.1 Biodegradation test results for EGBE 

Test 

# 

Type 

of test 

Detection Result Day Method Conc. Of 

TS 

Conc. Of 

inoculum 

Reference Validity 

1 BOD5-

test 

O2 uptake 73% 

(BOD/ThOD) 

5 APHA n°219, 

1971 

Unknown 10 mL2,c Bridié et al., 

1979a 

Valid with 

restrictions 

2 BOD5-

test 

O2 uptake 31% 

(BOD/ThOD) 

5 APHA n°219, 

1971 

Unknown 10 mL1,c Bridié et al., 

1979a 

Valid with 

restrictions 

3 BOD5-

test 

O2 uptake 8% 

(BOD/ThOD) 

5 DEV H5 A2 Unknown River water 

taken below 

a factory 

effluent 

Knappe and 

Popp, 1974 

Valid with 

restrictions 

4 BOD20

-test 

O2 uptake 75% 

70% 

64% 

29% 

(BOD/ThOD) 

20 

15 

10 

5 

APHA n°219, 

1971 

3, 7 and 

10 mg/L** 

3 mL4

(filtered 

seawater 

seed) 

Price et al., 

1974 

Valid with 

restrictions 

5 BOD20

-test 

O2 uptake 88% 

82% 

74% 

26% 

(BOD/ThOD) 

20 

15 

10 

5 

APHA n°219, 

1971 

3, 7 and 

10 mg/L** 

3 mL1,a Price et al., 

1974 

Valid with 

restrictions 

6 Inherent 

biodeg 

Test 

DOC 95% 

92% 

8% 

8 

7 

4 

Zahn-Wellens 

test 

(OECD 302B) 

750 mg/L 33 mg/L1,b BASF AG, 

1980 

Valid with 

restrictions 

Table 3.1 continued overleaf 

22 
CHAPTER 3. ENVIRONMENT

Table 3.1 continued Biodegradation test results for EGBE 

Test 

# 

Type 

of test 

Detection Result Day Method Conc. Of 

TS 

Conc. Of 

inoculum 

Reference Validity 

7 Inherent 

biodeg 

Test 

DOC 100% 28 OECD 302B 500 mg/L Unknown1,a Huels, 

1982* 

Lack of 

data 

8 Inherent 

biodeg 

Test 

DOC 100% 

63% 

22% 

5 

3 

1 

OECD 302B 450 mg/L Unknown1,a Hoechst, 

1976 

Valid with 

restrictions 

9 Ready 

biodeg 

Test 

O2 uptake 88% 28 OECD 301C 100 mg/L 30 mg/L3,b CEFIC, 

1993* 

Lack of 

data 

10 Ready 

biodeg 

Test 

DOC 95% 28 OECD 301E 10 mg/L 

DOC 

Unknown1,a Huels, 

1982* 

Lack of 

data 

11 Ready 

biodeg 

Test 

O2 uptake 72% 

57% 

5% 

(BOD/ThOD) 

20 

10 

5 

Unknown Unknown Inoculum 

from a 

WWTP 

Dow cited 

in Gonsior, 

1990 

Valid with 

restrictions 

12 Ready 

biodeg 

Test 

O2 uptake 96% 14 OECD 301C 100 mg/L 30 mg/L1,a CITI, 1992 Valid with 

restrictions 

13 Ready 

biodeg 

Test 

O2 uptake 75% 

70% 

47% 

28 

15 

5 

OECD 301D Unknown Unknown1 Waggy et 

al., 1994 

Valid with 

restrictions 

a) Activated (domestic) sewage 

b) Activated sludge from (industrial) STP 

c) Filtered effluent from STP 

1) Non-adapted inoculum 

2) Adapted inoculum 

3) No information on adaptation or non-adaptation of inoculum available 

4) Seed source maintained by adding small amounts of settled raw wastewater 

* Original report not available 

** Results of the biodegradation test were expressed taking into account all concentrations of EGBE used (“weighted average”) 

Several tests have been conducted according to APHA (American Public Health Association) 

method n°219. In tests 1 and 2, the Biological Oxygen Demand was measured after five days 

whereas in tests 4 and 5, the BOD was measured after 20 days. The four tests were performed 

with an inoculum constituted by filtered effluent from a STP excepted for test 4 in which the 

inoculum came from a filtered seawater seed. The domestic source of the inoculum was only 

specified for test 5. In tests 2, 4 and 5, the inoculum was reported as non-adapted contrarily to 

that used in test 1. In test number 4, the inoculum source was maintained by adding small 

amounts of settled raw wastewater. In the four tests, the biodegradation was quantified 

comparing the measured BOD to the Theoretical Oxygen Demand (ThOD). Concerning the three 

tests showing the biodegradation of EGBE in the presence of non-adapted inoculum (#2,4,5), 

they all show similar values after five days even for the one performed with artificial saltwater 

(#4), respectively 31, 29 and 26%. 

Tests number 6 to 8 carried according to the OECD guideline 302B (Zahn-Wellens test) tend to 

demonstrate the inherent biodegradability of EGBE. Biodegradation percentages were > 70% 

23
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

after 14 days in tests 6 (95% after 8 days) and 8 (100% degraded after 5 days) and reached 100% 

after 28 days in test 7. Each test was performed using a non-adapted inoculum from domestic 

sewage (#7 and 8) or industrial STP (#6). 

All ready biodegradation tests (#9-13) have shown a biodegradation > 60%. Excepted test 

number 9 where the inoculum were adapted and came from an industrial waste water treatment 

plant, all other tests have used a non-adapted inoculum from a domestic sewage (as specified in 

the OECD guidelines n°301). Tests 11, 12 and 13 where EGBE biodegradation reaches, 

respectively, 57% after 10 days, 96% after 14 days and 47% after 5 days (70% after 15 days), 

tend to confirm that the 10 days time window criterion is fulfilled. 

To conclude, according to standard tests on ready biodegradation and further experimental data 

which confirmed high biodegradation rates, EGBE can be regarded as readily biodegradable. 

As no result from biodegradation simulation tests in STP, surface freshwater, surface saltwater 

and soil is available, the degradation rates have to be estimated based on the “ready 

biodegradability” classification and the partition behaviour of EGBE according to the method 

described in the Technical Guidance Document for risk assessment of new and existing 

chemicals (TGD - EC, 2003). Results of this estimation are presented in Table 3.2. 

Table 3.2 Estimated biodegradation rate constants for EGBE in WWTP, surface water, soil and sediment 

Compartment / medium Biodegradation rate Half-life 

Activated sludge (WWTP) KSTP = 1 h-1 0.7 hour 

Surface water Kfreshwater = 4.7.10-2 d-1 15 days 

Marine water Ksaltwater = 1.4.10-2 d-1 50 days 

Soil* Ksoil = 2.3.10-2 d-1 30 days 

Marine and freshwater sediments ** Ksed = 2.3.10-3 d-1 300 days 

* Biodegradation rates in sediment and soil take account of adsorption to solid matter (Koc=27.3 L/kg, see below) 

** Biodegradation rate in sediment takes account of the aerobic fraction of this compartment (0.1) 

3.1.1.2 Distribution 

Volatilisation

Based on the measured Henry’s law constant of 0.08 Pa.m3

/mol at 25°C (Kim et al., 2000), the 

air-water partitioning coefficient (Kair-water) can be calculated. Kair-water of 3.23.10-5 indicates that 

volatilisation of EGBE from surface water and moist soil is expected to be very low. 

Adsorption/desorption

Using the log Kow of 0.8 and according to the TGD (EC, 2003) (QSAR for soil and sediment 

sorption for nonhydrophobic chemicals) a Koc of 27.3 L/kg can be estimated. The solid/water 

partition coefficients in each compartment can be calculated as supplied in TGD (EC, 2003) (see 

Table 3.3). 

24 
CHAPTER 3. ENVIRONMENT

Table 3.3 Estimated solids / water partition coefficients 

Water / Compartment OC fraction in solid 

phase (FOCcomp) 

Solids / water 

partition coefficients 

Total compartment water 

partition coefficient 

Soil / water 0.02 Kp_soil = 0.55 L/kg Ksoil-water = 1.03 m3/m3

Sediment / water 0.05 Kp_sed = 1.37 L/kg Ksed-water = 1.49 m3/m3

Suspended matter / water 0.1 Kp_susp = 2.73 L/kg Ksusp-water = 1.58 m3/m3

Distribution in the environment

The following theoretical distribution in the environment has been calculated using the 

multimedia fugacity model EQC (MacKay level I) and the physico-chemical properties given in 

Section 1.3. 

Table 3.4 Calculated distribution of EGBE in the different 

compartments of the environment 

Compartment % EGBE 

Air 0.24 

Water 99.20 

Soil 0.55 

Sediment 0.01 

Regarding these results, the hydrosphere is the preferential target of the substance in the 

environment. 

Distribution in STPs

Based on physical chemical properties discussed in this study (log H = -1.1 and Koc = 27.3 L/kg) 

as well as the biodegradation rate of 1 h-1 in STP, the elimination through biodegradation can be 

estimated with the model SIMPLETREAT in Table 3.5. 

Table 3.5 Estimated distribution in a STP (SIMPLETREAT) 

Designation % 

Air 9.54.10-2

Water 12.6 

Sludge 2.57.10-1

% degraded 87.0 

% removal 87.4 

3.1.1.3 Accumulation 

No experimental data on bioaccumulation is available. Therefore, BCF-values for fish and 

earthworm are estimated using the log Kow of 0.8. The estimated BCF-values amount to 0.97 

and 1.6 for, respectively, fish and worm. Another calculated value is quoted: a BCF of 2.5 has 

been estimated by Syracuse program (US EPA and Syracuse Research Corporation, 2001). 

In view of these BCF, EGBE is expected to have a low bioaccumulation potential. 

25
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

3.1.2 Environmental releases 

The regional and continental releases include all relevant life cycle stages of EGBE. For 

production, it is assumed that there is only one production site in the region. The exposure 

assessment is based on the EU Technical Guidance Documents (TGD - EC, 2003) applying the 

European Union System for the Evaluation of Substances, EUSES (EC, 2004). 

3.1.2.1 Release from production 

Ethylene oxide and anhydrous5 alcohol (n-butanol for the production of EGBE) are added 

continuously to a reactor containing catalyst that operates at elevated temperature and pressure. 

To keep the proportion of lower molecular weight products as a high percentage of total 

production, a large excess of alcohol is used. Unreacted excess alcohol is recycled using an 

alcohol recovery section (A and B parts in Figure 3.1). Some of the EGBE is also recycled as 

part of the production control process. The remaining reactor stream is drawn off for purification 

using a train of distillation columns (C, D and E parts in Figure 3.1). Relatively pure mono and 

diethylene glycol alkyl ether streams are drawn off along with a heavy stream (mainly tri but 

also containing penta and tetraethylene glycol alkyl ether). The remaining low volume polymeric 

residue stream is either burnt or sold. 

During normal operations, the process is entirely closed (due to physical properties of ethylene 

oxide: boiling point of 10.7°C) with little opportunity for environmental release. Such exposure 

is only likely during sampling, filling or loading operations along with campaign changes (most 

of producers revealed periodic campaigns of EGBE synthesis), when the plant is switched to a 

different glycol ether (different alcohol or using propylene rather than ethylene oxide). 

Figure 3.1 Basic schematic of the EGBE production units (OSPA) 

REACTOR

A B C D E 

Reaction section 

(Oxide-Alcohol-Catalyst) 

Alcohol removal Distillation section 

Alcohol + mono-GE Mono-GE Di-GE Tri-GE... 

Polymeric

stream

 

5 Presence of water during the reaction is not expected as this reacts violently with ethylene oxide under the 

prevailing conditions. 

26 
CHAPTER 3. ENVIRONMENT

Releases to water

Data of releases to water for EGBE production sites in Europe (see Table 2.2) are presented in 

Table 3.6 (Note: the site numbers in Table 3.6 do not directly correspond to the order of 

companies in Table 2.2). 

As there are only five EGBE production sites in Europe, the regional production will not be set 

at 10% of total EGBE production (TGD default) but at the maximum volume produced at one 

site. The amount remaining corresponds to the continental production. Fraction releases and 

number of days of emission are taken from Table A1.1 and Table B1.5 (TGD - EC, 2003; 

Chapter 3 – Appendix I), with Main Category equal to Ib (substances produced in a continuous 

production process) for EGBE is produced in a continuous production process. 

All EGBE producers reported that releases to water enter a sewage treatment plant. During this 

step, 87.4% of EGBE is expected to be removed by degradation processes. When more precise 

data were available (EGBE concentration in STP effluents) they were also used. 

Relevant data for the calculation of Predicted Environmental Concentrations (PEC) in a STP and 

local concentrations in the aquatic compartment are presented in Table 3.6. When such data was 

not available the effluent concentration leaving the STP (PECSTP) has been calculated according 

to Equation 3.1. This PECSTP is divided by a dilution factor so as to obtain the local 

concentration in surface water - Clocalaqua (see Equation 3.2). 

Equation 3.1 Calculation of PECSTP 

STP

water STPwater

STP EFFLUENT

Elocal F

PEC

6 ×× 10 =

Elocalwater local release to waste water during episode [kg/d] 

FSTPwater fraction of emission directed to water by STP 0.126 

EFFLUENTSTP effluent discharge rate of STP [L/day] 

PECSTP EGBE concentration in the STP effluent [mg/L] 

Equation 3.2 Calculation of Clocalaqua 

DILUTION

PEC Clocal STP

aqua =

PECSTP concentration of the substance in the STP effluent [mg/L] 

Clocalaqua local concentration in surface water during emission episode [mg/L] 

DILUTION (STP flow + river flow) / STP flow [-] 

The calculation of Clocalaqua is reduced to this equation due to the low adsorption of EGBE on 

suspended matter (Kpsusp = 2.73 L/kg). 

27
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.6 Aquatic emission data from production sites of EGBE in EU

Site # Emission in water 

1 Reference years: 2001 and 2002 

Release to wastewater treatment plant: 10* kg/day (300* days of production). Assuming 87.4% removal in STP: 

1.3* kg/day in STP effluent. Before discharge to receiving waters, the effluent undergoes a dilution by a factor of 100 

(mixing with seawater). Receiving water is the sea. The real dilution for marine environment is unknown. The releases 

of EGBE occur in a region where the tidal influences are really low. For those particular seas it is proposed in the TGD 

to use only a dilution factor of 10 instead of 100. 

Flow of STP = 7,000* m3/day. 

PECSTP = 0.18* mg/L and Clocalaqua = 1.8.10-4* mg/L 

2 Reference year: 1996 (production and processing volumes in 2000 were however comparable) 

Release to water: 1,680* kg/day in wastewater treatment plant influent and 8.7* kg/day in effluent (such values give 

99.5*% removal of EGBE in STP). Emissions are calculated as a worst case using 90th percentile of daily measured 

concentrations in effluent, average STP flow and 365* days/year. Both production and processing releases are 

included. 

Flow of STP = 5* m3/s; 10th percentile of receiving water flow = 734* m3/s; dilution in receiving water = 150*. 

PECSTP = 2.10-2* mg/L ; Clocalaqua = 1.3.10-4* mg/L 

Assuming 87.4% removal in STP: PECSTP = 0.49 mg/L ; Clocalaqua = 3.3.10-3 mg/L 

3 Reference year: 2000 

Release to water: 21.9* kg/day in STP influent (365* d/y). Assuming 87.4% removal in STP: 2.8 kg/day in STP 

effluent. 

Flow of STP = 0.83* m3/s; 10th percentile of receiving water flow = 18.4* m3/s; dilution in receiving water = 23*. 

PECSTP = 3.9.10-2 mg/L ; Clocalaqua = 1.7.10-3 mg/L 

4 Reference year: 2000 

Release to water: 7.1* kg/d in STP influent (140* d/y). Assuming 87.4% removal in STP: 0.9 kg/d in STP effluent. 

Concentration in STP influent = 0.2* mg/L; concentration in STP effluent = 1.10-3* mg/L. 

PECSTP = 8.9.10-2 mg/L ; Clocalaqua = 2.2.10-3 mg/L (assuming a dilution factor of 40 and a STP flow of 10,000 m3/day 

– default value for chemical industry, Cf. emission scenario documents, Chapter 7 of the TGD, E.C., 2003) 

5 No data but total annual site production for EGBE < 1kt. 

No process water discharge is reported 

* Original data provided by industry or calculated with original data. Other data is calculated using default TGD values 

Releases to air

Release data to air for EGBE production sites in EU are presented in Table 3.7. Local 

concentrations in air have been calculated based on information submitted by the producers, 

except for site 5. 

28 
CHAPTER 3. ENVIRONMENT

Table 3.7 Atmospheric emissions of EGBE from European producers 

Site # Release to air (kg/d) Clocalair,ann* (mg/m3) Year 

1 1.9 including emissions from plant 

and fugitive emissions 

(300 days/year) 

4.34.10-4 2002 

2 0.6 at production and 5.4 at 

processing (365 days/year) 

Total (production + processing) 6 

1.67.10-4 mg/m3 at production and 

1.50.10-3 at processing 

Total (production + processing) 1.67.10-3

1996 (production and 

processing volumes in 2000 

were however comparable) 

3 ~0.1 (365 days/year) 2.78.10-5 2000 

4 ~0.4 (140 days/year) 4.27.10-5 2000 

5 No data but total annual site production for EGBE < 1kt. 

* EGBE concentration in air calculated at a 100 m distance from the source (annual average) 

Releases to soil

Release data to soil for EGBE production sites in EU are presented in Table 3.8. Local 

concentrations in soil have been calculated based on information submitted by the producers, 

except for site 5. 

Table 3.8 Emissions of EGBE to soil from European producers 

Site 

# 

Total deposition 

flux during 

emission episode 

(μg.m-2.j-1) 

Annual 

average 

deposition flux 

(μg.m-2.j-1) 

Local concentration 

in agricultural soil 

after 

30 days (μg/kg ww) 

Local concentration 

in agricultural soil 

after 

180 days (μg/kg ww) 

Local concentration 

in grassland after 

180 days (μg/kg ww) 

1 0.76 0.63 33.40 10.30 3.84 

2* 2.40 2.40 0.27 0.27 0.50 

3* 0.04 0.04 0 (5.10-3) 0 (5.10-3) 0 (8.10-3) 

4* 0.16 0.06 0 (7.10-3) 0 (7.10-3) 0.01 

5 No data but total annual site production for EGBE < 1kt. 

* At this production site STP sludge is incinerated 

3.1.2.2 Release from formulation, processing and private use 

Generic exposure scenarios are used to estimate the releases from formulation, processing and 

private use of EGBE, as no actual data are available. The scenarios are based on the different use 

categories of EGBE (see Section 2.2). An overview of the various environmental exposure 

scenarios for formulation, processing and private use of EGBE is given in Table 3.9. 

Table 3.9 Environmental exposure scenarios for formulation, processing and private uses of EGBE 

Scenario names Designation Life cycle step 

Paints IF / Paints IP ¤ General industrial coatings (water-based) Formulation/Processing 

Paints IIF / Paints IIP ¤ Decorative trade coatings (water-based) Formulation/Processing 

Paints IIIF / Paints IIIU Decorative retail coatings (water-based) Formulation/Private use 

Paints IVF / Paints IVP ¤ Can coatings Formulation/Processing 

Table 3.9 continued overleaf 

29
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.9 continued Environmental exposure scenarios for formulation, processing and private uses of EGBE 

Scenario names Designation Life cycle step 

Paints VF / Paints VP ¤ Coil coatings Formulation/Processing 

Paints VIF / Paints VIP ¤ Automotive OEM coatings (water-based) Formulation/Processing 

Paints VIIF / Paints VIIP ¤ Anticorrosion coatings Formulation/Processing 

Paints VIIIF / Paints VIIIP ¤ Automotive OEM coatings Formulation/Processing 

Paints IXF / Paints IXP ¤ Wood coatings (water-based) Formulation/Processing 

Detergents IF / Detergents IP Industrial detergents Formulation/Processing 

Detergents IIF / Detergents IIU Domestic detergents Formulation/Private use 

Intermediates IP Intermediate for chemicals synthesis Processing 

Inks IF / Inks IP ¤ Printing inks Formulation/Processing 

Oilfield IF / Oilfield IP ¤ Oilfield chemicals Formulation/Processing 

Metal IF / Metal IP ¤ Metal cleaning Formulation/Processing 

Elec IP Electronic industry Processing 

Leather IP Leather treatment operations Processing 

Pharm IP Pharmaceuticals Processing 

Adhesives IF / Adhesives IP Adhesives Formulation/Processing 

Cosmet IF / Cosmet IU Cosmetics / Personal care Formulation/Private use 

Agri IF / Agri IP Agricultural products Formulation/Processing 

Fire IF / Fire IP Fire foams Formulation/Processing 

P Processing 

F Formulation 

U Private use 

¤ For these end uses there is a possibility that formulation and processing steps take place at a same site. 

 These cases will be treated during risk characterisation. 

3.1.2.2.1 Continental and regional releases 

The total continental and regional EGBE emissions from formulation, processing and private 

uses are given in Table 3.10. These results come from the calculation program EUSES (EC, 

2004) and do not integrate the emissions due to the scenario “Agri I” because of the scenario 

number limitations of the software. Nevertheless, the calculations have been made for this 

scenario in a separate file and it appears that the impact of emissions volumes for this scenario 

on total continental and regional emissions is negligible. 

Table 3.10 Total continental and regional EGBE emissions 

Air Water (total / waste water*) Soil 

Continental 9.69.104 kg/day 4.90.104 kg/day/3.92.104 kg/day 6.81.103 kg/day 

Regional 1.08.104 kg/day 5.56.103 kg/day/4.45.103 kg/day 7.59.102 kg/day 

* It is assumed that 80% of the waste water is treated in a biological STP and the remaining 

 20% released directly into surface waters 

30 
CHAPTER 3. ENVIRONMENT

3.1.2.2.2 Local releases: EGBE used in leather finishing operations 

For the industrial use of EGBE in leather industry the dedicated scenario “leather processing 

industry’ has been used (for water releases). The emission of EGBE to waste water can be 

calculated using Equation 3.3. Emissions to air and soil were estimated using A and B tables 

from Appendix 1 of the TGD (EC, 2003). 

Equation 3.3 Calculation of waste water emission in leather processing industry 

)1( Elocal _ water = product ×QQ chemical × − Ffix

Elocal_water daily amount released to waste water [kg/d] 

Qproduct mass of processed raw hide per day [15 t/d - default] 

Qchemical mass of substance used per mass of raw hide [3 kg/t6– 

personal 

communication 

from the 

European 

Leather Industry] 

Ffix proportion of the substance chemically concerted 

or fixed to the raw hide during processing [0.01- 

Table A3.6] 

3.1.2.2.3 Local releases: EGBE used for metal cleaning operations (processing) 

During the various steps of the production process, metal parts must be cleaned of oils, fluxes 

and grease. Due to the properties of EGBE it is used in cleaning formulations as a wetting agent 

and dispersant. Typical cleaning formulations contained 4% of this kind of additives (cleaning 

formulations used for soak). In the calculation of EGBE daily releases to wastewater it will be 

assumed that the metal parts are cleaned by a static soak process. During this step of the process, 

losses of substance will be by drag out into the rinse bath and subsequent release of the rinse 

water. Here, as a worst case, the following assumptions are made: 

− it is considered that there is no return of the rinse water (Frecycle = 0) 

− the amount of solution removed from treatment bath due to drag out is taken as 0.3 L/m2 as a 

worst case 

− is a same way, the surface area of metal processed is taken at 40 m2/hr 

If we consider a fraction of 0.04 for EGBE in the cleaning formulation, then, using a typical 

concentration of the formulation in the cleaning bath of 25-75 g/L, the concentration of 

substance in the treatment bath (Cbath) can be calculated as follow: 4% . 75 g/L = 3 g/L. 

Equation 3.4 is used to calculate the daily emission to wastewater: 

 

6 This scenario only takes into account the use of EGBE in aqueous degreasing systems for leather. The other use of 

EGBE in the leather processing can be put in the category of “coating” operations and consequently is already 

covered by the dedicated scenarios for paints. 

31
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Equation 3.4 Daily emission to wastewater during metal cleaning operations 

Elocal process water Qdragout type AREAprocess metal Cbath recycle TF process = ⋅ ⋅−⋅⋅⋅ − )1(10 3

, , ,

Elocalprocess water /792.022)01(103403.0 dkg 3

, =⋅−⋅⋅⋅⋅= −

Elocalprocess,water emission from process to water per day [kg/d] 

Qdragout,type amount of solution removed from treatment 

bath per unit area [0.3 L/m2] 

AREAprocess,metal surface area of metal processed per hour [40 m2/hr] 

Cbath concentration of substance in treatment bath [3 g/L] 

10-3 conversion factor for g to kg [kg/g] 

Frecycle fraction of drag out returned to treatment bath [0] 

Tprocess number of hours worked per day [22, worst case] 

In addition, it will be considered that the cleaning bath is disposed of every 4-8 weeks. Assuming 

a bath capacity of 1,000 L, the amount of substance released will be 3 g/L . 1,000 L = 3 kg – 

source OECD, 2004. This release will be considered an intermittent emission. 

Results are shown in Table 3.15 to Table 3.23. 

3.1.2.2.4 Local releases: EGBE used in oilfield chemicals (processing) 

a) Estimation of exposure concentrations using the CHARM model

EGBE has a wide range of uses as a solvent in paints and surface coatings, detergents and 

surface cleaners, inks or dyes. One of the reported end use is as oilfield chemicals used on 

offshore platforms. Oilfield chemicals are in general brines and brine components used as 

completion, workover and drill-in base fluids. A generic scenario has been applied in order to 

cover the use of EGBE in oilfield chemicals onshore (see Section 3.1.2.2.6). 

The CHARM model (Thatcher et al., 2004) takes into account four different types of application, 

since they might be introduced into the environment in a different way. Application groups are: 

− production chemicals (with injection chemicals and surfactants as special cases) 

− drilling chemicals (water based mud only) 

− cementing chemicals (i.e., spacer and mixwater) 

− completion and workover chemicals including well squeeze treatments and also pipeline 

hydrotest and preservation treating chemicals. 

Due to the range of usual uses of EGBE, the application group, to which it belongs, seems to be 

“completion and workover chemicals”. 

Two different scenarios are available depending on the use of the chemicals during those 

operations. A distinction has been made between surface/well cleaning and the other operations 

(other completion, workover, squeeze treatment and hydrotest chemicals). This is due to the fact 

that during cleaning operations, as noted in the CHARM manual, discharge is considered to be 

32 
CHAPTER 3. ENVIRONMENT

100% of the amount used, while for all other operations a fraction of the chemical is retained in 

the formation by, for example, adsorption to the formation matrix during the operation. 

The CHARM model considers that completion and workover chemicals are discharged in 

batches. Therefore a specific dilution factor has to be applied accounting for the discharge 

volumes. These releases should be considered as intermittent and compared to the appropriate 

PNEC for the risk characterisation. 

The equations used for the calculation of PECwater for completion, workover squeeze treatment 

and hydrotest chemicals are presented below. 

For surface and well cleaning chemicals, the PEC is calculated using: 

PECwater = Ci,cleaning × Dbatch, cleaning

With Ci, cleaning = initial concentration of chemical in cleaning fluid (dosage, mg.L-1) 

 Dbatch, cleaning = batchwise dilution factor for cleaning fluids 

For other completion, workover, squeeze treatment and hydrotest chemicals, the PEC is 

calculated using: 

PECwater = fr × Ci, completion × Dbatch, completion

With fr = fraction released - chemical 

Ci, completion = initial concentration of chemical in completion and workover 

including squeeze treatments and hydrotest fluids (dosage, mg.L-1) 

Dbatch, completion = batchwise dilution factor for completion and workover including 

squeeze treatments and hydrotest fluids 

Default values for the fraction released-chemical (fr) and the batchwise dilution factor (Dbatch) are 

reported in Table 3.11 as proposed in the CHARM model. For cleaning chemicals, the fraction 

released should be set at 1 as the model assumes that the chemical is entirely released. 

Table 3.11 Default values to be used for hazard assessment of completion and workover chemicals 

(specified as ‘cleaning chemicals’, other chemicals’, ‘squeeze treatments’ and ‘hydrotest 

chemicals’) (Thatcher et al., 2004). 

Parameter Symbol Cleaning chemicals Other chemicals 

Fraction released-chemical 

Dilution factor at 500 m 

fr

Dbatch

n. r. 

7.7 10-5 (1:13,000) 

0.1 

7.1 10-5 (1:14,000) 

Parameter Symbol Squeeze treatments Hydrotest chemicals 

Fraction released-chemical 

Dilution factor at 500 m 

fr

Dbatch

0.33 

7.1 10-5 (1:14,000) 

1 

0.001 (1:1,000) 

EGBE has been reported in a manufactured product at concentrations between 60 and 100%. 

Different dilution ratios are suggested for this product, depending on the type of use. The lowest 

dilution ratio (1:10) applies to the cleaning of drilling and construction equipment. This ratio will 

be used as a worst case and gives a concentration of EGBE in cleaning fluids of 100 g/L. With 

this figure, the different scenarios can be used. Results are presented in Table 3.12. 

33
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.12 PECintermittent for the use of oilfield chemicals containing 

EGBE on offshore platforms (marine compartment) 

PECseawater (mg/L) 

Surface and well cleaning chemicals 7.7 

Squeeze treatments 2.3 

Hydrotest chemicals 100 

b) Regulations on oilfield chemicals

In June 2000, OSPAR introduced Decision 2000/2 on a Harmonised Mandatory Control System 

for the Use and Reduction of the Discharge of Offshore Chemicals. At the heart of OSPAR 

Decision 2000/2 are two Recommendations: Recommendation 2000/4 on a Harmonised PreScreening Scheme for Offshore Chemicals, which facilitates the substitution of chemicals with 

certain characteristics by less hazardous alternatives and Recommendation 2000/5. 

Decision 2000/2 and its supporting Recommendations entered into force on 16 January 2001. 

The Decision requires offshore chemicals to be ranked according to their calculated Hazard 

Quotients (HQ - ratio of Predicted Environmental Concentration (PEC) to Predicted No Effect 

Concentration (PNEC). It also obliges authorities to use the CHARM “hazard assessment” 

module as the primary tool for ranking. 

Details on the national regulations put in place in order to comply with OSPAR 

recommendations have been found for two countries (United Kingdom and Norway). However 

other countries which have offshore oil and gas installations7 may have similar regulations in 

place following harmonisation of such schemes in 1996. 

− Norway 

The Norwegian State Pollution Control Authority (SFT) regulates the use of drilling fluids/muds 

through discharge permits. Water based muds are tested under OSPAR formats for bioaccumulation potential and bio-degradability and given a discharge permit if judged to be 

environmentally friendly. Synthetic muds are similarly evaluated and can be given a discharge 

permit according to their properties. All oil-based muds are injected or taken to shore for 

treatment. The discharge of solids containing more than 1% oil, by weight, is forbidden - 

whether the drilling fluid is water-, oil- or synthetic-based. The regulations also deal explicitly 

with well testing, workover and cementing. 

Norwegian regulations: 

1. Norwegian State Pollution Control Authority (SFT). 1998. Requirements for 

Ecotoxicological Testing and Environmental Assessment of Offshore Chemicals and 

Drilling Fluids. SFT, Oslo 

2. Norwegian State Pollution Control Authority (SFT). 1999a. Environmental Monitoring 

of Petroleum Activities on the Norwegian Shelf; Guidelines 99:01. SFT, Oslo 

3. Norwegian State Pollution Control Authority (SFT). 1999b. Pollution Control Act, 

1981. SFT, Oslo) 

 

7 In some countries there are no offshore oil and gas installations in the OSPAR maritime area under the jurisdiction 

of the Contracting Parties (i.e. Belgium, Finland, France, Portugal, Sweden). 

34 
CHAPTER 3. ENVIRONMENT

Norway uses the standard OSPAR “A” and “B” lists for offshore chemicals and requires that 

discharge of these “shall be reduced as much as possible, e.g., through recycling”. Operators are 

required to ensure the purity of the substances they use, with minimum contamination by other 

chemicals. Discharge of unused chemicals into the sea is expressly forbidden, even if they are on 

list A or B and their toxicity is therefore well known. All discharges must have a permit and 

chemicals not on the lists must be separately tested and notified. 

− United Kingdom 

The Offshore Chemical Notification Scheme (OCNS) was originally introduced in 1979. In 

1993, the UK Government introduced a revised scheme, which classified chemicals using test 

protocols approved by the Oslo and Paris Commissions (OSPAR). This was modified in detail, 

in early 1996, to meet the requirements of the OSPAR Harmonised Offshore Chemical 

Notification Format (HOCNF) which co-ordinates the testing requirements for oilfield chemicals 

throughout the NE Atlantic sector. 

In June 2000, OSPAR introduced Decision 2000/2 on a Harmonised Mandatory Control System 

for the Use and Reduction of the Discharge of Offshore Chemicals. In the UK this is to be 

administered under the Offshore Chemical Regulations 2002 (OCR 2002) which came into force 

on 15th May 2002. 

The UK operates the Offshore Chemical Notification Scheme (OCNS8). This is operated by the 

Centre for Environment, Fisheries and Aquaculture Science (CEFAS) on behalf of the 

Department for Trade and Industry (DTI). CEFAS is an Executive Agency of the UK 

Government's Department for Environment, Food and Rural Affairs (DEFRA). The OCNS 

requires the registration of all chemicals used in the actual exploration, exploitation and 

associated offshore processing of petroleum on the UK Continental Shelf. Dossiers on individual 

products rather than substances have to be notified. The required dossiers must include acute 

toxicity data on marine species from the three tropic levels as well as other data. Because 

individual products are notified, detailed information on usage rates and composition are 

available allowing more precise risk characterisations to be performed using the CHARM model 

and the products ranked according to the hazard they present. However, for reasons of 

commercial confidentiality, the toxicity data, usage and composition information is not 

published. 

EGBE has been registered by a number of suppliers to the industry under the name ‘EGMBE’ 

(ethylene glycol mono butyl ether), as shown below. 

Table 3.13 Examples of EGBE containing products registered in UK 

Product type or function Notifier Rating 

Carrier solvent Baker Petrolite Silver 

Drilling fluid Halliburton Gold 

Well stimulant (30% EGBE) Baker Petrolite Gold 

Gold is the highest possible rating out of 6 and is for products where the PEC:PNEC is less than 

one. Silver, the next highest, is for substances where the model PEC:PNEC is between 1 and 30. 

EGBE is also listed as a hydrotest chemical, but under a category of products where CHARM is 

 

8 http://www.cefas.co.uk/ocns/index.htm 

35
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

not regarded as suitable for assessing risk of use (not all uses are regarded as suitable for risk 

characterisation using CHARM). For this reason, it is only rated for hazard properties and falls 

into group E, the lowest hazard category available. It is also possible that EGBE and EGBE 

containing products may be registered under trade names (OSPA, personal communication). 

Because they use detailed information supplied under confidentiality terms, specific assessments 

performed under such regulations should be regarded as more precise than those carried out in 

this assessment. For instance, detailed information is provided by the manufactures on actual 

usage and emission rates. 

3.1.2.2.5 Local releases: EGBE used in oil spill dispersants (marine compartment) 

The development of oil spill dispersants has started in the beginning of the 70’s with the increase 

in oil transportation and as a consequence the increase in number of accidents. A summary of 

dispersants used today is provided in Table 3.14 and a list of agreed dispersants according to the 

Bonn Agreement is also available9. 

Table 3.14 Dispersants used today, application methods and dosages (Bonn Agreement, 2001) 

Standard name Generation Type Application method Solvent Dosage 

(dispersant / oil) 

Conventional 

dispersants Second 1 Not diluted on ships No aromatic hydrocarbons 30 –100% 

2 Diluted on ships Water-based (e.g., glycol 

ether) Concentrated 

dispersants Third 

3 Not diluted on ships or 

airplanes Hydrocarbon-based 

5 – 15% 

(concentrated 

products) 

It is difficult to know how often dispersants are used against oil spills. A survey of international 

uses of dispersants was performed by Lindgren et al., 2001. From this survey it seems clear that 

most of the time mechanical actions are recommended for combating oil spills and that the use of 

chemicals such as dispersants should be avoided as long as possible. This is particularly the case 

in the Baltic area, which falls under the HELCOM’s recommendations (Helsinki Commission; 

Denmark, Finland, Germany, Latvia, Lithuania, Poland, Sweden and the Russian Federation). 

In specific situations dispersants can nevertheless be used if the national authority approves it. 

This is also the case in the USA and Canada where mechanical methods are used unless weather 

prevents the use of these methods. 

In Belgium, France, United Kingdom and Germany, dispersants are used, particularly in England 

where the total number of approved dispersants is high in comparison with other countries. 

However dispersants in these different countries are applied under specific rules and 

recommendations and usually after approval by the government or the competent authority in 

this field. Apart from these theoretical considerations, it seems that dispersants are nevertheless 

used in a lot of situations because it is often the easiest way to manage an oil spill (personal com. 

Cedre). Therefore small oil spills resulting from usual practice are most of the time treated with 

dispersants when of course this is possible. 

 

9 This list of agreed dispersants will be updated in 2005. 

36 
CHAPTER 3. ENVIRONMENT

Lessard and Demarco, 2000 report that the rate of usage of dispersants has gone up over the past 

30 years and that dispersants have been successfully applied in half as many major spills during 

the 1990s as during the prior 25 years. 

Among all reported dispersants some contain EGBE in non-negligible concentrations. This is the 

case for example of Wellchem Wellaid 3315 (5% EGBE) and Corexit 952710 (38% EGBE). It 

was not possible to have the composition of all the agreed dispersants on the lists of the Bonn 

Agreement nevertheless from the two above examples it is clear that some of them contain high 

amounts of EGBE. Application rates of dispersants depend on the amount of oil to be treated. 

Usual application rates seem to be around 5 to 15% from information compiled on product 

fact-sheets (Cedre, 2004). 

EGBE sales for oil spill dispersant use have been reported in 2001 (191 tonnes). However, for 

the following years (2002 and 2003) no sale was reported. This is due to the mode of use of oil 

spill dispersant that required some stocks to be available. Indeed, oil spill dispersants are only 

used on occasions when an oil spill occurs in the environment and so may be used locally in 

relatively large amounts on very infrequent occasions. This should be consequently treated as an 

intermittent release. 

Figures on the number of oil spills detected are available for the Baltic Sea. Von Viebahn, 2002 

reports that 250 to 650 oil spills are detected each year. It is also stated in the same report that the 

number of oil spills in the North Sea is higher, however exact figures are not available. More 

information on annual oil spills would be useful in order to propose a regional scenario. 

However due to a lack of time this is not possible at present. 

In the same way, specific data (amount of oil released, dispersant used, etc.) are available on 

different oil spills, major as well as minor spills, in different locations. It is possible from the 

data available to construct worst case scenarios and assess local PEC, however this implies the 

development of specific and detailed emission scenario for major and minor spills. This could 

become difficult considering that a “typical oil spill” cannot be defined. Each presents unique 

circumstances, depending on location, quantity and type of oil, prevailing weather conditions etc. 

It has also to be taken into consideration that the decision whether to use a dispersant is made 

with the regulatory authorities case-by-case and on a risk benefit basis. It is generally accepted 

that dispersants create adverse effects but their use is intended to mitigate the even worse 

potential effects of environmentally hazardous oil products. For this reason, even if detailed 

composition information was available, it would still not be possible to carry out such a risk 

benefit analysis within the framework of the Existing Substances risk evaluation process. 

3.1.2.2.6 Local releases for other uses 

Note: Tables used for local release estimates refer to TGD (EC, 2003), Tables A and B, 

Chapter 3, Appendix I; other Tables, Chapter 7. 

The local release estimates for formulation, processing and use of EGBE are given in Table 3.15

to Table 3.20. 

 

10 This dispersant has been removed from the UK list of approved dispersant on 30/07/98 because it did not pass the 

Rocky Shore Test. However existing stock may have been used away from rocky shorelines in appropriate 

conditions. 

37
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.15 Local releases of EGBE from formulation of paints 

Scenario Paints IF Paints IIF and Paints IIIF Paints IVF Paints VF

Main category Multi-purpose 

equipment Multi-purpose equipment Multi-purpose equipment Multi-purpose equipment

Total fraction and 

connected tonnage 

0.1411/13,700 

tonnes 

0.2175 / 21,100 tonnes 0.0756/ 

7,340 tonnes 

0.0546 / 5,300 tonnes 

Regional tonnage 1,370 tonnes 2,110 tonnes 734 tonnes 530 tonnes 

Typical max. % of EGBE 3 3 7 7 

Number of days / Fraction 

of main source 

(Table B2.3) 

300 / 0.6 

(Table B2.3) 

300 / 0.4 

(Table B2.3) 

300 / 0.7 

(Table B2.3) 

300 / 0.8 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A2.1) 

0.005 

0.003 

0.0001 

(Table A2.1) 

0.005 

0.003 

0.0001 

(Table 3.6) 

0.03 

0 

0 

(Table 3.5) 

0.01 

0 

0 

Amount released (kg/d) 

- air 

- waste water 

- soil 

13.7 

8.2 

0.3 

14.1 

8.4 

0.3 

51.4 

0 

0 

14.1 

0 

0 

Table 3.16 Local releases of EGBE from formulation of paints (continue) 

Scenario Paints VIF Paints VIIF Paints VIIIF Paints IXF

Main category Multi-purpose 

equipment 

Multi-purpose 

equipment 

Multi-purpose 

equipment 

Multi-purpose 

equipment 

Total fraction and 

connected tonnage 

0.0546 / 

5,300 tonnes 

0.0218 / 

2,120 tonnes 

0.0109 / 

1,060 tonnes 

0.0109 / 

1,060 tonnes 

Regional tonnage 530 tonnes 212 tonnes 106 tonnes 106 tonnes 

Typical max. % of EGBE 8 1 2 2 

Number of days / Fraction 

of main source 

(Table B2.3) 

300 / 0.8 

(Table B2.3) 

300 / 0.7 

(Table B2.3) 

300 / 0.8 

(Table B2.3) 

300 / 0.8 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table 3.15) 

0.03 

0 

0 

(Table 3.19) 

0.02 

0.01 

0 

Amount released (kg/d) 

- air 

- waste water 

- soil 

7.1 

28.3 

0.1 

2.5 

9.9 

0 

8.5 

0 

0 

5.7 

2.8 

0 

38 
CHAPTER 3. ENVIRONMENT

Table 3.17 Local releases of EGBE from processing of paints 

Scenario Paints IP Paints IIP and 

Paints IIIU* 

Paints IVP Paints VP

Main category Non dispersive use Wide dispersive use Non dispersive use Non dispersive use 

Total fraction and 

connected tonnage 

0.1411 / 

13,700 tonnes 

0.2175 / 

21,100 tonnes 

0.0756 / 

7,340 tonnes 

0.0546 / 

5,300 tonnes 

Regional tonnage 1,370 tonnes 2,110 tonnes 734 tonnes 530 tonnes 

Typical max. % of EGBE 3 3 7 7 

Number of days / Fraction 

of main source 

(Table B3.13) 

300 / 0.05 

(Table B4.4) 

300 / 0.002 

(Table B3.13) 

300 / 0.1 

(Table B3.13) 

300 / 0.1 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A3.15 water 

based) 

0.8 

0.1 

0.001 

(Table A4.5 water 

based) 

0.8 

0.15 

0.01 

(Table 3.6) 

0.94 

0 

0.03 

(Table 3.5) 

0.01** 

0.01 

0 

Amount released (kg/d) 

- air 

- waste water 

- soil 

182.7 

22.8 

0.2 

11.3 

2.1 

0.1 

230.0 

0 

7.3 

1.8 

1.8 

0 

* It has been confirmed that application conditions vary little between professional and retail decorative paints. The only 

difference is the classification of the persons applying the paints (professionals or private consumers). Consequently, 

these scenarios have been merged and the default emission factors for consumer products will be used (wide 

dispersive use: use outside of a factory). 

** Assuming treatment of flue gases (98% if no treatment) 

Table 3.18 Local releases of EGBE from processing and private use of paints 

Scenario Paints VIP Paints VIIP Paints VIIIP Paints IXP

Main category Non dispersive use Non dispersive use Non dispersive use Non dispersive use 

Total fraction and 

connected tonnage 

0.0546 / 

5,300 tonnes 

0.0218 / 

2,120 tonnes 

0.0109 / 

1,060 tonnes 

0.0109 / 

1,060 tonnes 

Regional tonnage 530 tonnes 212 tonnes 106 tonnes 106 tonnes 

Typical max. % of EGBE 8 1 2 2 

Number of days / Fraction 

of main source 

(Table B3.13) 

300 / 0.1 

(Table B3.13) 

300 / 0.1 

(Table B3.13) 

300 / 0.1 

(Table B3.13) 

300 / 0.1 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A3.15 water 

based) 

0.01** 

0.1 

0.001 

(Table A3.15 

solvent based) 

0.9 

0.02 

0.001 

(Table 3.15) 

0.01** 

0 

0 

(Table 3.19) 

0.96 

0.01 

0 

Amount released (kg/d) 

- air 

- waste water 

- soil 

1.8 

17.7 

0.2 

63.6 

1.4 

0.1 

0.4 

0 

0 

33.9 

0.4 

0 

** Assuming treatment of flue gases (98% if no treatment for solvent-based coatings and 80% for water-based 

 coatings). Indeed, automotive producers operate VOC emission abatement systems to control air emissions 

39
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.19 Local releases of EGBE from formulation, processing and private use of detergents 

Scenario Detergents IF Detergents IP Detergents IIF Detergents IIU

Main category Multi-purpose 

equipment 

Non dispersive use Non dispersive use Wide dispersive use 

Total fraction and 

connected tonnage 

0.0850 / 

8,250 tonnes 

0.0850 / 

8,250 tonnes 

0.0283 / 

2,750 tonnes 

0.0283 / 

2,750 tonnes 

Regional tonnage 825 tonnes 825 tonnes 275 tonnes 275 tonnes 

Typical max. % of EGBE 10 10 10 10 

Number of days / Fraction 

of main source 

(Table B2.3) 

300 / 0.8 

(Table B3.3 for 

waste water only) 

200 / 0.002 

(Table B2.3) 

300 / 1 

(Table B4.1 for 

waste water only) 

365 / 0.002 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A2# - liquid)

0.00002 

0.0009 

0.0032 

(Table A3.5) 

0.0025 

0.9 

0.05 

(Table A2# - liquid) 

0.00002 

0.0009 

0.0032 

(Table A4.1) 

0 

0.99 

0.01 

Amount released (kg/d) 

- air 

- waste water 

- soil 

0 

2.0 

7.0 

- 

7.4 

- 

0 

0.8 

2.9 

- 

1.5 

- 

Table 3.20 Local releases of EGBE from formulation and processing of other uses 

Scenario Intermediates IP Inks IF Inks IP Pharm IP Elec IP

Main category Dedicated 

equipment 

Multi-purpose 

equipment 

Non dispersive 

use 

Non dispersive 

use 

Non dispersive 

use 

Total fraction and 

connected tonnage 

0.103/10,000 t. 

0.0927/9,000 t. 

(captive use) 

0.0257 / 2,500 t. 0.0257 / 2,500 t. 0.0051 / 500 t. 0.062 / 600 t. 

Regional tonnage 10,000* t. 

9,000* t. (captive 

use) 

250 t. 250 t. 50 t. 60 t. 

Typical max. % of 

EGBE 

100 (default value) 20 20 100 (default 

value) 

100 (default 

value) 

Number of days / 

Fraction of main source 

(Table B3.2) 

300 / 0.25 

(Table B2.3) 

300 / 1 

(Table B3.10 

large companies) 

300 / 0.333 

(Table B3.2) 

32 / 0.65 

(Table B3.2) 

12 / 0.5 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A3.3) 

0.00001 

0.007 

0.0001 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A3.12 – 

printing and allied 

processes) 

0.3 

0.001 

0.0001 

(Table A3.3) 

0.001 

0.02 

0.0001 

(Table A3.4) 

0.001 

0.005 

0.01 

Amount released (kg/d) 

- air 

- waste water 

- soil 

0.1 / 0.1** 

58.3 / 52.5** 

0.8 / 0.8** 

4.2 

16.7 

0.1 

83.3 

0.3 

0 

1.0 

20.3 

0 

2.5 

12.5 

25.0 

* The regional tonnage is the total tonnage (3 EGBEA producers in the EU) 

** Captive use: these amounts will be added to the releases at production 

40 
CHAPTER 3. ENVIRONMENT

Table 3.21 Local releases of EGBE from formulation and processing of other uses (continued) 

Scenario Leather IP Adhesives IF Adhesives IP Agri IF Agri IP

Non dispersive use Multi-purpose 

equipment 

Non dispersive 

use 

Multi-purpose 

equipment 

Wide 

dispersive use Main category 

Total fraction and 

connected tonnage 

0.0051 / 500 t. 0.0051 / 500 t. 0.0051 / 500 t. 0.0021 / 200 t. 0.0021 / 200 t. 

Regional tonnage 50 t. 50 t. 50 t. 20 t. 20 t. 

Typical max. % of 

EGBE 

5 10 10 100 (default 

value) 

1 

Number of days / 

Fraction of main 

source 

(Table B3.4 except for 

waste water release) 

200 / 0.5 

(Table B2.3) 

300 / 1 

(Table B3.3 only 

for waste water) 

50 / 0.002 

(Table B2.2) 

300 / 1 

(Table B3.1) 

2 / 0.001 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A3.6 except for 

waste water release) 

0 

specific scenario (see 

Section 3.1.2.2.2) 

0.01 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A3.5) 

0.05 

0.45 

0.45 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A3.1) 

1 

0 

0 

Amount released 

(kg/d) 

- air 

- waste water 

- soil 

- 

44.5 

1.3 

0.8 

3.3 

0 

- 

0.9 

- 

0.3 

1.3 

0 

10 

0 

0 

Table 3.22 Local releases of EGBE from formulation and processing of other uses (continued) 

Scenario Oilfield IF Oilfield IP Metal IF Metal IP

Main category Multi-purpose 

equipment 

Wide dispersive 

use 

Multi-purpose 

equipment 

Non-dispersive 

use 

Total fraction and 

connected tonnage 

0.0257 / 2,500 t. 0.0257 / 2,500 t. 0.0206 / 2,000 t. 0.0206 / 2,000 t. 

Regional tonnage 250 t. 250 t. 200 t. 200 t. 

Typical max. % of EGBE 100 100 4 4 

Number of days / Fraction 

of main source 

(Table B2.3) 

300 / 1 

(Table B3.3 only for 

waste water) 

50 / 0.002 

(Table B2.34) 

300 / 0.8 

(Table B3.6) 

300 / 0.7 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A3.5) 

0.05 

0.45 

0.45 

(Table A2.1) 

0.01 

0.02 

0.0001 

(Table A3.7) 

0.25 

Specific scenario 

(see point 

3.1.2.2.3) 

0.05 

Table 3.22 continued overleaf 

41
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.22 continued Local releases of EGBE from formulation and processing of other uses (continued) 

Scenario Oilfield IF Oilfield IP Metal IF Metal IP

Main category Multi-purpose 

equipment 

Wide dispersive 

use 

Multi-purpose 

equipment 

Non-dispersive 

use 

Amount released (kg/d) 

- air 

- waste water 

- soil 

4.2 

16.7 

0.1 

- 

4.5 (specific 

scenario for the 

marine 

compartment – see 

point 3.1.2.2.4) 

- 

5.3 

10.7 

0.1 

116.7 

0. 8 (3 kg – 

intermittent 

release) 

23.3 

Table 3.23 Local releases of EGBE from formulation and processing of other uses (continue)

Scenario Cosmet IF / Fire IF Cosmet IU Fire IP

Main category Multi-purpose equipment - Wide dispersive use 

Total fraction and 

connected tonnage 

0.0082 / 800 t. 0.0051 / 500 t. 0.0031 / 300 t. 

Regional tonnage 80 t. 50 t. 30 t. 

Typical max. % of EGBE 10 (cosmetic products) / 

16 (fire foam) 

10 0.5 

Number of days / Fraction 

of main source 

(Table B2.3) 

300 / 1 

(Table B4.1 only for 

waste water) 

365 / 0.002 

(Table B3.3 only for 

waste water) 

50 / 0.002 

Release estimates 

(fraction) 

- air 

- waste water 

- soil 

(Table A2.1) 

0.005 

0.02 

0.0001 

(Table A4.1) 

0 

0.8 

0.001 

(Table A3.5) 

0.05 

0.45 

0.45 

Amount released (kg/d) 

- air 

- waste water 

- soil 

1.3 

5.3 

0 

- 

0.2 

- 

- 

0.5 

- 

3.1.3 Continental and regional predicted environmental concentrations 

Continental and regional computations are done by means of multimedia fate models based on 

the fugacity concept. The standardised continental and regional environments of the TGD 

(EC, 2003) are used. Table 3.24 shows the calculated continental and regional PECs for air, 

water and soil using EUSES (EC, 2004). These results come from the calculation program 

EUSES (EC, 2004) and do not integrate the emissions due to the scenario “Agri I” because of the 

scenario number limitations of the software. Nevertheless, the calculations have been made for 

this scenario in a separate file and it appears that the impact of emissions volumes for this 

scenario on total continental and regional emissions is negligible. 

42 
CHAPTER 3. ENVIRONMENT

Table 3.24 Regional PECs in air, water and soil (calculations made by EUSES – SIMPLEBOX model) 

Compartment PEC continental PEC regional 

Air 1.10.10-5 mg/m3 1.15.10-4 mg/m3

Water 9.41.10-4 mg/L 7.35.10-3 mg/L 

Agricultural soil 8.44.10-5 mg/kg (ww) 8.74.10-4 mg/kg (ww) 

Pore water of agricultural soils 1.40.10-4 mg/L 1.45.10-3 mg/L 

Natural soil 1.82.10-4 mg/kg (ww) 1.89.10-3 mg/kg (ww) 

Industrial soil 4.39.10-3 mg/kg (ww) 4.30.10-2 mg/kg (ww) 

Sediment 1.16.10-3 mg/kg (ww) 9.08.10-3 mg/kg (ww) 

Seawater 6.09.10-7 mg/L 6.12.10-4 mg/L 

Marine sediment 7.54.10-7 mg/kg (dw) 7.57.10-4 mg/kg (ww) 

3.1.4 Local predicted environmental concentrations (PEClocal) 

3.1.4.1 Aquatic compartment 

3.1.4.1.1 PEClocal for production 

PECSTP

At production level, the local Predicted Environmental Concentration for micro-organisms in 

STP ranges from 0.02 to 0.49 mg/L (these values include emissions for EGBE processing, for 

on-site EGBEA production) (see Table 3.6) 

PECaqua

Emissions at production and processing levels (EGBE captive use for EGBEA synthesis) result 

in a PEC for surface water ranging from 0.8 to 10.6 μg/L (including the PEC regional for the 

aquatic compartment). The PECaqua for the different production sites are presented in Table 3.25. 

Table 3.25 Local PEC in water at production 

Production sites #1 (PEClocalmarine) #2 #3 #4 #5 

PEClocalaqua (μg/L) 0.8 10.6 9.0 9.6 - 

3.1.4.1.2 Calculation of PEClocal for formulation, processing and private use 

Concentrations of EGBE in water for formulation, processing and private use in the EU are 

estimated with a generic scenario which was carried out based on default values (TGD – 

EC, 2003) because no site specific data were available. 

It is assumed that the amounts released to water will enter a sewage treatment plant. During 

sewage treatment, 87.4% of EGBE is expected to be removed (see Table 3.5). The default flow 

rate of the treatment plant is 2,000 m3

/day. 

43
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

The effluent concentration leaving the STP (Predicted Environmental Concentration in a STP or 

PECSTP) is calculated according to Equation 3.1. This PECSTP is divided by a dilution factor (10: 

default value) to obtain the local PEC in surface water (see Equation 3.2). The daily amounts 

released for the generic scenarios are the basis for the calculation of the PECs. Table 3.26 gives 

the PECs for the aquatic compartment. PECseawater have been calculated with EUSES 2.0 (EC, 

2004). 

Table 3.26 Local PECSTP and PECaqua for EGBE 

Scenario Daily release 

to waste 

water (kg/d) 

PECSTP

(μg/L) 

Local 

PECaqua

(μg/L) 

Total local 

PECaqua* 

(μg/L) 

Total local 

PECseawater ¤

(μg/L) 

Paints IF / Paints IP 8.2 / 22.8 519 / 1,430 52 / 144 59 / 151 42 / 114 

Paints IIF & IIIF 8.4 533 54 61 43 

Paints IIP and Paints IIIU 2.1 132 13 21 11 

Paints IVF / Paints IVP 0 / 0 - / - - / - 7 / 7 1 / 1 

Paints VF / Paints VP 0 / 1.8 - / 110 - / 11 7 / 18 1 / 9 

Paints VIF / Paints VIP 28.3 / 17.7 1,780/1,090 179 / 109 186 / 116 142 / 87 

Paints VIIF / Paints VIIP 9.9 / 1.4 623 / 87 63 / 9 70 / 16 50 / 8 

Paints VIIIF / Paints VIIIP 0 / 0 - / - - / - 7 / 7 1 / 1 

Paints IXF / Paints IXP 2.8 / 0.4 178 / 22 18 / 3 25 / 10 15 / 2 

Detergents IF / Detergents IP 2.0 / 7.4 125 / 464 13 / 47 20 / 54 11 / 37 

Detergents IIF / Detergents 

IIU

0.8 / 1.5 52 / 93 6 / 10 13 / 17 5 / 8 

Intermediates** IP *** 58.3 736 19 26 59 

Inks IF / Inks IP 16.7 / 0.3 1,050 / 17 105 / 2 112 / 9 84 / 2 

Pharm IP *** 20.3 254 7 14 21 

Elec IP 12.5 791 80 87 63 

Leather IP 44.5 2810 281 288 223 

Adhesives IF / Adhesives IP 3.3 / 0.9 167 / 55 17 / 6 24 / 13 14 / 5 

Agri IF / Agri IP 1.3 / 0 82 / - 8 / - 15 / 7 7 / 1 

Oilfield IF / Oilfield IP

Surface and well cleaning 

Squeeze treatments 

Hydrotest chemicals 

16.7 / 4.5 

- 

- 

- 

1,050 / 276 

- 

- 

- 

105 / 28 

- 

- 

- 

112 / 35 

- 

- 

- 

84 / 23 

7,700 

2,300 

100,000 

Metal IF / Metal IP

Metal intermittent 

10.7 / 0.8 

3 

673 / 51 

1500 

68 / 5 

19 

75 / 12 

26 

54 / 5 

16 

Table 3.26 continued overleaf 

44 
CHAPTER 3. ENVIRONMENT

Table 3.26 continued Local PECSTP and PECaqua for EGBE 

Scenario Daily release 

to waste 

water (kg/d) 

PECSTP

(μg/L) 

Local 

PECaqua

(μg/L) 

Total local 

PECaqua* 

(μg/L) 

Total local 

PECseawater ¤

(μg/L) 

Cosmet IF & Fire IF/Cosmet IU 5.3 / 0.2 335 / 14 34 / 2 41 / 9 27 / 3 

Fire IP 0.5 32 4 11 2 

* Total local PECaqua = Local PECaqua + regional PECaqua 

¤ Total local PECseawater = Local PECseawater + regional PECseawater

** Captive use not included 

*** Dilution factor = 40 and EFFLUENTSTP = 10,000 m3/day (see scenario for IC3 chemicals used in synthesis) 

P Processing 

F Formulation 

U Private use 

3.1.4.1.3 Measured levels 

Contaminated sites

Ground water sample collections were performed in February 1974 near the “Valley of Drums”, 

Kentucky, USA, a contaminated site. EGBE was detected at a concentration of 2.3.10-2 mg/L in 

one sample out of seven (Stonebreaker and Smith, 1980). 

In April 1980, EGBE was detected by GC/MS at concentrations of 1.31 and 5.68 mg/L in the 

water of the Hayashida River, Hyogo prefecture, Japan. In this surface water, EGBE was 

considered as a contaminant from leather industry effluents. The values represent respectively 

levels after steams and vacuum distillation (Yasuhara et al., 1981). 

EGBE was measured in a municipal waste water treatment plant, in Göteborg, Sweden. Twentyfour hour flow proportional composite were taken in the influent (four samples during 1990 and 

eight samples in 1991) and the effluent (seven samples during 1989-1991). EGBE was separated 

by liquid – liquid extraction with dichloromethane and analysed by GC/MS. The range of 

influent concentrations goes from 0 (non detected) to 2.5.10-1 mg/L (average influent 

concentration in 1990 = 8.5.10-2 mg/L and in 1991 = 2.3.10-2 mg/L). The range of effluent 

concentrations was 0 (non detected) – 3.10-3 mg/L (Paxeus et al., 1992). 

In France, EGBE was detected in the influent of a domestic STP located in northern suburban 

Paris, in 1991 (November, 26). Analysis was performed with purge and trap – high resolution 

capillary GC/MS. EGBE was detected at a concentration of 3.5.10-2 mg/L Nguyen et al., 1994). 

Concentrations of EGBE have been measured with GC/MS in aqueous samples from a municipal 

and an industrial landfill in the United States. They were ranging from < 0.4 (detection limit) to 

84 mg/L. The municipal landfill consisted mostly of municipal waste (four wells were analysed). 

There, EGBE concentrations were detected in wells 3 (18 mg/L) and 4 (84 mg/L) which were 

located in the landfill. EGBE has not been detected in wells 1 and 2 which were located 

downgrade from the landfill. Industrial landfill consisted mostly in industrial waste and EGBE 

concentrations were detected: 2.5 mg/L (well 8), 3 mg/L (wells 5 and 6). In well 7, EGBE has 

not been detected. Wells 5, 6 and 7 were located on the landfill whereas well 8 is a downgrade 

interceptor well located between the landfill and the treatment plant (Beihoffer and Ferguson, 

1994). 

45
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

3.1.4.1.4 Comparison between predicted and measured levels 

Exposure during formulation, processing and private use

Measured concentrations are only available for some uses. In this section, they are compared to 

the concentrations predicted by exposure scenarios. 

EGBE concentrations measured by Yasuhara et al., 1981 can be put in parallel with values 

calculated with scenario “Leather IP”. The measured values, ranging between 1.31 and 

5.68 mg/L show factors 5 or 20 higher than the corresponding PEC calculated in surface water, 

0.288 mg/L. But we must also consider that these data come from a 20 year-old study and 

measurements were performed in a place where EGBE contamination from the leather industry 

was expected. Therefore, these results should be considered as a highly worst-case. 

EGBE was also measured in municipal waste water effluents. In the study by Paxeus et al., 1992 

EGBE concentrations reach 3.10-3 mg/L. In a first time this value can be compared to PECSTP

presented in Table 3.26. These concentrations range between 0.014 and 1.78 mg/L. 

3.1.4.2 Terrestrial compartment 

Different PECs can be determined to assess the exposure level in terrestrial compartment. The 

local PEC in soil is calculated according to the following equation: 

Equation 3.5 Calculation of PEClocalsoil 

soil soil natural soil PEClocal Clocal PECregional = + _

3.1.4.2.1 PEClocal for production 

The different PEClocal in soil at production and processing (EGBE captive use for on-site 

EGBEA production) levels are presented in Table 3.27. 

Table 3.27 PEClocalsoil at production and in situ processing (according to EUSES) 

Site 

# 

Local PEC in agricultural 

soil averaged over 30 days 

(μg/kg ww) 

Local PEC in agricultural 

soil averaged over 180 days 

(μg/kg ww) 

Local PEC in grassland 

averaged over 180 days 

(μg/kg ww) 

1 34.25 11.15 4.72 

2* 1.15 1.15 1.38 

3* 0.88 0.88 0.89 

4* 0.89 0.89 0.89 

5 No data but total annual site production for EGBE < 1kt. 

* At this production site STP sludge is incinerated 

3.1.4.2.2 Calculation of PEClocal for formulation, processing and private use 

The EUSES models (EC, 2004) take into account both the application of STP sludge on 

agricultural soil and deposition from air for the calculation of EGBE concentrations in the 

46 
CHAPTER 3. ENVIRONMENT

terrestrial compartment. Table 3.298 gives the terrestrial PECs at local scale for the various 

generic scenarios. 

Table 3.28 Local PECsoil for EGBE 

Scenario PECsoil - average 

concentration in 

agricultural soil over 

30 days (μg/kg ww) 

PECsoil - average 

concentration in 

agricultural soil over 

180 days (μg/kg ww) 

PECsoil - average 

concentration in 

grassland over 180 

days (μg/kg ww) 

Paints IF / Paints IP 30 / 84 11 / 32 6 / 23 

Paints IIF & IIIF 31 11 6 

Paints IIP and Paints IIIU 9 4 3 

Paints IVF / Paints IVP 4 / 10 4 / 10 5 / 17 

Paints VF / Paints VP 2 / 8 2 / 4 3 / 3 

Paints VIF / Paints VIP 96 / 59 31 / 20 13 / 8 

Paints VIIF / Paints VIIP 5 / 9 12 / 6 6 / 7 

Paints VIIIF / Paints VIIIP 2 / 2 2 / 2 2 / 2 

Paints IXF / Paints IXP 12 / 4 5 / 3 3 / 4 

Detergents IF / Detergents IP 9 / 26 4 / 9 3 / 5 

Detergents IIF / Detergents IIU 5 / 7 3 / 3 2 / 2 

Intermediates** IP 41 14 6 

Inks IF / Inks IP 57 / 6 19 / 5 8 / 8 

Pharm IP 15 6 3 

Elec IP 44 15 7 

Leather IP 150 47 19 

Adhesives IF / Adhesives IP 11 / 5 5 / 3 3 / 2 

Agri IF / Agri IP 7 / 3 3.8 / 2.5 3.0 / 2.5 

Oilfield IF / Oilfield IP 57 / 17 19 / 6 8 / 4 

Metal IF / Metal IP 38 / 9 13 / 7 6 / 10 

Cosmet IF & Fire IF / Cosmet 

I

U

20 / 3 7 / 2 4 / 2 

Fire IP 4 2 2 

* Captive use not included 

- PECregionalnatural_soil ~ 1.89 μg/kg (ww)Atmosphere 

P Processing 

F Formulation 

U Private use 

47
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

3.1.4.3 Atmosphere 

3.1.4.3.1 PEClocal for production 

Emissions of EGBE in air, both at production and processing (on-site EGBEA production), result 

in an average annual concentration in air (Clocalair,ann) of 1.67 μg/m3

 in the worst case (site 

specific information for site #2). This results in a PEClocalair,ann11 of 1.77 μg/m3. 

3.1.4.3.2 Calculation of PEClocal for formulation, processing and private use 

The calculated annual average EGBE concentrations in air are presented in Table 3.29 for the 

different use patterns. 

Table 3.29 Local PECair for EGBE 

Scenario Concentration 

during emission 

(μg/m3) 

Clocal_air,ann 100m 

from source 

(μg/m3) 

Annual deposition 

(μg/m2/d) 

PEClocal_air,ann* 

(μg/m3) 

Paints IF / Paints IP 3.81 / 50.40 3.13 / 41.40 4.51 / 59.60 3.25 / 41.50 

Paints IIF & IIIF 3.91 3.22 4.63 3.33 

Paints IIP and Paints IIIU 3.11 2.55 3.67 2.67 

Paints IVF / Paints IVP 14.30 / 62.00 11.70 / 51.00 16.90 / 73.40 11.90 / 51.10 

Paints VF / Paints VP 3.93 / 0.49 3.23 / 0.40 4.65 / 0.58 3.35 / 0.51 

Paints VIF / Paints VIP 1.97 / 0.48 1.62 / 0.39 2.33 / 0.57 1.73 / 0.51 

Paints VIIF / Paints VIIP 0.69 / 17.20 0.56 / 14.10 0.81 / 20.40 0.68 / 14.30 

Paints VIIIF / Paints VIIIP 2.35 / 0.10 1.93 / 0.08 2.78 / 0.11 2.05 / 0.19 

Paints IXF / Paints IXP 1.57 / 9.13 1.29 / 7.51 1.86 / 10.80 1.40 / 7.62 

Detergents IF / Detergents IP 0.12 / 7.12.10-4 0.10 / 3.90.10-4 0.14 / 5.62.10-4 0.22 / 0.12 

Detergents IIF / Detergents IIU 5.09.10-3 / 1.43.10-4 4.19.10-3 / 1.43.10-4 6.12.10-3 / 2.06.10-4 0.16 / 0.12 

Intermediates** IP 0.02 0.02 0.03 0.13 

Inks IF / Inks IP 1.16 / 22.50 1.00 / 18.50 1.37 / 26.60 1.07 / 18.60 

Pharm IP 0.30 0.02 0.04 0.14 

Elec IP 0.70 0.02 0.03 0.14 

Leather IP 4.29.10-3 2.33.10-3 3.35.10-3 0.12 

Adhesives IF / Adhesives IP 0.23 / 8.37.10-5 0.19 / 1.15.10-5 0.27 / 1.65.10-5 0.27 / 0.12 

Agri IF / Agri IP 0.09 / 27.6 0.08 / 0.15 0.10 / 0.22 0.23 / 0.30 

Oilfield IF / Oilfield IP 1.16 / 4.22.10-4 0.95 / 5.78.10-5 1.37 / 8.31.10-5 1.07 / 0.12 

Table 3.29 continued overleaf 

 

11 PEClocalair,ann = Clocalair,ann + PECregionalair

48 
CHAPTER 3. ENVIRONMENT

Table 3.29 continued Local PECair for EGBE 

Scenario Concentration 

during emission 

(μg/m3) 

Clocal_air,ann 100m 

from source 

(μg/m3) 

Annual deposition 

(μg/m2/d) 

PEClocal_air,ann* 

(μg/m3) 

Metal IF / Metal IP 0.93 / 32.4 0.76 / 26.7 1.10 / 38.4 1.33 / 26.80 

Cosmet IF & Fire IF / Cosmet IU 0.37 / 2.06.10-5 0.30 / 2.06.10-5 0.44 / 2.96.10-5 0.42 / 0.12 

Fire IP 4.82.10-5 6.60.10-6 9.49.10-6 0.12 

* PEClocal_air,ann = Clocal_air,ann + regional PECair 

** Captive use not included 

P Processing 

F Formulation 

U Private use 

3.1.4.3.3 Measured levels 

EGBE was detected at 8 μg/m3 in one out of six samples by GC/MS. Indoor air samples were 

collected from 14 homes and a small office in Italy (De Bortoli et al., 1986). 

Concentrations of EGBE have been included in the Environmental Protection Agency’s volatile 

organic compounds national ambient database. This database compiles data on indoor air but not 

industrial space. The average EGBE concentration for 14 samples was 1.05 μg/m3

(median = 0.37 μg/m3; lower quartile = 0.15 μg/m3; upper quartile = 1.77 μg/m3) (Shah and 

Singh, 1988). 

EGBE was also detected in the clean flue gas of a municipal waste incinerator in Germany, in 

1995. It has been measured using the technique of adsorption/thermodesorption and 

adsorption/elution on selected XAD resins and then detected with FID. EGBE concentration was 

0.23 μg/m3 (Jay and Stieglitz, 1995). 

EGBE background levels have been measured by Ciccioli et al., 1993 and Ciccioli, 1996 and 

Ciccioli et al., 1996, in Northern and Southern Europe, in Himalayas and in Antarctica. In the 

first study, concentrations ranged between 0.1 and 1.59 μg/m3

 and in the second between 1.26 

and 14.85 μg/m3

. Industrial or domestic emissions of EGBE were avoided in both studies. Major 

sources seemed to be vegetation in the first case and in the second, marine emission are proposed 

by the authors to explain the measurements. 

3.1.4.3.4 Comparison between predicted and measured levels 

The highest predicted EGBE concentrations in air take place during industrial uses of paints. For 

example, PEClocal_air,ann reaches 51 μg/m3 for the scenario “Paints IVP”. No measured data is 

available in the neighbourhood of plants using paints at high scale so comparison is not possible. 

EGBE emissions in air are often a ten order less at formulation than at processing with most of 

the scenarios used for paints (“Paints I-II/VI-IX”) and printing inks. For other uses, emissions to 

air are reduced: a maximum of ~ 41 μg/m3 occurred for the processing of general industrial 

coatings. Some indoor measured concentrations of EGBE are only presented to get an order of 

magnitude. 

EGBE background levels have been measured in appropriate places in order to avoid industrial 

and domestic sources (by Ciccioli et al., 1993 and Ciccioli, 1996). These studies show a natural 

49
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

production of EGBE by plants and the second study particularly underlined a possible marine 

source of EGBE. 

3.1.4.4 Secondary poisoning 

The bioconcentration factor for fish is very low, so it is not expected that there is a significant 

exposure for humans or predators via the local environment. Moreover, as EGBE is not 

classified as Very Toxic (T+), Toxic (T) or Harmful (Xn and R48), it is assumed that there is a 

low potential for the substance to cause toxic effects if accumulated in higher organisms. 

50 
CHAPTER 3. ENVIRONMENT

3.2 EFFECTS ASSESSMENT: HAZARD IDENTIFICATION AND DOSE 

(CONCENTRATION) - RESPONSE (EFFECT ASSESSMENT) 

Studies are classified as valid if they fully describe the test material used, the test organism, the 

test method and conditions and if the endpoint concentration is based upon measured levels. 

Where only some of these criteria are described the tests may be used with care or considered not 

valid. Moreover for some studies or results, some data are lacking, i.e. the original paper is not 

available but only a citation. 

3.2.1 Aquatic compartment (incl. sediment) 

3.2.1.1 Fish 

Acute toxicity

EGBE short term toxicity studies for fish are summarised in Table 3.30. 

Table 3.30 Short term fish toxicity data for EGBE 

Test 

# 

Species Duration Endpoint Result 

(mg/L) 

Method References Validity 

1 Poecilia 

reticulata 

7 days LC50 983 Not specified Konemann, 1981 Valid with 

restrictions 

2 Menidia 

beryllina*

96 hours LC50 1,125 Not specified Dawson et al., 1975 Valid with 

restrictions 

3 Lebistes 

reticulatus 

96 hours LC0

LC50

LC100

1,000 

1,400 

2,000 

Not specified Knappe and Popp, 

1974 

Invalid 

4 Oncorhynchus 

mykiss 

96 hours LC50 1,474 OECD 

guideline 203, 

1999 

INERIS, 1999b Valid 

5 Lepomis 

macrochirus 

96 hours LC50 1,490 Not specified Dawson et al., 1975 Valid with 

restrictions 

6 Leuciscus idus 

melanotus 

48 hours LC0

LC50

LC100

1,170-1,350 

1,395-1,575 

1,490-1,620 

DIN 38412 part 

15 

Juhnke and 

Lüdemann, 1978 

Valid with 

restrictions 

7 Carassius 

auratus 

24 hours LC50 1,700 APHA n°231 Bridié et al., 1979b Valid with 

restrictions 

8 Leuciscus idus 48 hours LC50 1,880 DIN 38412 part 

15 

Huels, 1982 Invalid 

9 Pimephales 

promelas 

96 hours LC50 2,137 US EPA Dow Chemical Co, 

1979 

Valid with 

restrictions 

10 Lepomis 

macrochirus 

96 hours LC50 2,950 Not specified Dow Chemical Co, 

1981, cited in 

Neely, 1984 

Invalid 

Table 3.30 continued overleaf 

51
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.30 continued Short term fish toxicity data for EGBE 

Test 

# 

Species Duration Endpoint Result 

(mg/L) 

Method References Validity 

- Fundulus 

heteroclitus* 

96 hours NOEC 

LC50

4 

6.7 

Stephan, 1974 Biospherics Inc., 

1981b 

Invalid** 

- Cyprinodon 

variegatus*

96 hours LC50 116 US EPA, 1975 Welchem Inc, 1984 Invalid** 

- Lepomis 

macrochirus 

96 hours LC50 127 Stephan, 1974 CIBA-GEIGY, 1976 Invalid** 

* Marine and estuarine species 

** Test performed with a preparation containing EGBE 

Konemann, 1981 studied the effect of EGBE on a fresh water fish species: Poecilia reticulata

(#1). This test lasted seven days in semi static conditions. The guideline followed was not 

recorded and the purity of EGBE was unknown. Groups of eight 2-3 month-old fish were 

exposed to each concentration tested in following conditions: 22 +/- 1°C, oxygen content 

> 5 mg/L, water hardness = 25 mg/L (CaCO3). Test solutions were renewed daily and a solvent 

vehicle was also employed (acetone or 2-propanol). A log EC50 was measured at 3.92 μmol/L 

(983 mg/L). 

In test #2, performed by Dawson et al., 1975, a chemically pure grade EGBE was tested on the 

marine fish Menidia beryllina. Test was conducted in synthetic seawater, at 20°C with mild 

aeration according to the oxygen demand, water hardness was 55 mg/L (CaCO3). For other test 

conditions see test #5 description. A LC50 of 1125 mg/L was obtained after 96 hours. 

The toxicity of EGBE has been tested on the fish Oncorhynchus mykiss by INERIS, 1999b - test 

#4, according to the OECD guideline No 203. A LC50 of 1,474 mg/L was obtained after 96 hours 

(95% confidence interval = 1,145-1,840 mg/L). Seven fish were disposed per aquarium, in 15 L 

test medium with continuous aeration. Water hardness was 250 mg/L (CaCO3), the range of pH 

during the test was 7.6-8.0 (measured before and after the test), DO between 8.6 and 10.1 and 

temperature was 16 +/- 1°C. Nominal concentrations tested in duplicates were as follow: 0, 100, 

500, 1,000, 2,000 and 4,000 mg/L. The loss of test substance during the test was between 3 and 

13%. 

The test #5 was also performed by Dawson et al., 1975 on the fish species Lepomis macrochirus

and a LC50 of 1,490 mg/L was obtained after 96 hours. The test was conducted in well water, at 

23°C with mild aeration supplied after 24 hours according to the oxygen demand measured daily. 

A range of pH has been measured between the beginning and the end of the test: 7.6-7.9. Fish 

have gone through an acclimation period of 14 days and have not been fed 48 hours prior to 

testing. A minimum of 1 L of test medium was used per gram of fish. No analytical monitoring 

was performed. Moreover, it is reported that some test substances in the study were diluted with 

distilled water or a solvent described as having relatively low toxicity but most samples did not 

required dilution (it can be expected that EGBE did not undergo dilution for its high water 

solubility). 

Test #6 is a static test performed on the fish Leuciscus idus melanotus in parallel in two different 

laboratories by Juhnke and Lüdemann, 1978. A continuous aeration is mentioned (mild 

bubbling) but no data on other test conditions is available. Different Lethal Concentrations were 

measured after 48 hours. Results for both laboratories are presented in Table 3.30. 

52 
CHAPTER 3. ENVIRONMENT

Standard methods for the examination of water and wastewater, APHA n°231: static tank acute 

toxicity test, have been used by Bridié et al., 1979b so as to measure the toxicity of EGBE (#7). 

A LC50 of 1,700 mg/L has been obtained after 24 hours. Ten fish (Carassius auratus) were used 

for each concentration, in 25 L test medium, at 20°C, pH 7 and solutions were aerated during the 

test period. Concentrations of EGBE in the test medium were determined using a total organic 

carbon analyser or by extraction and GC both at the beginning and at the end of the test but data 

are not available. 

Test #9, conducted with a fresh water fish, Pimephales promelas, by Dow Chemical Co, 1979 

gives a LC50 of 2,137 mg/L after 96 hours (95% confidence interval = 2,022-2,263 mg/L – 

moving average method). The method followed came from the US EPA. A static open system 

was used with raw lake water dechlorinated with activated carbon used in test aquariums 

containing 10 fish. Other test conditions were as follow: temperature = 12°C, pH between 7.6 

and 7.8, water hardness 101 mg/L (CaCO3), three test concentrations (1,550, 2,100, 2,800 mg/L) 

plus control. A water temperature between 20 and 24°C is recommended for Pimephales 

promelas in the directive 92/CEE, method C1 “acute toxicity to fish”. The low water temperature 

employed in this test is thus a reason to restrict its validity. 

Tests number 8 and 10 must be considered as invalid for available data are reduced to minimum 

and, in some cases, reports are not available but only a quotation. Test number 3 is also classified 

as invalid for there were both insufficient data on test conditions and an insufficient number of 

fishes per test concentration (5 fish). 

Long-term toxicity

A chronic toxicity test for EGBE is presented in Table 3.31. 

Table 3.31 Long term fish toxicity data for EGBE 

Test # Species Duration Endpoint Result (mg/L) Method References Validity 

1 Brachydanio 

rario 21 days NOEC > 100 

Project of 

OECD 

guideline 

INERIS, 2001 Valid 

Test conditions for this long term fish toxicity data are detailed in Section 3.2.1.5 of this study 

(endocrine disruption). Neither mortality nor endocrine disruption effect was observed at any test 

concentration. Thus a No Observed Effect Concentration can be set at > 100 mg/L, the highest 

tested concentration. 

3.2.1.2 Aquatic invertebrates 

Acute toxicity

Short term toxicity tests for EGBE are presented in Table 3.32. 

53
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.32 Short term invertebrate toxicity data for EGBE 

Test # Species Duration Endpoint Result 

(mg/L) 

Method References Validity 

1 Hydra attenuata

or H. vulgaris

(cnideria) 

72 hours EC50 540 

690 

Regeneration 

potential test 

Morphological 

characteristics 

Bowden et al., 

1995 

Valid with 

restrictions 

2 Crangon 

crangon

(crustacea) 

96 hours LC50 698 Not specified Blackman, 

1974 

Lack of data 

3 Daphnia magna 48 hours EC50 835 Not specified Dow Chemical 

Co, 1979 

Valid with 

restrictions 

4 Artemia salina* 

(crustacea) 

24 hours EC50 1,000 Not specified Price et al., 

1974 

Valid with 

restrictions 

5 Daphnia magna 24 hours EC0

EC50

EC100

1,140 

1,720 

2,500 

DIN 38412 part 11 Bringmann and 

Kühn, 1977 

Valid with 

restrictions 

6 Daphnia magna 24 hours EC0

EC50

EC100

1,283 

1,815 

2,500 

DIN 38412 part 11 Bringmann and 

Kuhn, 1982 

Valid with 

restrictions 

7 Daphnia magna 48 hours EC50 1,550 OECD guideline 

n°202 

INERIS, 1999a Valid 

8 Daphnia magna 24 hours EC50 5,000 DIN 38412 part 11 Hoechst, 1982 Lack of data 

- Palaemonetes 

pugio* 

(crustacea) 

96 hours NOEC 

EC50

2 

5.4 

US EPA, 1975 Biospherics 

Inc., 1981a 

Invalid** 

- Crassostrea 

virginica

(oyster)

96 hours LC50 80.5 US EPA, 1975 Welchem Inc, 

1984 

Invalid** 

- Panaeus 

setiferus (white 

shrimp)

96 hours LC50 117 US EPA, 1975 Welchem Inc, 

1984 

Invalid** 

* Marine species 

** Test performed with a preparation containing EGBE 

Bowden et al., 1995 have conducted their EGBE toxicity test on Hydra attenuata – test #1. The 

aim of this test was to measure the regeneration potential of this organism. Test conditions were 

as follow: 10 isolated digestive regions per test concentration, five test concentrations (90, 180, 

370, 740, 920 mg/L) plus one control, test solutions were renewed every 24 hours. At up to 

370 mg/L, the digestive region had regenerated some tentacles and in some cases the basal disc. 

Only normal wound healing was observed at 740 mg/L while at 920 mg/L the wounds were 

healed but the region expanded. A 72-hour EC50 was derived from these results: 540 mg/L. It 

should be noticed that there was no analytical monitoring and that the endpoint was unusual. 

Moreover no clear effect was shown in presence of the test substance. On the other side, another 

test performed with the same organism by the same team gives a 72-hour EC50 of 690 mg/L. 

Here, the endpoint was based on morphological characteristics. Test conditions were similar and 

10 adult polyps were used per test concentrations. Varying degrees of shortened tentacles and 

body were seen at 740 mg/L and at 920 mg/L polyps were totally contracted. 

54 
CHAPTER 3. ENVIRONMENT

Blackman, 1974 conducted a EGBE toxicity test on Crangon crangon and determined a LC50 of 

698 mg/L after 96 hours (test #2). 20 animals were used per test concentration and test solutions 

were renewed every 6, 12 or 24 hours. It should be noticed that the report is poorly documented. 

Dow Chemical Co, 1979 has conducted a test on Daphnia magna. A 48-hour EC50 was obtained: 

835 mg/L (95% confidence interval = 640-1,051 mg/L) – test #3. A static open system was used. 

Aquariums were filled with ten daphnids and raw lake water dechlorinated with activated 

carbon. Five test concentrations (370, 650, 1,000, 1,500, 2,800 mg/L) plus one control were 

tested in three replicates. Temperature was around 20°C, pH between 7.6 and 7.8 and water 

hardness was 101 mg/L (CaCO3). 

Test #4 has been performed with a crustacean, Artemia salina, by Price et al., 1974 and a 

24-hour EC50 of 1,000 mg/L has been determined. The test medium was constituted by artificial 

seawater, at 24.5°C. 30 to 50 shrimp / mL were disposed at the beginning of the test in 100 mL 

test solution. The following EGBE concentrations were tested: 100, 1,000, 10,000 mg/L. At the 

end of the test, the number of alive shrimp was determined with a colony counter. The lack of 

analytical monitoring and the insufficient number of test concentrations have to be deplored. 

In test #5, Bringmann and Kühn, 1977 have tested EGBE toxicity on Daphnia magna, according 

to the method DIN 38412 part 11: immobilisation in artificial fresh water. They noticed a 

24-hour EC50 of 1,720 mg/L. The test was conducted in an open system with 24 hour-old 

daphnids (wild species) in tap water. Temperature ranged between 20 and 22°C and pH was 

about 7.6-7.7. Test solution was saturated with oxygen at the beginning of the essay but no 

mention of O2 concentration at the end of the test was made. Three replicates were conducted for 

each concentration. 

Test #6 has also been performed by Bringmann and Kuhn, 1982 on 24 hour-old Daphnia magna

(IRCHA clone) with the same method as above. The EC50 after 24 hours was 1,815 mg/L (95% 

confidence interval = 1,698-1,940 mg/L). Other test conditions were as follow: open system, 

20°C, pH = 8 +/- 0.2, water hardness was 250 mg/L (CaCO3) and DO was measured at the end of 

the study to check that it was within the range tolerated by daphnids (2mg/L). Concentrations 

were tested in duplicate. 

INERIS, 1999a has tested the toxicity of EGBE on Daphnia magna according to OECD 

guideline 202 (test #7). Five daphnids were used per containers and four replicates were 

performed for each test concentration: 0, 650, 1,000, 1,500, 2,200, 3,350, 5,000 mg/L. Other test 

conditions were as follow: water hardness 250 mg/L (CaCO3), pH = 7.8 +/- 0.2, DO = 

5.3-6.8 mg/L. Less than 10% immobilisation was observed in the control after 48 hours and the 

analytical monitoring revealed a loss of substance during the test < 20%. A 48-hour EC50 of 

1,550 mg/L (95% confidence interval = 1,390-1,720 mg/L) was observed. 

We can notice another 24-hour EC50 result of 5,000 mg/L with Daphnia Hoechst, 1982) – test 

#8. But as no further data is available, this test is classified as invalid. 

Long-term toxicity

Chronic toxicity tests for EGBE are presented in Table 3.33. 

55
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.33 Long term invertebrate toxicity data for EGBE 

Test # Species Duration Endpoint Result (mg/L) Method References Validity 

1 Brachionus 

calyciflorus 48 hours 

EC10

EC20

EC50

7.2 

14.3 

164 

AFNOR NF T 

90-377 

DeVillers et 

al., 2002a 

Not useable 

for PNEC 

derivation 

2 

Daphnia 

magna 21 days 

NOEC 

EC10

EC20

EC50

100 

134 

175 

297 

OECD 

guideline 

n°211 

INERIS, 

1999d Valid 

3 Ceriodaphnia 

dubia 7 days EC10 134.9 Draft AFNOR 

NF T 90 – 376 

DeVillers et 

al., 2003 

Valid with 

restrictions 

The chronic toxicity of EGBE on the rotifer Brachionus calyciflorus has been determined by 

DeVillers et al., 2002a according to the French norm AFNOR NF T 90-377 – test #1. Cyst 

hatching was initiated in moderately hard water, about 20 hours before the beginning of the test 

(at 25°C under a light intensity of 3,000 lux.). pH was adjusted to 7.5. After 18 hours of 

incubation, cysts were regularly checked to ensure the removal of test organisms within two 

hours of hatching. The assay was performed in a 48-well microplate (five concentrations plus 

one control with eight replicates). Test media consisted in synthetic fresh water solution with a 

suspension of the green alga Chlorella vulgaris as food source. One rotifer was disposed per well 

(newly hatched rotifer) and the incubation occurred at 25°C in darkness, in a covered microplate. 

After 48 hours, the total number of rotifers per well was counted and EC10, EC20 and EC50 were 

determined by non-linear regression using a log logistic model. The respective confidence 

intervals for these endpoints were as follow: EC10 = 4.5-13.7 mg/L, EC20 = 8.8-37.8 mg/L and 

EC50 = 75-486 mg/L. No analytical monitoring was performed during the test. 

Several points can be highlighted so as to assess the validity of this test performed on 

Brachionus calyciflorus and showing the highest sensitivity. The pros and cons for the validation 

of the test are compiled in Table 3.34. 

Table 3.34 Pros and cons for the validation of the test performed with Brachionus calyciflorus

Issue Pros Cons 

Analytical monitoring The test compound is considered stable in 

similar testing conditions, during 48 hours. Under 

static conditions, the monitoring of test 

concentrations during test #7 for invertebrates 

(INERIS, 1999a) has revealed a loss of the 

substance less than 20%, after 48 hours. 

In order to prevent evaporation of the solvent or 

the test substance during the test (EGBE 

evaporates more slowly than water), the 

microplate was covered. Indeed, the French 

norm recommends that the incubation is carried 

out in a water saturated atmosphere in order to 

avoid losses of test solution by evaporation; for 

example, the microplate can be arranged in a 

closed dish at the bottom of which a film of water 

has been previously deposited. Furthermore, an 

interlaboratory test has been successfully 

performed with copper sulphate and no 

evaporation problem has been encountered. 

No analytical monitoring has been performed 

during the test. Consequently, neither a loss of 

test substance nor a loss of solvent (water) 

during the test duration could not have been 

followed. 

Table 3.34 continued overleaf 

56 
CHAPTER 3. ENVIRONMENT

Table 3.34 continued Pros and cons for the validation of the test performed with Brachionus calyciflorus 

Issue Pros Cons 

Concentration / effect 

relationship 

Glycol ethers often show non-conventional 

concentration/effect relationships. Such nonconventional curves have already been reported 

for Xenopus exposed to 2-methoxyethanol 

(Daston et al., 1991) or for Hydra attenuata

exposed to EGBE (Bowden et al., 1995). 

DeVillers et al., 2002b has also reported the 

same difficulty in finding clear relationships 

between the tested concentrations and the 

endpoints studied for their tests with 

EGME/EGMA performed on algae, rotifers, 

molluscs, daphnids and fish (some of these tests 

where repeated two or more times so as to 

ensure that they reached their respective 

conditions of acceptance). The authors also 

report that similar conclusions can be drawn from 

an analysis of published papers in which the 

ranges of tested concentrations of EGME are 

given. These tests were performed with 

Pimephales promelas and Drosophila 

melanogaster (Daston et al., 1991), D. 

melanogaster again in Lynch and Toraason, 

1996. 

The test results show a non-conventional 

concentration/effect relationship (a factor of 23 

can be calculated between EC50 and EC10). 

The studies from Daston et al., 1991 and 

Bowden et al., 1995 also show a conventional 

response for methoxyethanol with Hydra and, 

whilst the EGBE response is flat, it is still of 

conventional shape, which is not the case with 

the Brachionus. 

Even if there are indications that the non 

conventional dose-response curves may be a 

general reaction to glycol ethers, no explanation 

has been found to this phenomenon. 

Normalisation The test has been carried out following a French 

norm (NF T 90-377). An ISO norm is also in 

preparation. 

The methodology used for the test with rotifers is 

not as well normalised as the one for standard 

tests usually used for effect assessments of 

chemicals. 

Higher sensitivity For EGBE, the Brachionus test shows the 

highest sensitivity among all species tested. A 

ratio of 18.6 can be calculated between the 

ECs10 for Daphnia magna and Brachionus

indicating a greater sensitivity of Brachionus

compared to Daphnia magna. This is not in 

accordance with the trend generally observed 

when sensitivities of both species are compared, 

giving a ration of 2 between Brachionus and 

Daphnia indicating that Daphnia would be twice 

as sensitive as Brachionus (RIVM, 2004). 

Oxygenation of the 

test media 

Daphnia 48-hour test can be taken for 

comparison with the 48-hour Brachionus test. In 

an acute daphnids test (INERIS, 1999a), the 

measured dissolved oxygen concentration at the 

end of the test was between 5.3 and 6.8 mg/L. It 

can be expected that a similar DO level was 

maintained during the rotifer test. Although test 

solution volumes are lower in the rotifer test, both 

tests show similar conditions and the same 

duration 

Since no aeration took place and the test wells 

were covered during the test phase, it should 

have been reported how appropriate oxygen 

concentration was maintained during the whole 

test phase. This was not done. In fact, because 

EGBE is readily biodegradable, oxygen depletion 

is possible, which would mean that the low 

NOECs of the Brachionus calyciflorus studies 

were not caused by the toxic properties of 

EGBE. This may not occur to a significant 

degree over the timescale of the study but, with 

no oxygen data, it is not possible to be certain. 

Considering all the elements from Table 3.34 no clear reason can be found to fully invalidate the 

test. However, considering all the elements highlighted that have triggered off some concerns for 

the validation of this test, this study will be excluded from the PNEC derivation. This decision is 

also supported by the availability of standard toxicity test results such as the one performed with 

Daphnia magna (test #2). 

57
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Test #2 performed by INERIS, 1999d on Daphnia magna according to OECD guideline No 211, 

revealed a 21-day NOEC of 100 mg/L. Five concentrations (0.1, 1, 10, 100 and 500 mg/L + 

control) were tested and solutions were renewed three times a week. Other test conditions were 

as follows: pH = 7.5-7.8, temperature = 20 +/- 2°C, DO ranged between 6.9 and 8.2 mg/L and 

water hardness was 220 mg/L (CaCO3). A partial analytical monitoring has been performed, i.e. 

only for concentrations of 100 and 500 mg/L, and loss of substance over test duration was 

ranging between 3 and 14%. The confidence intervals for the endpoints measured were as 

follow: EC10 = 110-140 mg/L, EC20 = 150-180 mg/L and EC50 = 282-300 mg/L. 

DeVillers et al., 2003 – test #3 - have tested the toxicity of EGBE on Ceriodaphnia dubia

according to the draft of the method AFNOR NF T90-376 (equivalent to OECD guideline 

No 211). An EC10 of 134.9 mg/L has been determined after seven days. One daphnid was 

disposed per container, in ten replicates. The parental mortality and the number of offspring per 

living parent were used as endpoints. Other test conditions are shown here: eight concentrations 

plus control were tested at a temperature of 23 +/- 1°C and pH = 8-9, DO = 8.1-8.3 mg/L and 

water hardness was 200 +/- 40 mg/L (CaCO3). There was no analytical monitoring because 

preliminary studies showed that EGBE was stable at least during 24 hours, i.e. between two 

renewals. Furthermore, the dose – response curve was not clear and the 95% confidence interval 

for the EC10 ranged between 25.3 and 147.4 mg/L. A NOEC (55.6 mg/L) and an EC50

(138.2 mg/L) have also been calculated in another report (INERIS, 1999c) but methods used for 

calculation tend to give a preference to the EC10 value. 

3.2.1.3 Algae 

Toxicity tests for EGBE are summarised in Table 3.35. 

Table 3.35 Algae toxicity data for EGBE 

Test # Species Duration Endpoint Result 

(mg/L) 

Method References Validity 

1 Microcystis 

aeruginosa 

8 days EC3

(biomass) 

35 Cell multiplication 

inhibition test 

Bringmann and 

Kühn, 1976 

Not useable for 

PNEC derivation 

2 Pseudokirchn

eriella 

subcapitata 

72 hours NOEC 286 OECD guideline 

n°201 

INERIS, 1999e Valid 

3 Selenastrum 

capricornutum 

7 days NOEC 

LOEC 

EC50

125 

250 

>1,000 

US EPA 600/9-78-

018 

Dill and 

Milazzo, 1988 

Not useable for 

PNEC derivation 

4 Scenedesmus 

quadricauda 

8 days EC3

(biomass) 

900 Cell multiplication 

inhibition test 

Bringmann and 

Kühn, 1978 

Not useable for 

PNEC derivation 

Note: in some tests performed by Bringmann, TGK (Toxische GrenzKonzentration) values were 

established. As the percentage effect at the TGK is 3 or 5% (EC3 or EC5) these values are 

regarded as NOEC values. 

Bringmann and Kühn, 1976 have performed a EGBE toxicity test on the algae Microcystis 

aeruginosa – test #1. The test occurred in a closed static system and 14 concentrations were 

tested in duplicates (dilution with a pace of two from a stock solution). Agitation took place once 

a day and the test was conducted at 27°C in a neutral pH. The biomass was measured by 

spectrophotometry at 578 nm and an EC3 of 35 mg/L was determined after eight days. This EC3

can be considered as a NOEC. It should be noted that several drawbacks are associated with this 

58 
CHAPTER 3. ENVIRONMENT

study. Indeed, the test should have been performed during the exponential growth phase of algal 

cells. However, algal cells could not have been maintained in this phase within the duration of 

the study (7 days). There is also a deviation from more standard test results when converting the 

endpoint measured in the study (EC3) to a NOEC or EC10. Finally, the result obtained in this 

study is based on biomass and is comparable with test #2 and #3 results for biomass 

measurements too. Nevertheless, growth rate measurement is usually preferred when assessing 

the toxicity towards algae. Therefore, this study will not be used for the PNEC calculation. 

A NOEC of 286 mg/L was determined in test #2 (INERIS, 1999e) based on growth rate 

determination. This test was performed according to the OECD guideline No 201. Test 

conditions were as follow: 23 +/- 2°C, pH between 7.6 and 8.3, cell concentration = 

10,000 cells/mL, illumination was set at 7,000 lux. and test medium was subject to agitation. 

Nominal concentrations are presented hereafter: 0, 88, 159, 286, 514, 926, 1,667 and 

3,000 mg/L. There was an analytical monitoring and loss of substance over test duration ranged 

between 2 and 11%. Other endpoints have been measured during this test. A 72-hour NOEC of 

88 mg/L, an EC10 of 308 mg/L and an EC50 of 911 mg/L were determined as far as biomass is 

concerned. 

Test #3 has been performed by Dill and Milazzo, 1988 on the alga Selenastrum capricornutum. 

A 7-day NOEC of 125 mg/L has been determined according to a method from the US EPA: the 

Selenastrum capricornutum Printz algal assay EPA-600/9-78-018. Test conditions were as 

follow: pH was measured before and after the test and ranged between 7.4 and 7.8, temperature = 

24 +/- 2°C, initial cell concentration = 10,000 cells/mL, illumination was set at 4,304 +/- 

403 lux. Five concentrations (63, 125, 250, 500 and 1,000 mg/L) plus one control were tested in 

three replicates. Endpoints taken into account were the number of cells / mL and the total cell 

volume; two counts per replicate were performed with an electronic counter. It can be noticed 

that the highest concentration tested did not inhibit 50% growth. The comments made on test #1 

for its validation apply also for this test. 

Bringmann and Kühn, 1978 examined, in test #4, the toxicity of EGBE on Scenedesmus 

quadricauda. An EC3 of 900 mg/L (equivalent to a NOEC) was determined according to 

biomass measurement by spectrometry at 578 nm. Test conditions are shown hereafter: closed 

static system, neutral pH, temperature = 27°C, agitation once a day. 14 concentrations were 

tested in duplicate but no analytical monitoring took place. The comments made on test #1 for its 

validation apply also for this test. 

3.2.1.4 Micro-organisms 

EGBE toxicity studies with micro-organisms are presented in Table 3.36. 

59
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.36 Micro-organisms toxicity data for EGBE 

Test # Species Duration Endpoint Result 

(mg/L) 

Method References Validity 

1 Entosiphon 

sulcatum* 

(protozoa) 

72 hours EC5 91 Cell 

multiplication 

inhibition test 

Bringmann, 

1978 

Valid with 

restrictions 

2 Uronema 

parduzci

(protozoa)

20 hours EC5 463 Cell 

multiplication 

inhibition test 

Bringmann 

and Kühn, 

1980 

Valid with 

restrictions 

3 Pseudomon

as putida

(bacteria)

16 hours EC3 700 Cell 

multiplication 

inhibition test 

Bringmann 

and Kühn, 

1976 

Valid with 

restrictions 

4 Chilomonas 

paramecium* 

(protozoa) 

48 hours EC5 911 Cell 

multiplication 

inhibition test 

Bringmann

et al., 1980 

Valid with 

restrictions 

* Unciliated 

The toxicity of EGBE on protozoa has been evaluated in three tests (#1,2,4). To assess the 

toxicity of a chemical on micro-organisms tests conducted on ciliated protozoa are accepted but 

only one micro-organism is matching this characteristic: Uronema parduzci (#2), the others 

belong to unciliated protozoa. The cell multiplication inhibition was measured with an electronic 

cell counter. Tests were performed in a closed static system, at 25°C and neutral pH and 

14 concentrations were tested in duplicate. 

A test performed with the bacteria Pseudomonas putida (Bringmann and Kühn, 1976) is also 

quoted – test #3. The cell multiplication inhibition was retained as an endpoint (biomass 

measurement via extinction at 436 nm) and the test was performed in a closed static system 

during 16 hours, at 25°C and neutral pH. A range of 14 concentrations was tested in duplicate. 

3.2.1.5 Endocrine disruption 

A test has been conducted so as to identify a potential endocrine disrupting effect due to EGBE: 

INERIS, 2001. This study was performed according to an OECD guideline project based on the 

OECD guideline n°204. Fish (Brachydanio rerio) have been exposed during 21 days at three 

EGBE concentrations: 1, 10, 100 mg/L plus one control. No mortality was observed at any test 

concentration. Moreover the substance did not induce a higher vitellogenin synthesis (lower 

quantities than in the control were actually measured). Although a lower gonado-somatic index12

was observed at the lowest concentration this was not the case for higher ones. After an exposure 

to the positive control substance (oestradiol, an oestrogenic chemical) the brachydanio female 

presented genito-urinary buds which can normally only be found on the male. The percentage of 

apparition of genito-urinary buds was less than 10% in all treatment groups compared to 40% in 

the group of fish in contact with oestradiol. 

To conclude, EGBE has no potential for endocrine disruption. 

 

12 Ratio of weight of gonads compared to weight of total organism. 

60 
CHAPTER 3. ENVIRONMENT

3.2.1.6 PNEC for the aquatic compartment 

Three long term test results from three species representing three trophic levels will be used to 

derive the PNECaqua for EGBE. These tests are gathered in Table 3.37. 

Table 3.37 Toxicity tests retained for the derivation of PNECaqua

Species Duration Endpoint Result 

(mg/L) 

Reference Lowest short term toxicity result for 

the same trophic level 

Fish: Brachydanio 

rerio 21 days NOEC > 100 INERIS, 

2001 

Poecilia reticulata LC50 after 7 days = 

983 mg/L (Konemann, 1981). 

Invertebrates: 

Daphnia magna 21 days NOEC 100 DeVillers et al., 2002a 

Hydra attenuata EC50 after 72 hours 

=540 mg/L (Bowden et al., 1995) 

Daphnia magna EC50 after 48 hours = 

835 mg/L (Dow Chemical Co, 1979) 

Algae: 

Pseudokirchneriella 

subcapitata

72 hours NOEC 286 INERIS, 

1999e 

Pseudokirchneriella subcapitata EC50

after 72 hours = 911 mg/L (INERIS, 

1999e) 

An assessment factor of 10 is applied to the lowest test result in order to derive the PNECaqua: 

PNECaqua = 10 mg/L 

To support this assessment, we can add that EGBE is a non-polar narcotic (OECD, 1995) and 

acts with a non-specific mod of action in organisms. Although a smaller Assessment Factor can 

be used to derive a PNEC for non polar narcotics, a factor lower than 10 is not recommended by 

TGD (EC, 2003). Only additional studies, i.e. field data or model ecosystems, could result, after 

a review on a case by case basis, in a lower assessment factor. Nevertheless, the knowledge of a 

non-specific mode of action for EGBE seconds the PNECaqua proposal. 

Table 3.38 shows ecotoxicity results calculated from a log Kow of 0.8 and a molar weight of 

118.17 g/mol. The equations for non-polar narcotics given in Table 1 of Chapter 4, Part III of 

TGD (EC, 2003) were used to estimate QSAR ecotoxicity data. 

Table 3.38 QSAR ecotoxicity data for EGBE 

Species Enpoint Result (mg/L) 

Fish: Pimephales promelas 96-hour LC50 1,006 

Fish: Brachydanio rerio or Pimephales promelas 28, 32-day NOEC 113 

Daphnia: Daphnia magna 48-hour EC50 983 

Daphnia: Daphnia magna 16-day NOEC 242 

Algae: Selenastrum capricornutum 72, 96-hour EC50 1,103 

The close relationship between predicted ecotoxicity of EGBE and results from ecotoxicity tests 

confirms that EGBE does not act with a specific action manner. 

61
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

3.2.1.6.1 Calculation of the intermittent PNEC for freshwater 

For substances subject to intermittent release, long-term effects are not likely to occur. 

Consequently, the effect assessment for substances with intermittent release is based on the acute 

toxicity data set. For EGBE, these tests are gathered in Table 3.39. 

Table 3.39 Toxicity tests retained for the derivation of the intermittent PNECaqua

Species Duration Endpoint Result 

(mg/L) 

Reference 

Fish: Poecilia reticulata 7 days LC50 983 Konemann, 1981 

Invertebrates: Hydra 

attenuata 72 hours EC50 540 Bowden et al., 1995 

Algae: 

Pseudokirchneriella 

subcapitata

72 hours EC50 911 INERIS, 1999e 

Usually an assessment factor of 100 applies to the lowest result of toxicity tests performed on 

three trophic levels. It has been established that EGBE can be classified as non-polar narcotic. 

This could be a rationale for a lowering of the assessment factor. Consequently: 

PNECaqua,inter = 540 / 10 = 54 mg/L. 

3.2.1.6.2 Calculation of the PNEC for the seawater compartment 

Chronic toxicity data on three freshwater species representing three trophic levels are available. 

Only acute toxicity data on marine organisms (fish and invertebrates) are available. 

Nevertheless, the species tested do not show higher sensitivities than freshwater organisms. 

According to TGD (EC, 2003), both freshwater and seawater species are used to derive the 

PNEC for seawater. Thus the PNEC for marine organisms is determined from the lowest chronic 

test result (NOEC (21 days) = 100 mg/L on Daphnia magna) to which an assessment factor of 

100 is applied as proposed in the TGD. This gives a PNECsaltwater of 1 mg/L. 

3.2.1.6.3 Calculation of the intermittent PNEC for seawater 

An assessment factor of 1,000 should be applied on the lowest acute toxicity test result in order 

to determine an intermittent PNEC for seawater. For the same reason explained above, this factor 

could be lowered. Consequently: PNECsaltwater,inter = 540/100 = 5.4 mg/L. 

3.2.1.7 Calculation of a PNEC for the sediment compartment 

As no specific data is available for this compartment, the PNECsed will be calculated from the 

PNECaqua using the equilibrium partitioning method. 

62 
CHAPTER 3. ENVIRONMENT

Equation 3.6 Formula for the calculation of PNECsed using the equilibrium partitioning approach 

= × ×1000 −

aqua

susp

watersusp

sed PNEC

RHO

K

PNEC

PNECsed Predicted No Effect Concentration in sediment [mg/kg, wet weight] 

Ksusp-water partition coefficient suspended matter / water [~1.58 m3/m3] 

RHOsusp bulk density of wet suspended matter [~1150 kg/m3] 

PNECaqua Predicted No Effect Concentration in water [10 mg/L] 

This results in: PNECsed = 13.7 mg/kg (ww) 

3.2.1.7.1 Calculation of the PNEC for the marine sediment compartment 

No test is available on sediment dwelling organisms exposed via sediment. The PNEC for 

organisms living in marine sediments may provisionally be calculated using the equilibrium 

partitioning method from the PNEC for the marine aquatic compartment (PNECsaltwater). 

Thus, the PNECmarine sed = 1.4 mg/kg wet weight of marine sediment. 

3.2.1.8 PNEC for micro-organisms in STP 

Four EGBE toxicity tests on micro-organisms are quoted. Three tests were conducted with 

protozoa and one with an individual bacteria species. Studies testing ciliated protozoa can be 

used for the determination of a PNECmicro-organisms. That is why the test conducted on Uronema 

parduzci will be retained for the PNEC determination. 

PNECmicro-organisms = 463 mg/L. 

3.2.2 Terrestrial compartment 

Since there are no EGBE toxicity data for terrestrial organisms, no PNECsoil can be derived 

directly. Therefore, this PNEC was estimated from the PNEC for aquatic organisms using the 

equilibrium partitioning approach. 

Equation 3.7 Formula for the calculation of PNECsoil using the equilibrium partitioning approach 

= × ×1000 −

aqua

soil

watersoil

soil PNEC

RHO

K PNEC

PNECsoil Predicted No Effect Concentration in soil [mg/kg, wet weight] 

Ksoil-water partition coefficient soil water [~1.025 m3/m3] 

RHOsoil bulk density of wet soil [~1700 kg/m3] 

PNECaqua Predicted No Effect Concentration in water [10 mg/L] 

This results in: PNECsoil = 6 mg/kg (ww) 

63
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

3.2.3 Atmosphere 

No data are available in order to correctly assess the effect of EGBE for species living in the 

environment and exposed via the air compartment. In a first attempt to quantify the risk for this 

compartment, inhalation toxicity data from the human risk assessment have been reported in this 

section. 

In a repeat dose study with rats exposed by inhalation, a NOAEC value of 25 ppm (121 mg/m3) 

has been identified from a sub-chronic study. During these studies, haemolysis was consistently 

observed and sometimes associated with hepatic effects. Effects on body weight gain, on the 

forestomach and on the WBC sub-populations (T limphocyte) were also observed. In a separate 

study a LOAEC of 31 ppm (150 mg/m3) has been determined for mice and rats. Due to the 

closeness of the apparent LOAEC and NOAEC, it has been considered prudent to take the more 

conservative LOAEC of 31 ppm forward for the human health risk characterisation (with 

appropriate assessment factors). However, as the approach taken for the risk characterisation for 

the environmental section (atmospheric compartment) should be considered as a first tier, the 

NOAEC will be retained. 

3.2.4 Secondary poisoning 

No specific data available. 

64 
CHAPTER 3. ENVIRONMENT

3.3 RISK CHARACTERISATION 

3.3.1 Aquatic compartment (incl. sediment) 

STP and surface water (including seawater)

Table 3.40 presents the calculated PEC / PNEC ratios for the aquatic compartment. PECs for 

STP and surface water appear in Section 3.1.4.1 of this study whereas the corresponding PNECs 

are determined in Section 3.2.1: 463 mg/L for the PNECSTP and 10 mg/L for PNECaqua (1 mg/L 

for PNECsaltwater). 

Table 3.40 Risk characterisation for micro-organisms in STP and aquatic organisms 

Scenario RCRSTP RCRaqua RCRseawater

Production site #1 0 (3.10-5) n. a. 0 (8.10-4) 

Production site #2 0 (7.10-5) 0.001 n. a. 

Production site #3 0 (6.10-6) 0.001 n. a. 

Production site #4 0 (4.10-6) 0.001 n. a. 

Paints IF / Paints IP 0 (7.10-5) / 0 (2.10-4) 0.006 / 0.015 0.042 / 0.114 

Paints IIF & IIIF 0 (7.10-5) 0.006 0.043 

Paints IIP and Paints IIIU 0 (3.10-4) 0.002 0.011 

Paints IVF / Paints IVP - / - 0 (7.10-4) / 0 (7.10-4) 0.001 / 0.001 

Paints VF / Paints VP - / 0 (1.4.10-5) 0 (7.10-4) / 0.002 0.001 / 0.009 

Paints VIF / Paints VIP 0 (3.10-4) / 0 (1.4.10-4) 0.019 / 0.012 0.142 / 0.087 

Paints VIIF / Paints VIIP 0 (7.10-5) / 0 (1.4.10-5) 0.007 / 0.002 0.050 / 0.008 

Paints VIIIF / Paints VIIIP - / - 0 (7.10-4) / 0 (7.10-4) 0.001 / 0.001 

Paints IXF / Paints IXP 0 (3.10-5) / 0 (4.10-6) 0.003 / 0.001 0.015 / 0.002 

Detergents IF / Detergents IP 0 (2.10-5) / 0 (7.10-5) 0.002 / 0.005 0.011 / 0.037 

Detergents IIF / Detergents IIU 0 (7.10-6) / 0 (1.10-5) 0.001 / 0.002 0.005 / 0.008 

Intermediates** IP 0 (1.10-4) 0.003 0.059 

Inks IF / Inks IP 0 (1.10-4) / 0 (3.10-6) 0.011 / 0 (9.10-4) 0.084 / 0.002 

Pharm IP 0 (4.10-5) 0.001 0.021 

Elec IP 0 (1.10-4) 0.009 0.063 

Leather IP 0 (4.10-4) 0.029 0.223 

Adhesives IF / Adhesives IP 0 (3.10-5) / 0 (7.10-6) 0.002 / 0.001 0.014 / 0.005 

Agri IF / Agri IP 0 (1.10-5) / - 0.002 / 0 (7.10-4) 0.007 / 0.001 

Table 3.40 continued overleaf 

65
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.40 continued Risk characterisation for micro-organisms in STP and aquatic organisms 

Scenario RCRSTP RCRaqua RCRseawater

Oilfield IF / Oilfield IP 0 (1.10-4) / 0 (4.10-5) 0.011 / 0.004 0.084 / 0.023

Metal IF / Metal IP

Metal intermittent 

0.001 / 0 (1.10-4) 

0.003 

0.007 / 0.001 

0 (5.10-4) 

0.054 / 0.005 

0 (5.10-4) 

Cosmet IF & Fire IF / Cosmet IU 0 (5.10-5) / 0 (2.10-6) 0.004 / 0 (9.10-4) 0.027 / 0.003 

Fire IP 0 (5.10-6) 0.001 0.002 

* Captive use not included 

P Processing 

F Formulation 

U Private use 

For the risk characterisation at production and processing taking place on-site, the worst case 

based on site-specific data gives a risk characterisation ratio of 0.001 for freshwater and 8.10-4

for seawater. 

For some end uses, formulation and processing steps can be achieved at a same site. So, in order 

to characterise the total risk at such sites it is necessary to add the calculated risks for each step. 

According to Table 3.40 no risk is identified for all end uses even when both formulation and 

processing can be considered at a same site (dimmed lines of Table 3.40). 

No risk assessment could have been performed concerning the use of EGBE in oil spill 

dispersants (see Section 3.1.2.2.5). Nevertheless, no conclusion will be drawn concerning this 

use in the EU risk assessment since it has to be acknowledged that the decision whether to use a 

dispersant is made with the regulatory authorities case-by-case and on a risk benefit basis. It is 

generally accepted that dispersants create adverse effects but their use is intended to mitigate the 

even worse potential effects of environmentally hazardous oil products. For this reason, even if 

detailed composition information was available, it would still not be possible to carry out such a 

risk benefit analysis within the framework of the Existing Substances risk evaluation process. 

As far as the use of oilfield chemicals is concerned, specific regulations are in place in different 

EU countries. Because they use detailed information supplied under confidentiality terms, 

specific assessments performed under these regulations should be regarded as more precise than 

those carried out in this assessment. For instance, detailed information is provided by the 

manufactures on actual usage and emission rates. Consequently, in the knowledge that the use of 

such products is controlled by specific legislation, a Conclusion (ii) is appropriate. 

EGBE is readily biodegradable and has a low potential for accumulation in biota. Consequently, 

this substance will not remain in the environment and secondary poisoning is not expected. 

Based on the risk assessment performed for freshwater and on the lack of specific hazard 

identified for the marine environment, no risk is expected in the marine compartment. 

Sediment (freshwater and marine sediments)

As neither monitoring data on levels of EGBE in sediment nor ecotoxicity data for benthic 

organisms are available, no risk characterisation is conducted for this compartment. In addition, 

the partition coefficient between sediment and water for EGBE is low. So it can be assumed that 

the risk assessment for the sediment is covered by that for surface water (freshwater and 

seawater). 

66 
CHAPTER 3. ENVIRONMENT

Conclusions to the risk assessment for the aquatic compartment

Conclusion (ii). 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

3.3.2 Terrestrial compartment 

Risk characterisation for the terrestrial compartment has been performed calculating PEC/PNEC 

ratios. PECs for soil have been estimated in Section 3.1.4.2 and the corresponding PNEC has 

been determined in Section 3.2.2 (6 mg/kg, wet weight). Results are shown in Table 3.41. 

Table 3.41 Risk characterisation for terrestrial compartment 

Scenario RCRagricultural_soil_over_30_days RCRgrassland_over_180_days

Production site #1 0.006 0 (8.10-4) 

Production site #2 0 (2.10-4) 0 (3.10-4) 

Production site #3 0 (2.10-4) 0 (2.10-4) 

Production site #4 0 (2.10-4) 0 (2.10-4) 

Paints IF / Paints IP 0.005 / 0.014 0 (9.10-4) / 0.004 

Paints IIF and IIIF 0.005 / 0.013 0 (9.10-4) / 0.003 

Paints IIP and Paints IIIU 0.002 0 (6.10-4) 

Paints IVF / Paints IVP 0 (5.10-4) / 0.002 0 (8.10-4) / 0.003 

Paints VF / Paints VP 0 (3.10-4) / 0.001 0 (4.10-4) / 0 (3.10-4) 

Paints VIF / Paints VIP 0.016 / 0.011 0.002 / 0.003 

Paints VIIF / Paints VIIP 0.006 / 0.001 0 (9.10-4) / 0.001 

Paints VIIIF / Paints VIIIP 0 (2.10-4) / 0 (4.10-4) 0 (3.10-4) / 0 (6.10-4) 

Paints IXF / Paints IXP 0.002 / 0 (6.10-4) 0 (4.10-4) / 0 (6.10-4) 

Detergents IF / Detergents IP 0.001 / 0.004 0 (3.10-4) / 0 (7.10-4) 

Detergents IIF / Detergents IIU 0 (7.10-4) / 0.001 0 (2.10-4) / 0 (3.10-4) 

Intermediates** IP 0.032 0.004 

Inks IF / Inks IP 0.009 / 0 (9.10-4) 0.001 / 0.001 

Pharm IP 0.011 0.001 

Elec IP 0.007 0 (9.10-4) 

Leather IP 0.025 0.003 

Adhesives IF / Adhesives IP 0.002 / 0 (7.10-4) 0 (4.10-4) / 0 (2.10-4) 

Agri IF / Agri IP 0.001 / 0 (4.10-4) 0 (5.10-4) / 0 (4.10-4) 

Oilfield IF / Oilfield IP 0.009 / 0.003 0.001 / 0 (4.10-4) 

Table 3.41 continued overleaf 

67
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Table 3.41 continued Risk characterisation for terrestrial compartment 

Scenario RCRagricultural_soil_over_30_days RCRgrassland_over_180_days

Metal IF / Metal IP 0.008 / 0.002 0.001 / 0 (4.10-4) 

Cosmet IF and Fire IF / Cosmet IU 0.003 / 0 (3.10-4) 0 (5.10-4) / 0 (2.10-4) 

Fire IP 0 (4.10-4) 0 (2.10-4) 

* Captive use not included 

P Processing 

F Formulation 

U Private use 

For the risk characterisation at production and processing taking place on-site it can be noticed 

that no risk is expected. According to the figures presented in Table 3.41, no risk is expected at 

formulation, processing or private use stages. 

For some end uses, formulation and processing steps can be achieved at a same site (see Table

3.41). So, in order to characterise the total risk at such sites it is necessary to add the calculated 

risks for each step. According to Table 3.41 no risk is identified for all end uses where both 

formulation and processing are considered. 

Conclusions to the risk assessment for the terrestrial compartment:

Conclusion (ii). 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

3.3.3 Atmosphere 

No specific effect data are available in order to accurately assess the risk for the atmospheric 

compartment. However, due to the volatility of EGBE, direct emissions to air should not be 

overlooked. In a first attempt to quantify the risk for the air compartment, a NOAEC of 

121 mg/m3

 will be compared to the PECs calculated for air. This NOAEC has been determined 

in a study where rats where exposed via inhalation. 

The worst PEClocal_air,ann of 51 μg/m3 has been calculated for the scenario Paint IVP (use in can 

coatings). 

Using the methodology described in the risk characterisation for consumers, a minimal margin of 

safety (MOS) can be calculated as follows: 

Interspecies differences 0.1 (based on rationale described in the risk 

characterisation for workers part to account for relative 

species sensitivity to critical end point) 

Intraspecies differences 10 (default for consumers) 

Type of effect 1 

Confidence of the database 1 

Minimal MOS required 1 

68 
CHAPTER 3. ENVIRONMENT

The ratio between the threshold retained in the effect assessment and this worst case exposure is 

about a factor of 2,400. This rough risk characterisation for the air compartment leads to no 

concern by a sufficiently large margin that a more accurate assessment is not considered 

necessary. 

Conclusions to the risk assessment for atmosphere

Conclusion (ii).

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

3.3.4 Secondary poisoning 

Conclusions to the risk assessment for secondary poisoning

Conclusion (ii).

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

69
4 HUMAN HEALTH 

(to be added later). 

70 
5 RESULTS 

5.1 ENVIRONMENT 

Conclusions to the risk assessment for the aquatic compartment

Conclusion (ii) There is at present no need for further information and/or testing and no need 

for risk reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for the terrestrial compartment

Conclusion (ii) There is at present no need for further information and/or testing and no need 

for risk reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for the atmospheric compartment

Conclusion (ii) There is at present no need for further information and/or testing and no need 

for risk reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

Conclusions to the risk assessment for secondary poisoning

Conclusion (ii) There is at present no need for further information and/or testing and no need 

for risk reduction measures beyond those which are being applied already. 

Conclusion (ii) is applied to all levels of the life cycle of EGBE: production, formulation, 

processing and private use. 

5.2 HUMAN HEALTH 

(to be added later). 

71
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Van Nostrand Reinhold Company Inc. 1, 101-104. 

Shah JJ and Singh HB (1988) Distribution of volatile organic chemicals in outdoor and indoor air: a national VOCs 

database. Environ. Sci. Technol. 22 (12), 1381-1388. 

Shell Chemicals (2001) Data sheet for butyl oxitol, July: 4. 

SICOS (1999) Syndicat de l'Industrie Chimique Organique de Synthèse et de la biochimie. Personal 

communication., March 1999. 

Silverstein RM and Bassler GC (1963) Ultraviolet spectrometry. Spectrometric identification of organic compounds, 

John Wiley & Sons, Inc, New York, London, Sydney. 

SRC (1988) Syracuse Research Corporation calculated values. 

Staples CA, Boatman RJ and Cano ML (1998) Ethylene glycol ethers: an environmental risk assessment. 

Chemosphere 36 (7), 1585-1613. 

Stephan CE (1974) Methods for acute toxicity tests with fish, macroinvertebrates and amphibians, July 29, 1974, 

National Water Quality Laboratory, Environmental Protection Agency. 

Stonebreaker RD and Smith AJ (1980) Containment and treatment of a mixed chemical discharge from the "Valley 

of the Drums" near Louisville, Kentucky. Control of hazardous materials spills, Louisville, Kentucky. 

Tanii H and Hashimoto K (1982) Structure-toxicity relationship of acrylates and methacrylates. Toxicol. Letters 

11, 125-129. 

Thatcher M, Robson M, Henriquez LR, Karman CC and Payne G (2004) User guide for the evaluation of chemicals 

used and discharged offshore. A CIN revised CHARM III report, Version 1.3: 72 pp. 

75
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

Tuazon EC, Aschmann SM and Atkinson R (1998) Products of the gas-phase reactions of the OH radicals with 

1-methoxy-2-propanol and 2-butoxyethanol. Environ. Sci. Technol. 32 (21), 3336-3345. 

Ullmann (2000) Solvents. Ullmann's encyclopedia of industrial chemistry, VCH. A24: 476-497. 

US EPA (1975) EPA-660/3-75-009, Committee on Methods for Toxicity tests with Aquatic Organisms. 

US EPA and Syracuse Research Corporation (2001) EPI Suite, v.3.10, US EPA. 

Verschueren K (2001) Handbook of Environmental Data of Organic Chemicals, vol. New York, NY, Van Nostrand 

Reinhold Co. 

Von Viebahn C (2002) Oil spill statistics of German territories. Proc. EARSeL, Prague, Czech Republic, 4-6 June 

2002, Rotterdam. 

Waggy GT, Conway RA, Hansen JL and Blessing RL (1994) Comparison of 20-d BOD and OECD closed-bottle 

biodegradation tests. Environ. Toxicol. Chem. 13 (8), 1277-1280. 

Welchem Inc (1984) Acute toxicity studies on Wellaid 311 [2-butoxyethanol], unpublished report H-6954 submitted 

to the US EPA by Amoco Corporation, OTS 50533457; 86-920000096. Houston, Texas, 5 June 1984, MBA 

Laboratories. 

Yasuhara A, Shiraishi H, Tsuji M and Okuno T (1981) Analysis of organic substances in highly polluted river water 

by mass. Environmental Science & Technology 15 (5), 570-573. 

76 
ABBREVIATIONS 

ADI Acceptable Daily Intake 

AF Assessment Factor 

ASTM American Society for Testing and Materials 

ATP Adaptation to Technical Progress 

AUC Area Under The Curve 

B Bioaccumulation 

BBA Biologische Bundesanstalt für Land- und Forstwirtschaft 

BCF Bioconcentration Factor 

BMC Benchmark Concentration 

BMD Benchmark Dose 

BMF Biomagnification Factor 

BOD Biochemical Oxygen Demand 

bw body weight / Bw, bw

C Corrosive (Symbols and indications of danger for dangerous substances and preparations 

according to Annex II of Directive 67/548/EEC) 

CA Chromosome Aberration 

CA Competent Authority 

CAS Chemical Abstract Services 

CEC Commission of the European Communities 

CEN European Standards Organisation / European Committee for Normalisation 

CEPE European Committee for Paints and Inks 

CMR Carcinogenic, Mutagenic and toxic to Reproduction 

CNS Central Nervous System 

COD Chemical Oxygen Demand 

CSTEE Scientific Committee for Toxicity, Ecotoxicity and the Environment (DG SANCO) 

CT50 Clearance Time, elimination or depuration expressed as half-life 

d.wt dry weight / dw 

dfi daily food intake 

DG Directorate General 

DIN Deutsche Industrie Norm (German norm) 

DNA DeoxyriboNucleic Acid 

DOC Dissolved Organic Carbon 

DT50 Degradation half-life or period required for 50 percent dissipation / degradation 

DT90 Period required for 90 percent dissipation / degradation 

E Explosive (Symbols and indications of danger for dangerous substances and preparations 

according to Annex II of Directive 67/548/EEC) 

EASE Estimation and Assessment of Substance Exposure Physico-chemical properties [Model] 

77
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

EbC50 Effect Concentration measured as 50% reduction in biomass growth in algae tests 

EC European Communities 

EC10 Effect Concentration measured as 10% effect 

EC50 median Effect Concentration 

ECB European Chemicals Bureau 

ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals 

ECVAM European Centre for the Validation of Alternative Methods 

EDC Endocrine Disrupting Chemical 

EEC European Economic Communities 

EINECS European Inventory of Existing Commercial Chemical Substances 

ELINCS European List of New Chemical Substances 

EN European Norm 

EPA Environmental Protection Agency (USA) 

ErC50 Effect Concentration measured as 50% reduction in growth rate in algae tests 

ESD Emission Scenario Document 

EU European Union 

EUSES European Union System for the Evaluation of Substances [software tool in support of 

the Technical Guidance Document on risk assessment] 

F(+) (Highly) flammable (Symbols and indications of danger for dangerous substances and 

preparations according to Annex II of Directive 67/548/EEC) 

FAO Food and Agriculture Organisation of the United Nations 

FELS Fish Early Life Stage 

foc Organic carbon factor (compartment depending) 

GLP Good Laboratory Practice 

HEDSET EC/OECD Harmonised Electronic Data Set (for data collection of existing substances) 

HELCOM Helsinki Commission -Baltic Marine Environment Protection Commission 

HPLC High Pressure Liquid Chromatography 

HPVC High Production Volume Chemical (> 1000 tonnes/annum) 

IARC International Agency for Research on Cancer 

IC Industrial Category 

IC50 median Immobilisation Concentration or median Inhibitory Concentration

ILO International Labour Organisation 

IPCS International Programme on Chemical Safety 

ISO International Organisation for Standardisation 

IUCLID International Uniform Chemical Information Database (existing substances) 

IUPAC International Union for Pure and Applied Chemistry 

JEFCA Joint FAO/WHO Expert Committee on Food Additives 

JMPR Joint FAO/WHO Meeting on Pesticide Residues 

78 
ABBREVIATIONS

Koc organic carbon normalised distribution coefficient 

Kow octanol/water partition coefficient 

Kp solids-water partition coefficient 

L(E)C50 median Lethal (Effect) Concentration 

LAEL Lowest Adverse Effect Level 

LC50 median Lethal Concentration 

LD50 median Lethal Dose 

LEV Local Exhaust Ventilation 

LLNA Local Lymph Node Assay 

LOAEL Lowest Observed Adverse Effect Level 

LOEC Lowest Observed Effect Concentration 

LOED Lowest Observed Effect Dose 

LOEL Lowest Observed Effect Level 

MAC Maximum Allowable Concentration 

MATC Maximum Acceptable Toxic Concentration 

MC Main Category 

MITI Ministry of International Trade and Industry, Japan 

MOE Margin of Exposure 

MOS Margin of Safety 

MW Molecular Weight 

N Dangerous for the environment (Symbols and indications of danger for dangerous 

substances and preparations according to Annex II of Directive 67/548/EEC 

NAEL No Adverse Effect Level 

NOAEL No Observed Adverse Effect Level 

NOEL No Observed Effect Level 

NOEC No Observed Effect Concentration 

NTP National Toxicology Program (USA) 

O Oxidising (Symbols and indications of danger for dangerous substances and preparations 

according to Annex II of Directive 67/548/EEC) 

OC Organic Carbon content 

OECD Organisation for Economic Cooperation and Development 

OEL Occupational Exposure Limit 

OJ Official Journal 

OSPAR Oslo and Paris Convention for the protection of the marine environment of the Northeast 

Atlantic 

P Persistent 

PBT Persistent, Bioaccumulative and Toxic 

PBPK Physiologically Based PharmacoKinetic modelling 

79
EU RISK ASSESSMENT – 2-BUTOXYETHANOL (EGBE) FINAL REPORT, 2006 

PBTK Physiologically Based ToxicoKinetic modelling 

PEC Predicted Environmental Concentration 

pH logarithm (to the base 10) (of the hydrogen ion concentration {H+

} 

pKa logarithm (to the base 10) of the acid dissociation constant 

pKb logarithm (to the base 10) of the base dissociation constant 

PNEC Predicted No Effect Concentration 

POP Persistent Organic Pollutant 

PPE Personal Protective Equipment 

QSAR (Quantitative) Structure-Activity Relationship 

R phrases Risk phrases according to Annex III of Directive 67/548/EEC 

RAR Risk Assessment Report 

RC Risk Characterisation 

RfC Reference Concentration 

RfD Reference Dose 

RNA RiboNucleic Acid 

RPE Respiratory Protective Equipment 

RWC Reasonable Worst-Case 

S phrases Safety phrases according to Annex IV of Directive 67/548/EEC 

SAR Structure-Activity Relationships 

SBR Standardised birth ratio 

SCE Sister Chromatic Exchange 

SCHER Scientific Committee on Health and Envionment Risks 

SDS Safety Data Sheet 

SETAC Society of Environmental Toxicology And Chemistry 

SNIF Summary Notification Interchange Format (new substances) 

SSD Species Sensitivity Distribution 

STP Sewage Treatment Plant 

T(+) (Very) Toxic (Symbols and indications of danger for dangerous substances and 

preparations according to Annex II of Directive 67/548/EEC) 

TDI Tolerable Daily Intake 

TG Test Guideline 

TGD Technical Guidance Document

TNsG Technical Notes for Guidance (for Biocides) 

TNO The Netherlands Organisation for Applied Scientific Research 

ThOD Theoritical Oxygen Demand 

UC Use Category 

UDS Unscheduled DNA Synthesis 

UN United Nations 

80 
ABBREVIATIONS

UNEP United Nations Environment Programme 

US EPA Environmental Protection Agency, USA 

UV Ultraviolet Region of Spectrum 

UVCB Unknown or Variable composition, Complex reaction products of Biological material 

vB very Bioaccumulative 

VOC Volatile Organic Compound 

vP very Persistent 

vPvB very Persistent and very Bioaccumulative 

v/v volume per volume ratio 

w/w weight per weight ratio 

WHO World Health Organisation 

WWTP Waste Water Treatment Plant 

Xn Harmful (Symbols and indications of danger for dangerous substances and preparations 

according to Annex II of Directive 67/548/EEC) 

Xi Irritant (Symbols and indications of danger for dangerous substances and preparations 

according to Annex II of Directive 67/548/EEC) 

81

European Commission 

DG Joint Research Centre, Institute of Health and Consumer Protection 

European Chemicals Bureau 

EUR 22501 EN European Union Risk Assessment Report 

 2-butoxyethanol (EGBE) – Part I – Environment, Volume 68 

Editors: S.J. Munn, K. Aschberger, O. Cosgrove, S. Pakalin, A. Paya-Perez, B. Schwarz-Schulz, 

S. Vegro 

Luxembourg: Office for Official Publications of the European Communities 

2006 – VIII pp., 82 pp. – 17.0 x 24.0 cm 

EUR – Scientific and Technical Research series; ISSN 1018-5593 

The report provides the comprehensive risk assessment of the substance 2-butoxyethanol 

(EGBE). It has been prepared by France in the frame of Council Regulation (EEC) No. 793/93 on 

the evaluation and control of the risks of existing substances, following the principles for 

assessment of the risks to humans and the environment, laid down in Commission Regulation (EC) 

No. 1488/94. 

Part I – Environment 

The evaluation considers the emissions and the resulting exposure to the environment in all life 

cycle steps. Following the exposure assessment, the environmental risk characterisation for each 

protection goal in the aquatic, terrestrial and atmospheric compartment has been determined. 

The environmental risk assessment for 2-butoxyethanol (EGBE) concludes that there is at present 

no concern for the atmosphere, the aquatic ecosystem, the terrestrial ecosystem or for 

microorganisms in the sewage treatment plant. There is at present no need for further information 

and/or testing and no need for risk reduction measures beyond those which are being applied 

already. 

Part II – Human Health 

This part of the evaluation considers the emissions and the resulting exposure to human 

populations in all life cycle steps. The scenarios for occupational exposure, consumer exposure 

and humans exposed via the environment have been examined and the possible risks have 

been identified. 

This part of the evaluation will be added later. 

The mission of the JRC is to provide customer-driven scientific and technical support 

for the conception, development, implementation and monitoring of EU policies. As a 

service of the European Commission, the JRC functions as a reference centre of 

science and technology for the Union. Close to the policy-making process, it serves the 

common interest of the Member States, while being independent of special interests, 

private or national. 

European Commission – Joint Research Centre 

Institute for Health and Consumer Protection 

European Chemicals Bureau (ECB) 

European Union Risk Assessment Report 

2-butoxyethanol (EGBE) 

Part I – environment 

CAS No: 111-76-2 EINECS No: 203-905-0 

Series: 4th Priority List Volume: 68 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_HH_CLEAN

European Union Risk Assessment Report 

2-BUTOXYETHANOL 

Part II – Human Health 

CAS No: 111-76-2 

EINECS No: 203-905-0 

RISK ASSESSMENT 

FINAL APPROVED VERSION
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_HH_CLEAN

LEGAL NOTICE 

Neither the European Commission nor any person 

acting on behalf of the Commission is responsible for the use which might 

be made of the following information 

A great deal of additional information on the European Union 

is available on the Internet. 

It can be accessed through the Europa Server 

(http://europa.eu.int). 

Cataloguing data can be found at the end of this publication 

Luxembourg: Office for Official Publications of the European Communities, [ECB: 

year] 

ISBN [ECB: insert number here] 

© European Communities, [ECB: insert year here] 

Reproduction is authorised provided the source is acknowledged. 

Printed in Italy 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_HH_CLEAN

2-BUTOXYETHANOL 

Part II – Human Health 

CAS No: 111-76-2 

EINECS No: 203-905-0 

RISK ASSESSMENT 

FINAL APPROVED VERSION

August 2008 

France 

Rapporteur for the human health risk assessment of 2-BUTOXYETHANOL is BERPC. 

Contact point: 

BERPC 

60-62 rue d’hauteville 

75009 Paris 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 TITLE

RAPPORTEUR FRANCE R408_0808_HH_CLEAN

Date of Last Literature Search : 2003 

Review of report by MS Technical Experts finalised: 07-2007 

Final report: 2008 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 FOREWORD

RAPPORTEUR FRANCE R408_0808_ V HH_CLEAN

Foreword 

We are pleased to present this Risk Assessment Report which is the result of in-depth work 

carried out by experts in one Member State, working in co-operation with their counterparts in 

the other Member States, the Commission Services, Industry and public interest groups. 

The Risk Assessment was carried out in accordance with Council Regulation (EEC) 793/931

on the evaluation and control of the risks of “existing” substances. “Existing” substances are 

chemical substances in use within the European Community before September 1981 and 

listed in the European Inventory of Existing Commercial Chemical Substances. Regulation 

793/93 provides a systematic framework for the evaluation of the risks to human health and 

the environment of these substances if they are produced or imported into the Community in 

volumes above 10 tonnes per year. 

There are four overall stages in the Regulation for reducing the risks: data collection, priority 

setting, risk assessment and risk reduction. Data provided by Industry are used by Member 

States and the Commission services to determine the priority of the substances which need to 

be assessed. For each substance on a priority list, a Member State volunteers to act as 

“Rapporteur”, undertaking the in-depth Risk Assessment and recommending a strategy to 

limit the risks of exposure to the substance, if necessary. 

The methods for carrying out an in-depth Risk Assessment at Community level are laid down 

in Commission Regulation (EC) 1488/942, which is supported by a technical guidance 

document3. Normally, the “Rapporteur” and individual companies producing, importing 

and/or using the chemicals work closely together to develop a draft Risk Assessment Report, 

which is then presented at a meeting of Member State technical experts for endorsement. The 

Risk Assessment Report is then peer-reviewed by the Scientific Committee on Health and 

Environmental Risks (SCHER) which gives its opinion to the European Commission on the 

quality of the risk assessment. 

If a Risk Assessment Report concludes that measures to reduce the risks of exposure to the 

substances are needed, beyond any measures which may already be in place, the next step in 

the process is for the “Rapporteur” to develop a proposal for a strategy to limit those risks. 

The Risk Assessment Report is also presented to the Organisation for Economic Co-operation 

and Development as a contribution to the Chapter 19, Agenda 21 goals for evaluating 

chemicals, agreed at the United Nations Conference on Environment and Development, held 

in Rio de Janeiro in 1992 and confirmed in the Johannesburg Declaration on Sustainable 

Development at the World Summit on Sustainable Development, held in Johannesburg, South 

Africa in 2002. 

This Risk Assessment improves our knowledge about the risks to human health and the 

environment from exposure to chemicals. We hope you will agree that the results of this indepth study and intensive co-operation will make a worthwhile contribution to the 

Community objective of reducing the overall risks from exposure to chemicals. 

 

1 O.J. No L 084, 05/04/199 p.0001 – 0075

2 O.J. No L 161, 29/06/1994 p. 0003 – 0011

3 Technical Guidance Document, Part I – V, ISBN 92-827-801 [1234] 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 FOREWORD

RAPPORTEUR FRANCE R408_0808_ VI HH_CLEAN

Contact Details of the Rapporteur(s)

Rapporteur: France 

Human Health: BERPC 

 60/62 rue d’Hauteville 

 75010 Paris 

 France 

 Tel: + 33 1 55 07 89 89 

 Fax: + 33 1 47 70 63 13

The scientific work on the human health sections has been elaborated by: 

Effects assessment, Institut National de Recherche et de Sécurité (INRS) 

Exposure and Risk Expertise and Technical Advice Division - RCB 

Characterisation for 30 rue Olivier Noyer 

Workers 75680 Paris Cedex 14 

 France 

And 

 BERPC 

 60/62 rue d’Hauteville 

 75010 Paris 

 France 

Exposure and Risk Institut National de l'Environnement Industriel et

Characterisation for des Risques (INERIS) 

Man via the environment Département TEC 

 Parc Technologique ALATA 

 BP n° 2 

 60550 Verneuil-en-Halatte 

 France 

And 

 BERPC 

 60/62 rue d’Hauteville 

 75010 Paris 

 France 

Exposure and Risk Centre Anti-Poison de Lille 

Characterisation for 5, avenue Oscar Lambret 

Consumers 59037 Lille cedex France 
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ 1 HH_CLEAN

0 OVERALL RESULTS OF THE RISK ASSESSMENT4

CAS Number: 111-76-2 

EINECS Number: 203-905-0 

IUPAC Name: 2-butoxyethanol 

Human health 

Human health (toxicity)

Workers 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already.

Consumers 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

Conclusion (ii) applies to all scenarios and all toxicological end-points 

Humans exposed via the environment 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

Human health (physico-chemical properties)

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

 

4 Conclusion (i) There is a need for further information and/or testing. 

Conclusion (ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond 

those which are being applied already. 

Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into 

account.
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

RAPPORTEUR FRANCE R408_0808_ 2 HH_CLEAN

CONTENTS 

1 GENERAL SUBSTANCE INFORMATION ............................................................................................. 7

1.1 IDENTIFICATION OF THE SUBSTANCE...................................................................................... 7

1.2 PURITY/IMPURITIES, ADDITIVES ............................................................................................... 7

1.3 PHYSICO-CHEMICAL PROPERTIES............................................................................................. 7

1.3.1 Physical state ........................................................................................................................... 8

1.3.2 Melting point ........................................................................................................................... 8

1.3.3 Boiling point............................................................................................................................ 9

1.3.4 Relative density ....................................................................................................................... 9

1.3.5 Vapour pressure....................................................................................................................... 9

1.3.6 Surface tension ........................................................................................................................ 9

1.3.7 Water solubility ....................................................................................................................... 9

1.3.8 Partition coefficient n-octanol/water........................................................................................ 10

1.3.9 Granulometry........................................................................................................................... 10

1.3.10 Flash point ............................................................................................................................... 10

1.3.11 Autoflammability..................................................................................................................... 10

1.3.12 Flammability............................................................................................................................ 10

1.3.13 Explosive properties ................................................................................................................ 10

1.3.14 Oxidising properties................................................................................................................. 10

1.3.15 Viscosity .................................................................................................................................. 11

1.3.16 Henry’s constant...................................................................................................................... 11

1.4 CLASSIFICATION ............................................................................................................................ 12

1.4.1 Current classification (28th TPA of directive 67/548/EEC)..................................................... 12

1.4.2 Proposed classification ............................................................................................................ 12

2 GENERAL INFORMATION ON EXPOSURE ......................................................................................... 13

3 ENVIRONMENT........................................................................................................................................ 14

4 HUMAN HEALTH..................................................................................................................................... 15

4.1 HUMAN HEALTH (TOXICITY)...................................................................................................... 15

4.1.1 Exposure assessment ............................................................................................................... 15

4.1.1.1 General discussion..................................................................................................... 15

4.1.1.2 Occupational exposure............................................................................................... 16

4.1.1.2.1 Scenario 1 : Manufacture and use as intermediate.................................... 20

4.1.1.2.2 Scenario 2 : Formulation of products containing EGBE ............. 24

4.1.1.2.3 Scenario 3 : Use of products containing EGBE............................. 32

4.1.1.2.4 Summary of occupational exposure.......................................................... 54

4.1.1.3 Consumer exposure ................................................................................................... 55

4.1.1.3.1 Exposure from uses................................................................................... 55

4.1.1.3.2 Summary of consumer exposure............................................................... 63

4.1.1.4 Humans exposed via the environment....................................................................... 64

4.1.2 Effects assessment: Hazard identification and dose (concentration)- response (effect) assessment67

4.1.2.1 Toxicokinetics, metabolism and distribution............................................................. 67

4.1.2.1.1 Studies in animals..................................................................................... 67

4.1.2.1.2 Studies in humans..................................................................................... 87

4.1.2.1.3 Other data ................................................................................................. 94

4.1.2.1.4 Summary of toxicokinetics, metabolism and distribution......................... 101

4.1.2.2 Acute toxicity ............................................................................................................ 103

4.1.2.2.1 Studies in animals..................................................................................... 103
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

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4.1.2.2.2 Data from human exposure....................................................................... 120

4.1.2.2.3 Specific toxicity: haematotoxicity ............................................................ 125

4.1.2.3 Irritation 153

4.1.2.3.1 Skin........................................................................................................... 153

4.1.2.3.2 Eye............................................................................................................ 155

4.1.2.3.3 Respiratory tract........................................................................................ 160

4.1.2.4 Corrosivity................................................................................................................. 161

4.1.2.5 Sensitisation............................................................................................................... 161

4.1.2.5.1 Studies in animals..................................................................................... 161

4.1.2.5.2 Studies in humans..................................................................................... 162

4.1.2.5.3 Summary of sensitisation.......................................................................... 162

4.1.2.6 Repeated dose toxicity............................................................................................... 163

4.1.2.6.1 Studies in animals..................................................................................... 163

4.1.2.6.2 Studies in humans..................................................................................... 189

4.1.2.6.3 Mechanistic studies of liver pathology ..................................................... 190

4.1.2.6.4 Mechanistic studies on forestomach pathology ........................................ 195

4.1.2.6.5 Summary of repeated dose toxicity........................................................... 199

4.1.2.7 Mutagenicity.............................................................................................................. 200

4.1.2.7.1 Studies in vitro.......................................................................................... 200

4.1.2.7.2 Studies in vivo (table 4.81) ....................................................................... 208

4.1.2.7.3 Summary of mutagenicity......................................................................... 210

4.1.2.8 Carcinogenicity.......................................................................................................... 212

4.1.2.8.1 Studies in animals..................................................................................... 212

4.1.2.8.2 Studies in humans..................................................................................... 217

PROPOSED MECHANISMS OF ACTION ASSESSED WITHIN THE IPCS FRAMEWORK.................... 229

4.1.2.9 Toxicity for reproduction........................................................................................... 235

4.1.2.9.1 Effects on fertility ..................................................................................... 235

4.1.2.9.2 Developmental toxicity............................................................................. 240

4.1.2.9.3 Summary of toxicity for reproduction ...................................................... 255

4.1.3 Risk characterisation ............................................................................................................... 256

4.1.3.1 General aspects.......................................................................................................... 256

4.1.3.2 Workers 259

4.1.3.2.1 Acute toxicity............................................................................................ 260

4.1.3.2.2 Irritation and corrosivity ........................................................................... 263

4.1.3.2.3 Sensitisation.............................................................................................. 265

4.1.3.2.4 Repeated dose toxicity.............................................................................. 265

4.1.3.2.5 Mutagenicity............................................................................................. 270

4.1.3.2.6 Carcinogenicity......................................................................................... 270

4.1.3.2.7 Toxicity for reproduction.......................................................................... 270

4.1.3.2.8 Summary of risk characterisation for workers.......................................... 273

4.1.3.3 Consumers................................................................................................................. 273

4.1.3.3.1 Acute toxicity............................................................................................ 273

4.1.3.3.2 Irritation and corrosivity ........................................................................... 274

4.1.3.3.3 Sensitisation.............................................................................................. 275

4.1.3.3.4 Repeated dose toxicity.............................................................................. 275

4.1.3.3.5 Mutagenicity............................................................................................. 278

4.1.3.3.6 Carcinogenicity......................................................................................... 278

4.1.3.3.7 Toxicity for reproduction.......................................................................... 279

4.1.3.4 Humans exposed via the environment....................................................................... 280

4.1.3.4.1 Summary of risk characterisation for exposure via the environment........ 280

4.2 HUMAN HEALTH (PHYSICO-CHEMICAL PROPERTIES) ......................................................... 280

5 RESULTS ................................................................................................................................................... 281

5.1 INTRODUCTION .............................................................................................................................. 281

5.2 ENVIRONMENT ............................................................................................................................... 281
EU RISK ASSESSMENT –2-BUTOXYETHANOL CAS 111-76-2 CONTENTS

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5.3 HUMAN HEALTH ............................................................................................................................ 281

5.3.1 Human health (toxicity)........................................................................................................... 281

5.3.1.1 Workers 281

5.3.1.2 Consumers................................................................................................................. 281

5.3.1.3 Humans exposed via the environment....................................................................... 281

5.3.2 Human health (risks from physico-chemical properties)......................................................... 282

6 REFERENCES............................................................................................................................................ 283

ABBREVIATIONS .......................................................................................................................................... 301

Appendix A....................................................................................................................................................... 308

Apppendix B..................................................................................................................................................... 313

EUSES Calculations can be viewed as part of the report at the website of the European Chemicals Bureau: 

http://ecb.jrc.it

TABLES 

Table 1.1.1: Summary of physico-chemical properties..................................................................................... 8

Table 1.1.2: Henry’s law constant values (at 25°C) in EPI suite (US EPA and SRC, 2001)........................... 11

Table 1.1.3: Henry’s law constant values as measured in Kim et al., 2000...................................................... 11

Table 4.1: Concentration of EGBE in the main use categories in the Danish product register (2001) ............. 16

Table 4.2: Concentration of EGBE in the main use categories in the French product register SEPIA (INRS, 

2003) ................................................................................................................................................................. 16

Table 4.3: OEL values (Ariel Research, 2003) ................................................................................................. 19

Table 4.4: Biological exposure levels (BEL) .................................................................................................... 20

Table 4.5: Measured inhalation exposure for EGBE manufacture (SIDS, 1996, slightly modified with NICNAS, 

1996) ................................................................................................................................................................. 21

Table 4.6: Personal air measurements in Producer 1 enterprises (1995-2000) ................................................. 22

Table 4.7: Personal air measurements in Producer 2 plant (1995-2000)........................................................... 22

Table 4.8: Personal air measurements in Producer 3 plant (2002).................................................................... 23

Table 4.9: Distribution of potential exposure duration (NICNAS, 1996) ......................................................... 25

Table 4.10: Exposure frequency and duration in the EU paints and inks manufacturing industry (CEPE, 2002) 26

Table 4.11: Atmospheric and biological monitoring during formulation of products (8 hr-TWA personal 

inhalation exposure and BAA urinary concentration)....................................................................................... 26

Table 4.12: Personal inhalation exposure in the MEGA database, 1996-2000 (BGAA, 2001) ........................ 27

Table 4.13: Results of measurements of potential hand exposure to DEGBE in loading mixers and filling 

containers with products containing DEGBE (after RISKOFDERM, 2002a and Gijsbers et al., 2004) .......... 28

Table 4.14: Contents of EGBE in paints (CEPE, 2002).................................................................................... 33

Table 4.15: Relative frequencies of application techniques in painting/surface coating (CEPE, 2002) ........... 33

Table 4.16: Atmospheric and biological monitoring during painting (8 hr-TWA inhalation exposure and BAA 

urinary concentration)....................................................................................................................................... 34

Table 4.17: Personal exposure in painting activities for measurements 60-480 minutes, years 1987-1998 

(Vincent, 1999) ................................................................................................................................................. 36

Table 4.18: Personal exposure measurements in the MEGA database, 1996-2000 (BGAA, 2001) ................. 36

Table 4.19: Dermal exposure rates during brushing ( Gijsbers et al, 2004)...................................................... 38

Table 4.20: Atmospheric and biological monitoring during printing (8 hr-TWA inhalation exposure and BAA 

urinary concentration)....................................................................................................................................... 40

Table 4.21: Personal air measurements during screen printing (BP, 2002a)..................................................... 42

Table 4.22: Personal exposure in printing activities made for measurements 60-480 minutes, years 1987-1998 

(Vincent, 1999). ................................................................................................................................................ 43

Table 4.23a: Potential dermal exposure measurements during silkscreening (KRIOH, 2003) ......................... 44

Table 4.23b : Workers’internal exposures (Urany butoxyacetic acid (BAA) excretion, μmol/mol creatinine) 45

Table 4.24: Main uses of EGBE containing cleaners in Australia (NICNAS, 1996)........................................ 47

Table 4.25: Measured atmospheric exposure (TWA) in cleaning activities. .................................................... 48

Table 4.26: Exposure estimates for use of cleaning products (SIDS, 1996) ..................................................... 49

Table 4.27: Dermal exposure rates during spraying and wiping (RISKOFDERM, 2003b).............................. 50
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Table 4.28: Characterisation of exposure by job category (Vincent et al., 1993)............................................. 52

Table 4.29: Summary of proposed reasonable worst case exposures................................................................ 54

Table 4.30: summary of studies about exposure by household cleaning products............................................ 57

Table 4.31: summary of studies about exposure by indoor air.......................................................................... 59

Table 4.32: Summary of studies about exposure by paints............................................................................... 61

Table 4.33: Summary of proposed “reasonable worst-case” exposures in the main scenarios ......................... 62

Table 4.34: Summary of combined worst case scenarios................................................................................. 63

Table 4.1: exposure assessment for human via the environment (concentrations in foodstuff, air and drinking 

water and connected daily doses)...................................................................................................................... 64

Table 4.35: Average tissues concentrations of EGBE and BAA following 20 ppm exposure.......................... 66

Table 4.36: Excretion of [14C]EGBE ................................................................................................................ 67

Table 4.37: Urinary metabolites of EGBE ........................................................................................................ 68

Table 4.38: Comparison of EGBE elimination between rats and mice at the same dose level tested............... 69

Table 4.39: Comparison of BAA urinary excretion between rats and mice...................................................... 70

Table 4.40: Effect of time and dose on the excretion of BE metabolites in urine............................................. 77

Table 4.41: Quantities of EGBE analysed in the different system compartments for the 2 tested concentrations 

(5 and 10 %)...................................................................................................................................................... 88

Table 4.42: Kinetic parameters measured in the Jakasa study .......................................................................... 92

Table 4.43: Intra and inter-individual variation of dermal fluxes after dermal exposure to 50 % EGBE in water.92

Figure 4.44: First PBPK developed for EGBE.................................................................................................. 95

Figure 4.45: Improvement of the PbPk model (from Dow, 1993) .................................................................... 97

Figure 4.46: Last PbPk model (Lee et al., 1998) .............................................................................................. 99

Figure 4.47: Metabolism of EGBE (Patty, 2001).............................................................................................. 101

Table 4.48: Summary inhalation route.............................................................................................................. 104

Table 4.49: Summary acute toxicity via dermal route. ..................................................................................... 110

Table 4.50: Summary of the LD50 reported in Carpenter et al., 1956.............................................................. 113

Table 4.51: Summary of LD50 values after oral route administration.............................................................. 115

Table 4.52: Summary other routes .................................................................................................................... 118

Table 4.53: Summary human acute toxicity data.............................................................................................. 121

Table 4.54: Summary of the erythrocyte toxicity in Carpenter et al., 1956...................................................... 124

Table 4.55: Haemolytic activity of various concentrations of butoxyacetic acid incubated with erythrocytes from 

rat, human, dog and rabbit ................................................................................................................................ 125

Table 4.56: Haemolytic activity of various concentrations of EGBE incubated with erythrocytes from rat, human, 

dog and rabbit. .................................................................................................................................................. 126

Table 4.57: Haemolysis of human and rat erythrocytes by EGBE.................................................................... 126

Table 4.58: Haemolysis of human and rat erythrocytes by BAA...................................................................... 127

Table 4.59: Effects on rat blood........................................................................................................................ 131

Table 4.60: Effects on rabbit blood................................................................................................................... 132

Table 4.61: Incidence of haemoglobinuria induced in rats of various ages by gavage administration of EGBE. 134

Table 4.62: In vivo and in vitro results.............................................................................................................. 140

Table 4.63: Effects of BAA on blood from different species............................................................................ 144

Table 4.64: Effect of external sucrose on BAA-induced cell swelling ............................................................. 146

Table 4.65: Effect of replacement of external sodium with potassium on haemolysis and MCV changes induced 

by 2.0 mM BAA. .............................................................................................................................................. 147

Table 4.66: Effects of divalent cathions and Ca chelation on BAA-induced haemolysis ................................. 148

Table 4.67: Dose response relationship for BAA induced haemolysis and cell swelling ................................. 148

Table 4.68: Effect of Ca on haemolysis ............................................................................................................ 150

Table 4.69: Irritation scores in the Parent, 1992 Jacobs, 1992 study ................................................................ 155

Table 4.70: Draize scores in the Kennah study................................................................................................. 156

Table 4.70 bis: Individual score for Safepharm laboratories study, 1994b)...................................................... 157

Table 4.70 ter: Individual score for BASF study, 2000 .................................................................................... 158

Table 4.71: Experimental pattern of exposure for Gage study, 1970................................................................ 163

Table 4.71 bis: Incidence of non-neoplastic lesions in the urogenital system of male mice following 2-years 

exposure to EGBE............................................................................................................................................. 172

Table 4.72 : Summary of the studies on animals performed by inhalation route.............................................. 175

Table 4.73: Summary of the studies performed by dermal route ...................................................................... 180

Table 4.74: Summary of studies performed by oral route................................................................................. 186

Table 4.75: Effects of EGBE on indicators of oxidative stress in B6C3F1 mice, mean & standard deviation & 

percentage of the control (100 %) (Kamendulis et al., 1999) ........................................................................... 189
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Table 4.76: Effects of EGBE, BAA and ferrous sulphate on indicators of oxidative stress in male B6C3F1 mouse 

and male F344 rat hepatocytes, mean & standard deviation & percentage of the control (100 %) (Kamendulis et 

al., 1999; Park et al., 2002a) ............................................................................................................................. 193

Table 4.77: Effects of haemolysed erythrocytes (RBCs) on oxidative stress parameters in mouse hepatocytes 

(Park et al., 2002a)............................................................................................................................................ 194

Table 4.78: Halve lives and AUC in blood, saliva and tissues. (Poet et al., 2003). ......................................... 197

Table 4.79: Test for gene mutation induction in bacteria by EGBE and its metabolites................................... 201

Table 4.80: Tests for genetic and related effects in cultured mammalian cells by EGBE and its metabolites.. 204

Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and 

its metabolites ................................................................................................................................................... 205

Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and 

its metabolites ................................................................................................................................................... 206

Table 4.81: In vivo tests in mammals for the genotoxicity of EGBE and its metabolites ................................. 210

Table 4.82: Neoplastic and non-neoplastic lesions of the adrenal medulla in female F344/N rats.................. 212

Table 4.83: Neoplastic and non-neoplastic lesions of the forestomach in male and female B6C3F1 mice...... 214

Table 4.84: Neoplastic and non-neoplastic lesions of the liver in male and female B6C3F1 mice .................. 215

Table 4.85: Summary of relationship between chemicals inducing haematoxicity and haemangiosarcomas in 

B6C3F1 mice and F344 rats Incidences are for control, low, middle and high dose groups, respectively. ..... 221

Table 4.85 bis: Data on estrous cycling for rats exposed to EGBE for 13 weeks in drinking water................. 236

Table 4.85 ter: Vaginal cytology during the estrous cycle of the rat1 ............................................................... 236

Table 4.86: effects of EGBE on the development of rat embryos explanted on day of gestation and cultured for 

48h. ................................................................................................................................................................... 246

Table 4.87: Effects of BAA on the development of rat embryos explanted on day 9 of gestation and cultured for 

48 hr. ................................................................................................................................................................. 247

Table 4.88: absorption coefficients taken into account for the calculations of internal doses .......................... 254

Table 4.89: Summary of proposed reasonable worst case exposures (see also table 4.29 in & 4.1.1.2.4)........ 257

Table 4.90: Assessment factors applied for the calculation of minimal MOS for acute toxicity (for inhalation and 

dermal route)..................................................................................................................................................... 258

Table 4.91: Occupational risk assessment of EGBE for acute toxicity............................................................. 259

Table 4.91 bis: Occupational combined risk assessment for acute toxicity ...................................................... 261

Table 4.92: Occupational risk assessment of EGBE for eye and respiratory irritation by vapour exposure..... 262

Table 4.92a: Equivalent human doses for LOAEL(C) / NOAEL(C) ................................................................ 264

4.92b: Assessment factors for repeated dose toxicity ....................................................................................... 264

Table 4.92c: Risk assessment for inhalation and dermal exposure for RDT..................................................... 265

Table 4.92d: Risk characterisation for combined exposure and for RDT ......................................................... 267

Table 4.93: Assessment factors applied for the calculation of minimal MOS fertility effects.......................... 268

Table 4.93bis: Risk characterisation for reprotoxicity ...................................................................................... 270

Table 4.94: Internal dose exposure depending on scenarios ............................................................................. 271

Table 4.95: Assessment factors applied for the calculation of minimal MOS for acute toxicity and for inhalation 

and dermal route. .............................................................................................................................................. 272

Table 4.96: MOSs and conclusion for acute toxicity ........................................................................................ 272

Table 4.97: Consumer risk assessment of EGBE for eye and respiratory irritation by vapour exposure.......... 273

Table 4.97 bis: Equivalent human doses for NOAEL(C) / LOAEL(C) ............................................................ 274

Table 4.97 ter: Assessment factors for oral exposure........................................................................................ 274

Table 4.98: Internal dose exposure depending on scenarios average over a year ............................................. 275

Table 4.99: MOSs and conclusion for repeated dose toxicity........................................................................... 276

Table 4.99 bis: Risk characterisation for carcinogenicity ................................................................................. 277

Table 4.100: Assessment factors applied for the calculation of minimal MOS fertility effects........................ 277

Table 4.101: MOSs and conclusion for reproduction ....................................................................................... 277
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1 GENERAL SUBSTANCE INFORMATION 

1.1 IDENTIFICATION OF THE SUBSTANCE 

CAS Number: 111-76-2 

EINECS Number: 203-905-0 

IUPAC Name: 2-butoxyethanol 

Molecular formula: C6H14O2

Structural formula: CH3-CH2-CH2-CH2-O-CH2-CH2-OH 

Molecular weight: 118.17 g.mol-1

Synonyms: EGBE (this synonym will be used in the present study to refer to the 

chemical 2-butoxytehanol). Other synonyms: butoxyethanol ; 2-butoxy1-ethanol ; n-butoxyethanol ; butyl ethoxol ; 3-oxa-1-heptanol ; o-butyl 

ethylene glycol ; butyl glycol ; butyl monoether glycol ; ethylene glycol 

butyl ether ; 2-BE ; ethylene glycol n-butyl ether ; ethylene glycol 

monobutyl ether ; glycol butyl ether 

Commercial trade names : Dowanol EB ; Butyl Cellosolve ; Butyl 

Icinol ; Butyl Oxitol ; Eastman EB Solvent 

1.2 PURITY/IMPURITIES, ADDITIVES 

Purity : the purities quoted in IUCLID were all ≥ 99 % w/w 

Impurities : 

- as the synthesis reaction produces other glycol ethers, small concentrations of 

them can be found. 

- 2-butoxyethoxyethanol (CAS 112-34-5) ≤ 0.3 % w/w 

- 1,2-ethanediol (CAS 107-21-1) ≤ 0.3-0.5 % w/w 

- 1-butanol (CAS 71-36-3) ≤ 0.1-0.2 % w/w 

- water < 0.1-0.2 % w/w 

Additives : 0.008-0.012 % w/w 2,6-bis(1,1-dimethylethyl)-4-methylphenol (CAS 

128-37-0) added to prevent the formation of peroxides. 

1.3 PHYSICO-CHEMICAL PROPERTIES 

The physico-chemical properties are discussed below and summarised in Table 1.1. 

H3C O

OH
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Table 1.1.1: Summary of physico-chemical properties 

Property Value 

Physical state Liquid 

Melting point -73.4°C 

Boiling point 170.5°C 

Relative density 0.9 at 20°C 

Vapour pressure 1 hPa at 20°C 

Surface tension 26.6 mN/m at 20°C 

Water solubility Miscible (~1.106 mg/L) 

Partition coefficient 

n-octanol/water (log value) 

0.8 

Granulometry Not applicable 

Flash point 63.2°C 

Autoflammability 244.5°C 

Flammability Upper limit: 12.7 % (volume) 

Lower limit: 1.1 % (volume) 

Explosive properties Not explosive 

Oxidising properties No oxidising properties 

Viscosity 3.28 mPa.s at 20°C 

Henry’s constant 0.08 Pa.m3/mol at 25°C 

Conversion factors (101 kPa, 20°C) 1 ppm = 4.9 mg/m3

1 mg/m3 = 0.204 ppm 

1.3.1 Physical state 

EGBE is a neutral, colourless liquid with a very weak pleasant odour (Ullmann, 2000). An 

absolute perceptible limit of 0.1 ppm (50 % recognition = 0.35 ppm and 100 % recognition = 

0.48 ppm) was referred to EGBE (Verschueren, 2001). 

1.3.2 Melting point 

Several values are reported in literature. The value of -77°C appears in several studies 

(ECETOC, 1994 ; OECD SIAR, 1997 ; Dow, 2001) and handbooks show values ranging 

between -74.8 and -70°C (Lewis, 1999 ; Ullmann, 2000 ; Howard, 1989 ; Eastman, 2001 ; 

BASF, 2002 ; Verschueren, 2001). 

The mean of all the values found is retained for this study : -73.4°C. 
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1.3.3 Boiling point 

Most of the values found in literature are in the following range: 168-172°C (Ullmann, 2000). 

A boiling point of 170.8°C for EGBE can be found (ECETOC, 1994) which is close to those 

appearing in handbooks (170°C in Kirk-Othmer, 1983 and Verschueren, 2001). Two other 

handbooks show values between 171 and 172°C (Lewis, 1999 ; Howard, 1989). Boiling point 

of 171°C was found in technical data sheets or product information reports (BP, 2002b; Dow, 

2001). 

The mean of all the values above is 170.5°C. This value will be used for the risk evaluation. 

1.3.4 Relative density 

Several relative density values are available too. Most of them are about 0.9 (Verschueren, 

2001 - value measured at 20°C ; Merck, 1996). Technical data sheets or product information 

reports give values of 0.9011 and 0.902, at 20°C (BP, 2002b; Dow, 2001). 

A rounded value of 0.9 will be chosen for this study. 

1.3.5 Vapour pressure 

Vapour pressures ranging from 0.79 to 1.3 hPa, at 20°C have been reported. Data come from 

technical data sheets: 0.8 hPa (Shell Chemicals, 2001), 0.89 hPa (Dow, 2001), 1.3 hPa (BP, 

2002b). The same range appears in Sax (1986) and other chemical handbooks give almost the 

same values: 0.89 hPa (Verschueren, 2001), 1 hPa (Ullmann, 2000). 

The mean of these values gives a vapour pressure of 1 hPa, at 20°C. It will be used in the risk 

assessment. 

1.3.6 Surface tension 

Several values of surface tension appear in technical data sheets for commercial EGBE: Dow 

Chemical Company (2001) reports a value of 27.4 mN/m at 25°C, Eastman (2001) gives a 

value of 26.6 mN/m at 20°C and Shell Chemicals (2001) another of 28.9 mN/m. A value of 

27.4 mN/m at 25°C is also reported in a handbook (Kirk-Othmer, 1983). 

The value reported in Eastman (2001) will be retained for the study: 26.6 mN/m, at 20°C, the 

temperature recommended in the OECD guideline No 115. 

1.3.7 Water solubility 

EGBE is often reported as miscible with water in several data sheets and product information 

reports (Hoechst, 1992 and 1993 ; BP, 2002b). A solubility of about 50000 mg/L is reported 

by Eckel et al. (1996), 63500 mg/L by ASTER (1996). A value of 1.106

 mg/L has been 

measured (Riddick et al., 1985 cited in HSDB). 

EGBE can be considered as highly miscible in water and the maximum value permitted by 

EUSES (EC, 1996), 1.105

 mg/L, is retained for modelling purposes. 
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1.3.8 Partition coefficient n-octanol/water 

Several QSAR calculations of partition coefficient n-octanol/water for EGBE have been 

performed. They are presented hereafter using an ascending order of logarithmic values: 0.57 

calculated with KOWWIN v.1.66. – (US EPA and SRC, 2001) ; 0.81 (Verschueren, 2001), 

0.84 (ASTER, 1996). 

Partition coefficient measurements have been performed (Tanii and Hashimoto, 1982): 0.77 is 

the mean value of three determinations. Measurements have been carried out with gas 

chromatography. 

Other measured log Kow can be found in literature: 0.74 (Verschueren, 2001), 0.81 (Staples et 

al., 1998), 0.83 (Korenman and Dobromyslova, 1975 ; Hansch and Leo, 1985 ; Staples et al., 

1998). 

Mean of all measured data gives a log Kow of 0.8 that is retained for this study. 

1.3.9 Granulometry 

Not applicable: the substance is a liquid. 

1.3.10 Flash point 

Values reported in literature range from 60 (INRS, 1996 and Merck, 1996) to 68°C (BP, 

2002b). Both values are measured with closed cup method. Two other values were obtained 

using this method: 67°C (Shell Chemicals, 2001) and 61°C (Sax, 1986), while methods 

leading to values of 65°C (Dow Chemical, 2001) and 67°C (Ullmann, 2000) are not 

mentioned. 

The mean of the values measured using a closed cup is retained: 63.2°C. 

1.3.11 Autoflammability 

Two temperatures of autoflammability have been found in technical data sheets: 244°C (Dow 

Chemical Company, 2001) and 245°C (BP, 2002b). The mean value of 244.5°C is retained. 

1.3.12 Flammability 

Limits of flammability are reported by BP (2002b) for EGBE. The upper limit is 12.7 % 

(volume) and the lower one is 1.1 % (volume). 

1.3.13 Explosive properties 

Not explosive. 

1.3.14 Oxidising properties 

No oxidising properties. 
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1.3.15 Viscosity 

The range of viscosity values found in technical data sheets for EGBE goes from 2.9 (at 25°C, 

Dow, 2001) to 3.3 (Shell Chemicals, 2001) mPa.s. A value of 3.28 mPa.s is also reported by 

BP (2002b), at 20°C. BP (1998) also reports a range of viscosity values for several 

concentrations of EGBE in water (between 20 and 100 % volume): 2-6 mPa.s at 20°C. 

Several values have been found in handbooks: 3.26 mPa.s, at 20°C (Ullmann, 2000), 2.83 

mPa.s at 25°C (Prager, 1995) and a catalogue of commercial substances gives a viscosity of 

5.83 mPa.s, at 20°C (Merck, 1996). 

The most common values are rounded and a viscosity of 3.3 mPa.s (at 20°C) is retained for 

this study. 

1.3.16 Henry’s constant 

Different QSAR estimation methods calculated with HENRYWIN v.3.10. (US EPA and SRC, 

2001) give values of Henry’s law constant presented in Table 1.1.2. 

Table 1.1.2: Henry’s law constant values (at 25°C) in EPI suite (US EPA and SRC, 2001) 

Method Value (Pa.m3 / mole) 

Bond method 0.009 9 

Group method 0.002 1 

Experimental value quoted in EPI suite database 0.162 1 

Calculated with VP.M / WSol ratio using EPI suite estimated values (QSAR) 0.116 1 

Henry’s law constant values have also been calculated using another program (MPBPVP), at 

25°C. Depending on the method used, values of 0.01 and 0.002 Pa.m3/mol are reported (SRC, 

1988). 

Another study shows a calculated Henry’s law constant of 0.551 Pa.m3

/mol at 25°C (ASTER, 

1996). 

Henry’s law constant can also be estimated from the ratio of the vapour pressure to the water 

solubility using selected values from this study: 100 Pa for vapour pressure and 1.106

 mg/L 

for water solubility. Calculation gives a Henry’s law constant of 0.012 Pa.m3

/mol. 

Experimental measurements of Henry’s law constant have also been reported (Kim et al., 

2000). Two different methods were used to determine Henry’s law constant for the 

equilibrium partitioning of EGBE contained in an aqueous solution and the air. Results of 

measurements are presented in Table 1.1.3. 

Table 1.1.3: Henry’s law constant values as measured in Kim et al., 2000 

Bag method for equilibrium partitioning Batch stripping method 

T = 20°C ; H = 0.041 Pa.m3/mol T = 22°C ; H = 0.063 Pa.m3/mol 

T = 25°C ; H = 0.081 Pa.m3/mol T = 23°C ; H = 0.068 Pa.m3/mol 

T = 25°C ; H = 0.082 Pa.m3 T = 30°C ; H = 0.101 Pa.m /mol 3/mol 

T = 30°C ; H = 0.135 Pa.m3/mol 
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In the previous study, there is a good correlation between both method used, bag method for 

equilibrium partitioning and batch stripping method, and particularly at 25°C. Moreover, 

direct measurement of the Henry’s law constant is recommended in TGD (EC, 2003) for 

water miscible compounds. So a Henry’s law constant of 0.08 Pa.m3/mol will be used. 

1.4 CLASSIFICATION 

1.4.1 Current classification (28th ATP of directive 67/548/EEC) 

Classification: Xn; R 20/21/22 

 Xi; R 36/38 

Labelling: Xn; R 20/21/22, 36/38 

 S 2 – 36/37 - 46 

1.4.2 Proposed classification 

Unchanged.

 
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2 GENERAL INFORMATION ON EXPOSURE 
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3 ENVIRONMENT 
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4 HUMAN HEALTH 

4.1 HUMAN HEALTH (TOXICITY) 

4.1.1 Exposure assessment 

4.1.1.1 General discussion 

Humans may be exposed to EGBE at workplace, via consumer products and indirectly via the 

environment. The highest potential exposure is likely to occur during occupational exposure. 

Workers and consumers are primarily exposed via inhalation and dermal routes. EGBE is 

readily absorbed through the skin including absorption from direct contact with liquid or 

aerosol form or contact with vapours. Because this compound has a relatively low vapour 

pressure (0.1 kPa at 20°C), dermal absorption after direct contact with the liquid may be 

predominant or may contribute significantly to overall exposure.

Environmental monitoring of breathing zone or air concentrations in the work area has been 

found to be inadequate to assess overall exposures: the total exposure to EGBE should take 

into account the respiratory uptake of vapours and aerosols and the dermal absorption of 

EGBE in liquid, vapour and aerosol form. Biological monitoring of the common toxic urinary 

metabolite, 2-butoxyacetic acid (BAA), is considered necessary for a complete assessment 

(Angerer et al., 1990 ; Johanson et al., 1988 ; Sohnlein et al., 1993 ; Vincent et al., 1993). 

Limited data on urinary BAA concentrations are available for occupationally exposed groups 

of the population. Biomonitoring data in the general population are not available. 

Exposure may occur during manufacture and use as intermediate in the chemical and 

pharmaceutical industries, and during formulation and use of products. EGBE is a solvent 

used in many industrial activities or consumer applications. The main uses are in paints or 

surface coatings (solvent-based or water-based), followed by cleaners and printing inks. 

In the Swedish product register (KEMI, 2002), 882 products containing EGBE have been 

identified, of which 58 % are paints or inks, 16 % cleaning agents, 8 % antirust and 8 % 

polishing agents. 

In the Danish product register (Arbejdstilsynet, 2001), 1 204 products containing EGBE have 

been identified, of which 76 were private household products. The most common uses were 

paints and varnishes (37 %), cleaning/washing agents (20 %), surface treatment (8 %), 

corrosion inhibitors (6 %), surface active agents (3 %), adhesives/binding agents (3 %), 

solvents (2 %).

Other data extracted from the French product register SEPIA (INRS, 2003) showed that 368 

products out of the 10 345 products registered between 1997 and 2003 contained EGBE. The 

main use categories were: paints, varnishes and inks (39 %), cleaning agents (37 %) and 

products for metallurgical and mechanical sectors, e.g. grease cleaners (14 %). 

The distribution of concentration intervals in the main type of products is presented in the 

tables 4.1 and 4.2. 
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Table 4.1: Concentration of EGBE in the main use categories in the Danish product register (2001)

Content 

% Total Cleaning agents Solvents Paints Corrosion inhibitors 

Surface 

treatment 

Surface 

active 

agents 

Adhesives

[0-1] 526 51 278 23 46 4 21 

]1-5] 276 74 72 36 14 4 

]5-10] 176 56 5 53 8 10 6 

]10-20] 147 40 8 31 27 4 6 

]20-50] 51 13 6 4 8 

]50-80] 10 9 6 11 

]80-100] 17 4 12 6 

Table 4.2: Concentration of EGBE in the main use categories in the French product register SEPIA (INRS, 2003) 

Concentration 

(%) 

Products for 

metallurgical and 

mechanical sectors

Paints, 

varnishes 

and inks 

Cleaning agents 

[0-1] 2 36 11 

]1-5] 36 41 53 

]5-10] 6 41 21 

]10-20] 2 9 13 

]20-50] 4 5 

]50-80] 2 

]80-100] 

Other minor uses are reported: adhesives, metal cleaning, electronic industry, cutting oils, 

agricultural products, fire fighting foams, leather treatment, oil field chemicals and cosmetics 

(hair dyes) (SIDS, 1996 and OSPA, 2002). 

4.1.1.2 Occupational exposure 

Definitions and sources 

In this document, unless otherwise stated, the term exposure is used to denote external 

personal exposure as measured or otherwise assessed without taking into account the 

attenuating effect of any personal protective equipment (PPE) which might have been worn. 

This definition permits the effects of controls, other than PPE, to be assessed and avoids the 

considerable uncertainty associated with attempting to precisely quantify the attenuation of 

exposure brought about by the proper use of PPE. Furthermore, inappropriate use of gloves 

may even increase dermal uptake. 

The worst-case estimates generated in this exposure assessment are considered to be 

reasonable worst-case estimates, as they describe high-end or maximum exposures in feasible 

but not unrealistic situations. They are not intended to account for extreme or unusual use 

scenarios. The majority of exposures are expected to be well below these estimates. 

Available recent high-quality documentation prepared by national or international bodies has 

been largely used in this report to avoid duplication of efforts. The following documents 

deserve here a specific mention: ATSDR, 1998 ; NICNAS, 1996 ; SIDS, 1996. 
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Air sampling data are presented in this section from a number of sources and have been 

tabulated, where practicable. There is in general little or no information on the activities 

carried out while the sampling was running, the concentration of EGBE in the products, the 

control measures, and other important matters, such as sampling strategy and measurement 

methods, mean and 90th or 95th percentile of results; this is most often a serious difficulty for 

interpreting the data correctly. 

Measured exposure data are compared with that predicted from the EASE (Estimation and 

Assessment of Substance Exposure) model version 2. EASE is a general purpose predictive 

model for workplace exposure assessments. It is an electronic, knowledge based, expert 

system which is used where measured exposure data is limited or not available. The model is 

in widespread use across the European Union for the occupational exposure assessment of 

new and existing substances. 

A few measured dermal exposure data are available for EGBE or analogous substances. They 

will be considered together with modelling to predict occupational dermal exposure to EGBE. 

Many of the references stress the importance of dermal exposure, particularly during use of 

products. All sections on dermal exposure deal with liquid exposure. 

All models are based upon assumptions. Their outputs are at best approximate and may be 

wrong. EASE is only intended to give generalised exposure data; it predicts inhalation 

exposure as ranges for concentrations for continuous exposure at the process under 

consideration. Dermal exposure estimates are provided by EASE as the quantity of a product 

adhering to the skin due to a task, they do not take into account evaporation of the product. 

In the present assessment all inhalation exposures are expressed in parts per million (ppm), 

although the figures in the original publication are sometimes given as mg/m3. All mg/m3

have been converted to ppm using the following approximation: 

ppm = mg EGBE/m3 x 24.05/118.2 = mg EGBE/m3 x 0.2 

mg EGBE/m3 = ppm x 118.2/24.05 = ppm x 4.9 

A number of biological monitoring data are available for potentially exposed workers. 

Although the dataset is not extensive, they give useful indications about the total exposure and 

are presented in this section. 

Routes of exposure and relevant scenarios

The major occupational routes of exposure to EGBE are inhalation and skin contact. 

Assuming proper hygiene measures are applied, oral exposure would normally not occur in 

the workplace. 

Extremely low exposures have been reported for non-industrial office settings. Data from the 

National ambient VOCs database indicate that for 14 samples from non-industrial offices, the 

daily mean and median indoor atmospheric concentrations of EGBE were 0.214 ppb (1.03 

μg/m3

) and 0.075 ppb (0.36 μg/m3), respectively (Shah and Heyderdahl, 1988 ; Shah and 

Singh, 1988 in ATSDR, 1998). Due to the very low atmospheric exposure levels (comparable 

to the general population), non-industrial office settings will not be dealt with thereafter. 

Workers may be significantly exposed during the production of EGBE, its processing as an 

intermediate or during the formulation and use of EGBE containing products. 
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Occupational exposure assessment will be carried out through three main categories of 

scenarios: 

(a) the manufacture of EGBE and its use as an intermediate; 

(b) the formulation of products containing EGBE; 

(c) the use of products containing EGBE. 

The third category will focus on particular sub-scenarios for exposure in the most frequent 

type of use, or particular pattern of use, when relevant. 

Number of workers exposed

Due to the wide range of products containing EGBE, it is assumed that a large number of 

workers in many professional sectors may be exposed daily or occasionally. 

Data from the National Occupational Exposure Survey (NOES) conducted by NIOSH from 

1980 to 1983 indicate that an estimated 2,139,292 workers in 2,259 industry/occupation 

categories were potentially exposed to EGBE in the United States from 1981 to 1983. These 

numbers do not include workers potentially exposed to trade-name compounds that contain 

EGBE (ATSDR, 1998). In Denmark, a survey of chemical exposure in the workplace 

estimated that there were a total of 55,000 exposures to EGBE in 1989 (Brandorff et al., 

1995). Although these quantitative data are quite old, they confirm that EGBE is a widely 

used chemical with many workers potentially exposed. 

Other data specific to some activities are reported further in this section. 

Occupational exposure limits (OELs) 

OELs apply to workplace air concentrations of chemicals. They are normally intended to 

protect workers against short-term adverse effects (irritation, acute CNS effects) or long-term 

effects (e.g. on liver, lungs, kidneys, or chronic CNS effects) after months or years of 

exposure. When applicable, a "short-term exposure limit" (STEL) may be proposed or 

imposed for the first ones, and/or a "time-weighted average" (TWA) for the second. The first 

value ordinarily refers to a 15 minutes or so duration, the second to a shift (generally 

considered as an 8-hour shift). 

Table 4.3 details the OELs recommended for EGBE in various countries. They are provided 

for information and are not an indication of the level of control of exposure achieved in 

practice in workplaces. All of them add a “skin” notation. 
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Table 4.3: OEL values (Ariel Research, 2003) 

 8-hour TWA STEL, 15 min 

Country mg/m3 ppm mg/m3 ppm 

EU* 98 20 246 50 

Austria 100 20 200 40 

Belgium 98 20 246 50 

Denmark 98 20 - - 

Finland 98 20 250 50 

France 9.8 2 147.6 30 

Germany 98 20 392 80 

Ireland 100 25 

Italy 97 20 

Netherlands 100 20 246 50 

Norway 50 10 - - 

Spain 98 20 - - 

Sweden 50 10 100 20 

Switzerland 100 20 200 40 

United Kingdom 25 - 50 

USA (ACGIH) - 20 - - 

USA (NIOSH) 24 5 

USA (OSHA) 120 25 - - 

*Directive 2000/39/CE of 8 June 2000

Biological exposure levels (BELs) 

Biological monitoring is recommended when applicable to measure both inhalation and skin 

exposure and can be carried out by determination of total urinary BAA (free and conjugated). 

Sensitive methods for the quantitative analysis of total urinary BAA were developed from the 

late 1980’s and adapted by successive improvements in analytical chemistry. Sampling at the 

end of the shift would be the most appropriate (Johanson and Johnsson, 1991 ; Laitinen, 1998 

; Sakai et al., 1993). 

BELs proposed to characterize occupational exposure to EGBE are presented in table 4.4. 
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Table 4.4: Biological exposure levels (BEL)

Country Determinant Sampling time BEL Reference 

Germany BAA in urine Post-shift at the 

end of the working 

week 

100 mg/l DFG, 2002 

USA BAA in urine Post-shift 60 mg/g 

creatinine 

NIOSH, 1990 

France BAA in urine 80 mg/g 

creatinine 

INSERM, 

1999 

Finland BAA in urine 70 mmol/mol 

creatinine 

Laitenen, 

1998 

Nota :1 mmol/mol creatinine = 1.17 mg/g creatinine = 0.83 mg/l 

The correlation between inhalation exposure to EGBE and urinary excretion of BAA was 

studied with persons mainly exposed through the respiratory system (Laitinen, 1998). The 

results indicate that an 8-hr inhalation exposure of 5 ppm in the absence of dermal exposure 

would correspond to a BAA excretion of about 75 mmol/mol creatinine (88 mg/g creatinine) 

in postshift urine samples. 

4.1.1.2.1 Scenario 1 : Manufacture and use as intermediate

EGBE is manufactured continuously, either full-time during the year or within periodic 

campaigns of several weeks or months. Workers may be exposed approximately 8 hours for 5 

days per week of campaign but typically there are no personnel working constantly on the 

plant, with only occasional visits from fitters, engineers and other technical staff. The process 

is enclosed as extensive precautions are taken to prevent and minimize exposure to workers in 

the production area, due to the toxicity of the ethylene oxide feedstock (SIDS, 1996). 

There is the potential for exposure to the chemical in control rooms but this is minimal (2 to 4 

people per shift per production facility) (ECETOC, 1994). 

Exposure during transfer to tankers or drums is generally minimized by the use of automated 

filling, where the operator is segregated from the area during transfer, and the use of local 

exhaust ventilation. Accidental exposure may occur when the process is breached or when 

spills occur. Exposure may also occur during maintenance and cleaning activities; however, 

the purging of plant and equipment is generally standard practice (SIDS, 1996 and NICNAS, 

1996). 

Inhalation exposure

Measured data 

Airborne measurements provided by manufacturers of EGBE were collected for the OECD 

assessment in 1996. They are reported in table 4.5. No indication are available in the SIDS 

document whether they represent task measurements or 8 hour TWA exposures. Clarifications 

were found and they are given for the Australian plant in the NICNAS report (NICNAS, 

1996). 
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Table 4.5: Measured inhalation exposure for EGBE manufacture (SIDS, 1996, slightly modified with NICNAS, 1996) 

Manufacturer Monitoring area No of readings Mean (ppm) Maximum 

(ppm) 

BASF (EU) Production 97 0.09 1.2 

 Filling 66 1.3 5.3* 

 Technical unit 9 0.25 1.2 

 Laboratory 14 1.3 11* 

 Various 8 0.5 2.7 

BP (EU) All 311 <0.1 1.6 

Eastman (US) Production 16 <0.04 

 Tanker loading 11 <0.25 1.8 

Hüls (EU) Production 30 <0.14 <0.31 

 Filling 10 <0.14 0.22 

 Laboratory 

sampling 

20 <0.38 1.1 

ICI (Australia) All (STEL and 

TWA, personal) 

Maintenance 

(TWA, area) 

 

0.1 

1.8 

*These values are reported as outliers by the department of Work Safety (Germany). 

Older data are reported in open literature: 

- measurements in a US plant showed that the highest results were obtained during drum 

filling, with a TWA of 1.7 ppm in area monitoring. The highest personal monitoring 

reading was 0.1 ppm (Clapp et al., 1984 ; SIDS, 1996). 

- TWA personal exposures in the manufacture of EGBE in Europe were in the range of 

0.01-0.4 ppm (production), 0.03-0.3 ppm (drum filling), 0.5-2.7 ppm (blending), <1.6 

ppm (roadcar filling) (ECETOC, 1985). 

- personal exposures at a number of production sites during 1988-1993 gave results in the 

range 0.1-1.6 ppm and a mean of typically 0.13 ppm (data received from CEFIC, 1993 in 

ECETOC, 1994). 

More recent measurements were received in the framework of this assessment from 4 EU 

producers of EGBE: 

Producer 1: in 24 production and subsequent processing enterprises, analysis of 126 

measurements (personal air sampling with exposure duration >1hour) carried out during the 

period 1995-2000 leads to a 90th percentile of 0.2 ppm. The highest results are obtained in the 

laboratory with a 90th percentile of 2.4 ppm. Details are presented in table 4.6. 
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Producer 2: 198 whole-shift personal measurements carried out in the production plant 

between 1995 and 2000 result in 8-hr TWA exposure <0.1 ppm in most operations. Details 

presented in table 4.7 show that personal exposure may be quite occasionally higher. 

Producer 3: personal air measurements were carried out in 2002 in two departments of the 

production plant. Shift TWA exposures were all <0.1 ppm, task levels were slightly higher in 

the despatch department (up to 0.6 ppm). Frequency and duration of exposure of the main 

tasks were given. Details are presented in table 4.8. 

Producer 4: 5 measurements made in the production plant in 2000 result in exposure <0.05 

ppm (time span 420 to 690 minutes). No more details are available. 

Table 4.6: Personal air measurements in Producer 1 enterprises (1995-2000) 

Type of 

processing/workplace 

No of 

results 

No of 

enterprises

50 % value

(ppm) 

90 % value 

(ppm) 

95 % value

(ppm) 

All 126 24 0.08 0.2 0.38 

Production 

(closed system)

21 1 0.02 0.06 0.07 

Subsequent users 

(closed system)

75 11 0.08 0.09 0.11 

Laboratory 

(with LEV)

12 8 0.2 2.4 6.4 

Filling/storage 

(with LEV)

18 4 0.08 0.6 0.68 

Table 4.7: Personal air measurements in Producer 2 plant (1995-2000) 

Job function No of results Average 

TWA (ppm) 

Max TWA 

(ppm) 

TWA (ppm) 

5 to 95 % confidence range 

 Lower Upper 

Head of shift 106 0.2 9.0* <0.1 0.4 

Chemist 10 <0.1 <0.1 <0.1 <0.1 

Engineer 6 <0.1 <0.1 

Fitter 2 <0.1 <0.1 

Operator 57 0.3 9.7* <0.1 1.1 

Physicist 4 <0.1 <0.1 

Technician 13 <0.1 <0.1 <0.1 <0.1 

* Statistical analysis of the raw data shows that these two high measurements should be 

considered as outliers. 
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Table 4.8: Personal air measurements in Producer 3 plant (2002) 

Job type No of samples1 Sampling type Range2 (ppm) Median 

(ppm) 

90 % percentile 

(ppm) 

Outside production 

operator 

12 (3 <LOD) Shift TWA 0.002-0.03 0.01 0.024 

Mechanical 

maintenance operator 2 (1 <LOD) Task 0.01-0.136 

Mechanical 

maintenance operator

1 Task 0.01 

Road gantry operator 10 (9 <LOD) Shift TWA 0.002-0.008 0.002 0.004 

Road gantry operator 8 (7 < LOD) Task 0.006-0.052 0.022 0.042 

Road gantry operator 1 (< LOD) Task 0.003 

Road gantry operator 1 (<LOD) Task 0.038 

Ship/jetty outside 

operator 

2 (1 <LOD) Shift TWA 0.02-0.3663

 

Ship/jetty maintenance 

operator 

1 (<LOD) Task 0.064 

Ship/jetty maintenance 

operator 

1 Task 0.628 

Road/jetty outside 

operator 

2(2 < LOD) Shift Both 0.002 

Road/jetty outside 

operator 

2(2 < LOD) Task Both 0.006 

Note 1: LOD = lower limit of detection (5μg which equates to 0.042 ppm for a 24 liter 

sample). 

Note 2: for results <LOD, a value of 50 % of the LOD has been applied. 

Note 3: one task ran over two shifts. Measurement was completed for the whole task and was 

therefore split between two operators, thus exposure approximates to 50 % of the level 

recorded. 

Modelled data 

The EASE model used to predict exposure during production in closed system with full 

containment provides an exposure estimation of 0 - 0.1 ppm. If the system is breached in 

some activities (like maintenance, sampling, cleaning, filling), concentrations could be in the 

range of 0.5 - 1 ppm (non dispersive use, low tendency to become airborne, presence of 

LEV). 

Summary/statement of the exposure level 

The results of monitoring data during production show very low workplace air levels. Due to 

automated processes for feeding reactors and for drum and tanker filling as well, typical 

inhalation exposure is < 0.5 ppm (using personal sampling) in most situations. 

Higher exposure may occur during non-routine maintenance activities or during rare incidents 

such as spills or leaks. Such incidents are presented as exceedingly rare by industry, adding 
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that workers would wear PPE in such circumstances. Some measurement results show 

concentrations up to 2.7 ppm (SIDS, 1996) or 2.4 ppm (90 % of laboratory results of 

producer 1). 

SIDS (1996) retained a maximum atmospheric concentration of 3 ppm. Recent data confirm 

that exposure is lower in nearly all situations. It is proposed to adopt the value of 2.4 ppm 

(12 mg/m3

) as a reasonable worst-case TWA atmospheric concentration in production 

activities. 

Dermal exposure

Due to the enclosure of the process and control measures taken to minimize skin contact, for 

example, during transfer to tankers, dermal exposure at the plant is incidental and therefore 

likely to be low. The main source of potential exposure is during maintenance activities. 

Purging of plant and equipment is standard practice on site. Maintenance personnel are 

provided with butyl (or nitrile) rubber gloves and long-sleeved overalls, so exposure is not 

expected to be significant (NICNAS, 1996). 

Questioned on the possibility of real skin exposure, industry answered that: "Operating 

controls are such that accidental spills and leaks are extremely rare. Campaign changes would 

not normally result in breaking of confinement. Equipment for transfer to drums/trucks use 

engineering controls of exposure; equipment is designed to virtually eliminate the possibility 

of exposure during such operations. The only real possibility for exposure would be during 

maintenance, but even in such a situation, the plant is normally purged to remove hazardous 

products before containment is broken and gloves would be recommended." 

SIDS (1996) retained incidental contact time of 1 % of the working day with a 1000 cm2 skin 

area exposed (a hand and a forearm). Incidental contact with the liquid seem appropriate for 

this scenario because exposure will be occasional (not daily) and intermittent contact would 

probably overestimate the exposure. The EASE model estimated a dermal exposure in the 

range of 0 - 0.1 mg/cm2

/day (non dispersive use with direct handling and incidental contact). 

Assuming exposed skin surface area is 420 cm2 (palms of hands for consistency with other 

EU occupational risk assessments), maximum external dermal exposure would be 42 mg/day. 

This exposure will be mitigated by the use of suitable gloves. 

4.1.1.2.2 Scenario 2 : Formulation of products containing EGBE 

During the formulation of products containing EGBE, workers may be exposed during preweighing before mixing, during transfer to the mixing tank, during mixing and during the 

filling of containers with products. The whole operation is generally carried out at room 

temperature. Because of the similarity of scenarios, it will be assumed that exposure during 

formulation is the same whatever the final use of products is. 

Quite a high number of workers are likely to be exposed during formulation of products. In 

Australia, during the assessment of EGBE only in cleaning products, 82 companies were 

identified, some producing cleaning products at more than one site, with at least 200 workers 

involved in formulation (NICNAS, 1996). An enquiry was recently conducted by CEPE 

(European council of the paint, printing and artists’ colours industry) on the industrial uses of 

4 glycol ethers in paints or inks, one of which is EGBE: 109 answers were received from all 

over Europe, 65 users and 44 non-users. They comprise both multinationals and small or 

medium size enterprises from most of the EU countries. The number of workers exposed was 
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indicated by 43 user companies out of the 65, the answers were in the range of 1 to 250 and 

represent a total number of 1760 workers (CEPE, 2002). 

Exposure strongly depends on the process, which may be enclosed or relatively open. When 

the transfer of EGBE to the mixing vessel is carried out in a sealed system, potential exposure 

will be minimal, but when the operator adds the raw materials directly by drum to the mixing 

tank, exposure may be greater due to possible splashing and vapour and/or aerosol generation. 

Information obtained from the national assessment of EGBE in Australia indicated that a 

number of formulators of cleaning products containing EGBE use the latter procedure and 

that approximately 50 % of formulators carry out mixing in open top tanks, with greater 

potential for exposure (NICNAS, 1996). The situation is likely to be similar in the EU where 

many formulators are small or medium size enterprises. 

Exposure will also strongly depend on the quantities handled, the concentration in the 

products and the duration and frequency of exposure. 

While during preweighing and transfer to the mixing tank, workers are potentially exposed to 

pure EGBE, they are exposed to a more dilute form during filling. However the frequency and 

duration of exposure may be greater. As operators may be involved in both mixing and filling, 

assessment of exposure is for the formulation process as a whole.

From responses to a questionnaire sent to formulators in Australia (NICNAS, 1996), workers 

are potentially exposed to EGBE for an average of 3 hours/week (range 0.1-20). For most 

formulators, EGBE is an ingredient in only some of their products, so exposure is not 

continuous on a daily or weekly basis. From 74 responses to the questionnaire, the 

distribution for potential exposure duration was as follows: 

Table 4.9: Distribution of potential exposure duration (NICNAS, 1996) 

Duration/week Number % 

< 1 hour 30 41 

1 hour 11 15 

2 hours 11 14 

3-4 hours 8 11 

5-8 hours 6 8 

Greater than 8 hours 8 11 

This suggests that 89% of formulators use EGBE for 8 hours or less per week. 

Information about exposure frequency and duration has also been recently collected in the EU 

by CEPE (table 4.10) which suggests more frequent and/or longer exposures. 
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Table 4.10: Exposure frequency and duration in the EU paints and inks manufacturing industry (CEPE, 2002) 

 Exposure in days/year 

(41 answers) 

Exposure in hours/day 

(42 answers) 

Arithmetic mean 173 5.1 

Median 200 6.0 

Range 2-250 0.5-8 

Inhalation exposure

Measured data 

There are a few measured data published for assessment of exposure during formulation. 

They are presented in table 4.11. Control measures are generally unknown.

Table 4.11: Atmospheric and biological monitoring during formulation of products (8 hr-TWA personal inhalation 

exposure and BAA urinary concentration) 

Products 

No 

of samples 

Mean (range) 

(ppm) 

No of postshift urinary 

samples 

Mean (range) 

(mg/g creatinine) d

Source 

Printing 

inks 

9 <1 Winchester, 

1985 

Varnishes (12)a

 1.1 (0.1-8.1) (12)a 14.8 (0.8-42.7) 

(post-shift) 

Angerer et al.,

1990 

(12)a 0.5 (<0.1- 1.4) b (12)a 0.30 (<0.03-1.83) b 

Varnishes (post-shift)

(12)a 0.6 (<0.1- 1) b (12)a 23.1 (1.13-85.4) c 

(post-shift)

Söhnlein et al.,

1993 

Paints 4 1.26 (0.41-3.13) Foo et al., 

1994 

Unknown 15 0.7 (max 1.5) SIDS, 1996 

(industry) 

Paints 328 0.4 (max 44.7) 300 3.9 (<2-59.6) 

(post-shift) 

Vincent et al., 

1996 

Paints 179 0.1 (0.0-1.4) 

 Wesolowski 

and Gromiec, 

1997 

a

 No of workers (No of samples not stated) 

b

 Measurements on Monday 

c

 Measurements on Tuesday 

d

 BAA mg/l converted into BAA mg/g creatinine using the relation mentioned in nota of 

table 4.4 

In the inquiry of CEPE (2002), 20 facilities out of the 65 EGBE users who answered the 

questionnaire said they performed workplace monitoring (11 replied negatively to the 
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question while the others did not answer at all). Twenty seven users indicated typical 

concentrations: 10 answers were <0.2 ppm, 5 in the range 0.2-2 ppm and 6 in the range 2-

5 ppm (10-25 mg/m3). No details are available. 

Information from database 

In the German MEGA database, 165 exposure measurements have been registered between 

1996 and 2000, which were obtained in the paint formulation industry. The results 

(measurement values with an exposure duration ≥1 hour and a sampling duration ≥1 hour 

converted to 8-hour weighted averages) are presented in table 4.12. When possible, a 

distinction is made on the basis of whether or not control measures (LEV) were taken. In this 

regard, the results present an apparent paradox that the workplaces with LEV frequently do 

not exhibit lower exposures than those without LEV and the exposures may even be higher. 

Technical measures are mostly taken in place where the situation may result in a higher 

release of vapours, for instance when large quantities of substance are handled or when 

process occurs at high temperature. By contrast, the release is comparatively low during use 

of small quantities or processing at ambient temperature. In most cases, control measures 

create a situation where the exposure level of workplaces with large release approximately 

reaches the level of workplaces with only low release but without control measures. 

Table 4.12: Personal inhalation exposure in the MEGA database, 1996-2000 (BGAA, 2001) 

Paint production 

activity No of results No of companies 

50% 

value 

(ppm) 

75 % 

value 

(ppm) 

90 % 

value 

(ppm) 

95 % 

value 

(ppm) 

Raw EGBE 

handling, mixing 

and filling 

- without LEV 

- with LEV 

165 

71 

92 

39 

23 

28 

a 

a 

a 

a 

a 

a 

1.4. 

1.9 

1.4 

2.4 

2.5 

2.4 

Cleaning of 

containers 

- without LEV 

- with LEV 

74 

23 

49 

36 

15 

23 

a 

a 

a 

a 

a 

a 

2.7 

3.2 

2.2 

7.6 

6.8 

6.6 

a : measurement value < analytical determination limit 

Modelled data 

Using the EASE model (non dispersive use, low tendency to become airborne), the exposure 

estimate would be in the range of 0.5-1 ppm with LEV and 10-20 ppm in case of direct 

handling with dilution ventilation. 

Summary/statement of the exposure level 

SIDS (1996) and NICNAS (1996) estimated that exposure during formulation of products 

would be 2 ppm for formulations including up to 10 % EGBE, and 10 ppm for those including 

30 % or more. As very few measured data were available for formulation, this estimate was 

based on monitoring data of Vincent et al. (1993) and Salisbury and Bennett (1987) during 

cleaning activities. 
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Although some of the published data are limited, results during formulation show that 

airborne concentrations are generally low. Recent data provided by industry indicate that 

exposure would not be higher than 5 ppm but very little information is available to the context 

of this measurements. Based on the 90th percentile of the MEGA database values, we propose 

in a first approach that worst case inhalation exposure during formulation of products 

containing EGBE would be 3.2 ppm (15.7 mg/m3). Typical exposure levels are probably 

much lower (<1 ppm). 

Information presented in tables 4.9 and 4.10 show that frequency and duration of exposure 

may considerably vary. A continuous exposure for full shift (8 hours per day) will be 

assumed, although the data available suggest that this is unlikely to be a daily exposure. 

Dermal exposure

Measurements

As part of the “RISKOFDERM” project, a study was performed by the TNO 

(RISKOFDERM, 2002a ; Gijsbers et al., 2004) to directly assess dermal exposure to products 

containing 2-(2-butoxyethoxy)ethanol (DEGBE), a chemical of the same family as EGBE, but 

much less volatile (vapour pressure 2.7 Pa at 20 °C compared to 100 Pa at 20 °C for EGBE). 

Hand exposure was measured during loading (typically at the beginning of the formulation 

process, handled product is the substance, short task duration ranged between 1 and 15 

minutes) and during filling (typically at the end of the formulation process, handled product is 

the formulation, task duration ranged between 22 and 125 minutes). The measurements were 

made using cotton sampling gloves which were worn over new protective gloves, where 

present. Exposure was mainly due to exposure on the hands. The most important source of 

variabilitywas due to between-company variability, rather than to either between-worker or 

within-worker variability. Results (given in DEGBE and product) are presented in table 4.13. 

Table 4.13: Results of measurements of potential hand exposure to DEGBE in loading mixers and filling containers 

with products containing DEGBE (after RISKOFDERM, 2002a and Gijsbers et al., 2004) 

Exposure to DEGBE 

or product 

N Range 

(mg) 

AM 

(mg) 

GM 

(mg) 

GSD 

(mg) 

AM 

(μg/cm2

/min) 

GM 

(μg/cm2/min) 

GSD 

(μg/cm2/min) 

Loading (pure DEGBE) 

Hands DEGBE 28 0.28-28300.0 3313.6 218.9 19.9 727.4 52.9 17.2 

Hands Product* 28 0.31-27745.1 3215.0 217.0 19.3 708.8 52.4 16.7 

Filling (all data) 

Hands DEGBE 30 0.062 – 19000.0 1955.8 35.9 42.0 45.1 0.75 42.6 

Hands Product* 30 4.1 – 18269.2 2726.6 555.4 9.4 58.5 11.5 9.6 

Filling (only products < 10% DEGBE); data not published, but calculated from original data 

Hands Product* 21 4.1-11146 1216 249 8.5 15 4.1 7.6 

N = number of measurements 

AM = arithmetic average 

GM = geometric average 

GSD = geometric standard deviation) 

* recalculated towards the full product by dividing the value measured for DEGBE by the fraction of DEGBE as analysed in the product 
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The 90th percentile from the measured data for loading was approximately 11,000 mg on 820 

cm

2

 (expressed as total product), approximately 11,100 mg on 820 cm2 for filling (all 

products, including (almost pure) DEGBE) and approximately 3,300 mg for filling of 

products containing less than 10% of DEGBE (not published data, derived from original 

data). This value would lead to a level of 330 mg for DEGBE if the percentage of DEGBE 

would be 10%. It appears that the situations with handling (almost) pure products lead to 

higher exposure levels. That can be caused by the fact that products with small percentages of 

DEGBE, such as paints can be packaged in cans by highly automated equipment, while 

(almost) pure DEGBE is often packaged in larger containers with more handling of the 

container by the workers. 

Although assessed in a direct way and in actual working situations, these results should not be 

given too much weight for the main following reasons: 

- this is an isolated study, and it is known that there is an extreme variability in skin (and 

especially hand) exposure (Kromhout et al., 2004), depending of a number of most often 

qualitative factors that have been divided in six categories (Marquart et al., 2003); 

- measurement durations are relatively short (range 1-139 min, arithmetic mean range 6-

74 min, all tasks counfounded; Gijsbers et al., 2004) compared to a shift, so the results may 

represent rather task-associated exposure measurements than shift exposure assessments. 

- although from the same chemical family as EGBE, DEGBE has very different 

physicochemical properties, among which volatility, viscosity and the octanol/water partition 

coefficient may play a significant role. 

Biomonitoring studies

In this context, biomonitoring studies might be considered as an alternative means to assess 

skin exposure because: i) biomonitoring allows an assessment of the exposure on the whole 

shift, although it integrates dermal and inhalation routes; ii) several studies have been 

published on EGBE itself, so there is no need to extrapolate from other more or less related 

chemicals with the corresponding uncertainties. Technically, only studies where total (i.e. free 

and conjugated) urinary butoxyacetic acid (BAA) has been evaluated are sufficiently reliable 

(Jones and Cocker, 2003; Franks et al., 2006). In order to properly correct for EGBE 

absorption via the inhalation route, the latter must have been separately assessed on the same 

day and for the same worker and task as for dermal exposure. Difficulties may however arise, 

among which: i) too short sampling times when compared to a shift (e.g. more than 50% 

workers are exposed for 1 hour or less, according to table 4.9); ii) sampling conditions often 

unspecified (e.g. was it area sampling or personal sampling? were there incidents when 

sampling was running? how was the mean inhalation exposure concentration calculated?); 

iii) some skin absorption of vapours may occur (up to 39% of the total uptake in extreme 

conditions, after Jones et al., 2003); (note: these difficulties may lead to an overestimation of 

inhalation exposure with the associated underestimation of skin exposure) iv) different values 

for the urinary excretion of total BAA per ppm EGBE inhalation exposure have been 

proposed (e.g. 12 mmol/mol creat per ppm, Laitinen et al., 1998; 15 mmol/mol creat per ppm, 

Laitinen, 1998; 10 mmol/mol creat per ppm, Franks et al., 2006). 

Keeping these difficulties in mind, evaluations are tentatively proposed, based on biological 

monitoring with EGBE in real working conditions. If A is the result of biomonitoring (total 

BAA, mg/g creatinine) and B the corresponding inhalation exposure (ppm), a “dermal 

equivalent” (mg/day) may be evaluated using the formula: 

[(A x 5/88) – B] x 88/5 /0.6 [which is equal to (5A – 88B) / 3 ] 
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where 5 and 88 are the correspondence between inhalation exposure (5 ppm during a shift) 

and the excreted BAA (88 mg/g creatinine, post shift; after Laitinen, 1998), and 0.6 is the 

absorption efficiency by inhalation. 

Among the studies from Angerer et al. (1990), Söhnlein et al. (1993) and Vincent et al.

(1996), only peak measurements from Söhnlein show significant dermal absorption 

(85.4 mg/g creatinine with 1 ppm as a peak measured inhalation exposure), leading to a 

dermal equivalent absorption of 113 mg/day, that is to say a dermal load of 377 mg/day 

assuming 30% absorption. There is a high proportion of results entirely explained by the inhalation 

route or even leading to calculation of a negative dermal exposure, i.e. the biological monitoring 

results are (much) lower than would be expected due to the inhalation exposure data. This suggests a 

negative bias, especially for a chemical being known to be easily absorbed through the skin; 

this may originate from the many unknowns in sampling conditions, as already mentioned, 

and also from remaining uncertainties in the biological disposition of 2-BE (Franks et al., 

2006). 

If this bias is estimated as 30% (Franks et al., 2006, estimate a 31% discrepancy solely due to 

sampling time differences), the dermal load would be re-assessed as ca. 500 mg/day.

Modelling 

Considering the process and the tasks where exposure may occur, SIDS (1996) retained 

intermittent contact time of 20 % of the working day with a 1000 cm2 skin area exposed (a 

hand and a forearm). 

Taking into account the same assumption, the EASE model estimates a dermal exposure in 

the range of 0.1-1 mg/cm2/day (non dispersive use with direct handling and intermittent 

contact). Assuming exposed skin surface area is 420 cm2 (for consistency with other EU 

occupational risk assessments and default assumptions recommended in table 3 of Appendix I 

of the Technical Guidance), maximum external exposure would be: 

- 42-420 mg/day for loading (pure substance) 

- 21-210 mg/day for filling (assuming 50% EGBE in the product as a worst case : in 

most situation, EGBE content will be much lower) 

Assessment 

Dermal exposure can occur during part of the working day during loading of mixers (short 

periods, up to approximately 20 minutes) and during packaging of products containing EGBE 

(up to 120 minutes per day). Measured data for this scenario for EGBE are not available. Data 

from DEGBE for similar processes may be relevant. The measured values, using cotton 

gloves as samplers, for DEGBE are generally high compared to the estimates by EASE. 

Geometric mean exposure levels to products, for situations where formulations were made, so 

excluding the filling of (almost pure) DEGBE, were in the order of 200-250 mg and the 90th

percentile was in the order of 3,300 mg for filling of products with less than 10% DEGBE and 

11,000 mg for loading of DEGBE into mixers, both on 820 cm2

. The measured data for 

DEGBE may be an overestimate of potential dermal exposure for EGBE for two reasons. 

Firstly, the measurement method may have led to overestimation of dermal exposure, because 

cotton gloves are considered to retain more liquid than the skin would do. Secondly, EGBE is 

much more volatile than DEGBE and therefore, more of the substance may evaporate from 

the skin and not be available for uptake. The effect of both factors is difficult to estimate. The 
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estimate based on measured data is substantially higher than that based on EASE that is 

considered a weak model for dermal exposure. Although the measured data have a number of 

uncertainties, they should not be disregarded for risk characterisation. Therefore, a rounded 

value of 10,000 mg EGBE/day will be used for the loading step of the process. In the 

evaluation of the Margin of Safety (MOS) it will be considered that this may be a (substantial) 

overestimation. 

For the purpose of determining the evaporation time, the following equation can be used 

(TGD Appendix I.E): 

T(s)= (mRT/MβpA)K 

This equation leads for EGBE to an estimate of evaporation time of 3 hours, with the 

following input values: m = 10 000 mg, R = 8.134 J.K-1.mol-1, T = 305 Kelvin, K = 3.6 * 104

, 

M = 118.17, β = 8.7 m.h-1 (default), p = 100 Pa, A = 820 cm2. This indicates that in 

approximately 3 hours all EGBE would be evaporated from the skin.. For comparison, the 

same calculation with DEGBE (vapour pressure = 2.7 Pa) leads to an evaporation time of 

more than 100 hours. 

The filling of packages with products leads to substantially lower exposure levels. Assuming 

a product with 50% EGBE and a 90th percentile exposure level for the product of 3,300 mg 

leads to a reasonable worst case exposure level of approximately 1,600 for EGBE. The related 

evaporation time would be approximately half an hour. 

Conservatively, it may be assumed that both tasks are done by the same workers, leading to a 

total exposure of approximately 11 600 mg that will take approximately 3,5 hours to 

evaporate from the skin. Due to the sampling method for the measured data and the higher 

vapour pressure of EGBE compared to DEGBE, these are considered to be overestimates of 

the real exposure levels, which should be taken into account in the evaluation of the MOS. 

Biological monitoring data

On the one hand, exposure data have been obtained in real use conditions, which is a very 

strong argument to take them in consideration, but in an isolated work and with a chemical 

presenting physico-chemical properties very different from those of EGBE, which are strong 

arguments not to accept them at face value. On the other hand, limited biomonitoring data are 

available for EGBE itself, also in real conditions of use, with the advantage of not requiring 

any extrapolation of uncertain validity from another chemical substance. Its main weakness 

lies in the important penetration of EGBE through the inhalation route, which must then be 

assessed with sufficient reliability. Since neither of these sources can be ignored, a final 

assessment must be based on both. The difficulty to do so lies here in assessments that differ 

by a factor of 23 (11,600 mg/day for Risk of Derm, 500 mg/day for biomonitoring). This 

factor is in fact relatively small considering the extreme variability evidenced in assessments 

made by RISKOFDERM in a variety of situations. Moreover, there are good reasons to think 

that data obtained with DEGBE may be overestimates when transposed to EGBE. On the 

other hand, based on extensive data from this RISKOFDERM project, Marquart et al. (2006, 

table 5) consider that 550 mg is more a typical exposure value than a reasonable worst-case. 

According to them, “individual mean dermal exposure levels were on average within a 4-fold 

range”. In this context, it is proposed to re-evaluate the assessment of the limited data based 

on biomonitoring by a factor of 4. This eventually leads to propose a skin penetration of 

2,000 mg/day for this scenario. 
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4.1.1.2.3 Scenario 3 : Use of products containing EGBE 

EGBE is used in a wide variety of products. The following scenarios are considered as 

representative: 

- use of paints and coatings 

- use of printing inks 

- use of cleaners 

Cleaning related to painting and printing activities are included in the first and second 

scenarios. 

Measured exposure levels in general 

Exposures to EGBE were found in a 1983 survey of 336 Belgian businesses (Veulemans et 

al., 1987a). In this study, EGBE was found in 25 of 94 air samples from sites using printing 

pastes; 10 of 81 samples where painting was done; 1 of 20 samples from automobile repair 

shops; and 17 of 67 samples from various other industrial sites where materials such as 

varnishes, sterilization agents and cleaning agents were used. The geometric mean 

atmospheric concentrations and ranges of EGBE at various sites were as follows: printing 

shops, 0.8 ppm, range 0.3-3.7 ppm; painting areas, 3.9 ppm, range 0.7-19.4 ppm; automobile 

repair shops, 1.2 ppm (one sample); various other industries, 1.8 ppm, range <0.1-367 ppm 

(this data are considered further when relevant to a specific scenario).

In a survey on industrial solvents conducted in 1994 to 1996 by Yasugi et al. (1998) in 95 

different plants (196 unit work areas), EGBE was found 59 times (8 during printing and 51 

during painting) with a median atmospheric concentration of 0.5 ppm and a maximum 

concentration of 1.3 ppm (10-l bag sample, analysed the day of sampling). 

From 1987 to 1998, the French COLCHIC database collected 10,593 personal sampling 

results of glycol ethers for 602 facilities (Vincent, 1999). EGBE was found 1,195 times; the 

arithmetic atmospheric mean value of the 60 to 480 minutes samplings (622 results) was 2.3 

ppm (median 0.6 ppm; range 0.02-110 ppm; 95th percentile 6.2 ppm). It should be noted that, 

globally, atmospheric concentrations have significantly decreased from 1987-92 (arithmetic 

mean of 147 personal samplings 1.1 ppm, median 0.6 ppm, range 0.02-10.7 ppm) to 1993-98 

(arithmetic mean of 178 personal samplings 0.28 ppm, median 0.1 ppm, range 0.02-3.8 ppm) 

(Vincent and Jeandel, 1999). 

For the years 1999 to 2002, the COLCHIC database collected 116 personal atmospheric 

sampling results of EGBE. The arithmetic mean value of 60 to 480 minutes samplings was 

found 0.52 ppm (median 0.14 ppm, range 0.02-14.84, 95th percentile 1.22 ppm) (Vincent, 

2003). 

• Scenario 3-1 Painting/Surface coatings 

EGBE is used as a solvent in paints and surface coatings, particularly in water-based type. It 

is the main application of EGBE and due to the high volume use, a large number of workers 

are potentially exposed. 

Twenty seven water-based paints and formulations used in the automotive industry have been 

analysed (Jargot et al., 1999). EGBE was found 18 times, with concentrations ranging from 

traces to 40.4 %; in thinners, its concentration ranged from 0 to 24.7 %. 
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SIDS (1996) reports that coatings may typically contain 1 % up to 8 % EGBE. 

Analysis of the answers collected in the paint formulating industry by CEPE (2002) shows 

that the concentrations of EGBE range from traces to 39 % with an arithmetic mean up to 8 % 

(see table 4.14). Taking into account this data together with the information collected in 

European products registers (tables 4.1 and 4.2), a worst case EGBE content of 20 % in 

industrial paints and surface coatings and 3 % in decorative paints will be assumed in this 

assessment. Therefore the conclusions in this section refer to solvent-based paints. Exposure 

from use of water-based paints (lower EGBE content) would be much lower. 

Table 4.14: Contents of EGBE in paints (CEPE, 2002) 

 Industrial paints Decorative paints 

 Water-based Solvent-based Water-based Solvent-based

Number of 

answers 

33 31 10 9 

Arithmetic mean 7.8 % 7.1 % 1.4 % 1.6 % 

Median 3.5 % 5 % 1.8 % 1 % 

Range* 0.06-22.5 % 0.01-38.7 % 0.03-3.0 % 0.01-3.0 % 

* minimum values probably correspond to incorporation of EGBE through additives.

Coatings and paints are applied by brushing, rolling, spraying or dipping in different industrial 

and skilled trade sectors, e.g. coating of metal and wood, vehicle production and repair, 

building trade. Application techniques inventoried in the CEPE enquiry are presented in table 

4.15 (CEPE, 2002). 

Table 4.15: Relative frequencies of application techniques in painting/surface coating (CEPE, 2002) 

Application 

technique No of mentions 

Spray 40 

Roll 22 

Brush 21 

Dipping 6 

Roller coaters 3 

Printing 3 

Falling curtain 1 
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Inhalation exposure

Measured data 

The data reported in the previous assessments (SIDS, 1996 ; NICNAS, 1996 ; ATSDR, 1998) 

completed by further references available in open literature (Collinot et al., 1996 ; Delest and 

Desjeux, 1995) are presented in table 4.16. Details have been added when available. 

Table 4.16: Atmospheric and biological monitoring during painting (8 hr-TWA inhalation exposure and BAA urinary 

concentration) 

Activity 

No/type 

of 

sampling 

EGBE air 

concentration 

Mean 

(range)(ppm)

No of 

post-shift 

urinary 

samples 

Post-shift 

BAA urinary 

excretion 

Mean (range) 

(mg/g 

creatinine) 

Reference 

Furniture 

production/finishing 

64/p 

1.5 

(0.07-9.9) 

 Zaebst, 1984 

Staining/varnishing 9 

5 

(≤71) 

Denkhaus et al.,

1986 

Car repair 1/a 1.2 Veulemans et 

al., 1987a 

General painting 10/a 

3.9 

(0.7-19.4) 

Veulemans et 

al., 1987a 

House painting 

(water-based <1.4 % 

EGBE) 

15/p (0.4-12) Hansen et al., 

1987 

Cabinet finishing 6/p Nd1-0.4 Newman and 

Klein, 1990 

Spray painting 11/p 

0.4 

(0.2-0.6) 

Winder and 

Turner, 1992 

Painting (waterbased paints <5 % 

EGBE) 

54 

0.1 

(0.1-0.3) (<2-13.2) 

Delest and 

Desjeux, 1995 

Indoors house 

painting 

(water-based paints)3

202/p 

0.01 

(<0.01-0.15) 

Norbäck et al.,

1996 

Cataphoresis a (≤7.5) Collinot et al.,

1996 
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Can coating 

- external deco 

- internal decor 

20/p 

11/p 

0.73 

(0.35-1.23) 

0.44 

(0.15—0.67)

 

12.2 

(0.3-51.4) 

9.2 

(0.6-20.4) 

Haufroid et al., 

1997 

Painting of plastic 

hup caps 79/p 

<0.1 

(<0.1-0.8) 

19 

n.d. 

Cataphoresis 66/p 

0.8 

(<0 .1-6.2) 

51 

17.9 (<2-210) 

Can coating 

168/p 

0.2 

(<0 .1-2.4) 

79 

5 (<2-33.9) 

New car painting 39/p 

<0.1 

(<0.1-0.5) 

40 

n.d. 

Coil coating 261/p 

0.1 

(<0.1-1.0) 

213 

2.3 (<2-28.4) 

Painting of metal 

frame 50/p 

<0.1 

(<0.1-0.3) 

46 

9.4 (<2-63) 

Painting of buildings 63/p 

<0.1 

(<0.1-0.2) 

63 

<2 (<2-13.2) 

Furniture varnishing n.d. 50 2.9 (<2-31.2) 

Printed circuit 

boards varnishing 57/p 

<0.2 

(<0.1-2.8) 

56 

4.6 (<2-30.4) 

Vincent et al.,

1996 

p: personal, a: area, 

Note 1: Limit of detection not reported. 

Note 2 : No of workers (No of samples not stated) 

Note 3: EGBE found in 2 samples. 

Information from database 

In the HSE exposure database (Brown et al., 1994), 12 results are available for painting and 

20 for lacquering; the ranges (and arithmetic means) were 0 – 1.4 ppm (0.15 ppm) and 0.2 – 7 

ppm (1.69 ppm) respectively. The single high exposure was taken during the spraying of 

lacquer onto wooden furniture and local exhaust ventilation was provided; this high level was 

considered as probably due to a failure in the LEV system. 

Exposure measurements (sampling period 60-480 minutes) registered between 1987 and 1998 

in the COLCHIC database were analysed by Vincent (1999). Results related to painting and 

coating are presented in table 4.17. 
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Table 4.17: Personal exposure in painting activities for measurements 60-480 minutes, years 1987-1998 (Vincent, 

1999) 

Type of work No of 

results 

Mean 

(ppm) 

Range 

(ppm) Median (ppm) 

95th 

percentile 

(ppm) 

Pneumatic spraying of 

paint or varnish 58 0.46 0.04-4.2 0.2 2.2 

Varnishing (curtain) 19 0.14 0.02-0.6 0.1 0.6 

Brush or roll coating 

of paint or varnish 21 0.18 0.04-0.6 0.12 0.42 

Cataphoresis 3 0.2 0-0.6 - - 

In the German MEGA database, 441 exposure measurements have been registered between 

1996 and 2000, which were obtained during application of paints or coatings, mainly in the 

plastic or metal processing industries, in electrical engineering or woodworking. The results 

(measurement values with an exposure duration ≥1 hour and a sampling duration ≥1 hour 

converted to 8-hour weighted averages) are presented in table 4.18. When possible, a 

distinction is made on the basis of whether or not control measures (LEV) were taken (see 

comments in scenario 2). 

Table 4.18: Personal exposure measurements in the MEGA database, 1996-2000 (BGAA, 2001) 

Type of 

company/working 

area 

No of results No of 

companies 

50% 

value 

(ppm) 

75 % 

value 

(ppm) 

90 % 

value 

(ppm) 

95 % 

value 

(ppm) 

Painting,brush and 

roller application, 

filling work 

- without LEV 

- with LEV 

40 

26 

10 

24 

16 

6 

a 

a 

a 

a 

a 

a 

a 

a 

a 

2.4 

2.2 

a 

Spraying (compressed 

air, airless, airmix) 

- without LEV 

- with LEV 

224 

23 

189 

112 

14 

98 

a 

a 

a 

a 

2.4 

a 

a 

11.6 

a 

3.8 

24.6 

2.6 

Surface coating, 

mechanical 

- without LEV 

- with LEV 

98 

44 

53 

47 

22 

26 

a 

a 

a 

a 

a 

a 

a 

a 

a 

a 

a 

a 

Surface coating, 

general 

- without LEV 

- with LEV 

79 

26 

50 

8 

21 

28 

a 

a 

a 

a 

a 

a 

a 

a 

a 

2.7 

1.5 

3.9 

a : measurement value < analytical determination limit 
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Modelled data 

Exposure to vapours during the use of paints or surface coatings is estimated by EASE to be 

in the range 100-140 ppm for wide dispersive, low tendency to become airborne, direct 

handling and dilution ventilation. 

The model overestimates exposure levels, particularly because of non-consideration of the 

content of EGBE in the mixtures. The estimates cannot be corrected for the partial vapour 

pressure because the composition of the formulations is not known. A simple approach based 

on a reduction of the exposure by a factor equivalent to the EGBE concentration in the 

mixtures (up to 20 % for industrial paints and 3 % for decorative paints) would lead to 

exposure levels of 20-28 ppm for industrial paints and 3-4 ppm for decorative paints. 

However the validity of these extrapolations is rather questionable. 

Summary/statement of the exposure level 

Exposure to EGBE during painting may be extremely variable, due to differences in 

frequency and duration of use, concentration of EGBE in the paint, method of application and 

precautions taken during use. To some extent, this variation is reflected in the atmospheric 

monitoring data available for EGBE during painting and surface treatment. 

SIDS proposed a maximum atmospheric concentration of 10 ppm for use of a paint/surface 

coating containing 10 % EGBE, the justification for this estimation is not clear. On the basis 

of the available data, results of air monitoring are generally much lower except during 

spraying. 

For spraying, the available measured data are mainly extracted from the COLCHIC and the 

MEGA databases. The 90th percentile of the MEGA (11.6 ppm) database values seems to be 

the most representative worst case inhalation exposure. 

For other application techniques, inhalation exposure is likely to be lower. The 95th percentile 

of the COLCHIC and MEGA databases results are lower than 4 ppm. The highest result from 

the largest study (Vincent et al., 1996) is 6.2 ppm. This value will be used for risk 

characterisation to ensure that highly exposed workers are represented. 

In conclusion, we propose in a first approach the following worst case inhalation exposures: 

- 11.6 ppm (58.1 mg/m3) for spray application of paint 

- 6.2 ppm (30.4 mg/m3

) for other application techniques 

Dermal exposure

Measurements 

In a study performed by TNO (RISKOFDERM, 2002a and Gijsbers et al., 2004), a part of the 

RISKOFDERM project, potential hand exposure to an analogous but less volatile glycol ether 

DEGBE (2-(2-butoxyethoxy)ethanol) was measured during indoors application of paint by 

brushing over of periods of 57-149 minutes. The sampling was ended when the painters (who 

usually painted for most of the working day) took a break for coffee or lunch, resulting in the 

measurement durations mentioned. The measurements were made using cotton sampling 

gloves which were worn over new protective gloves, where present. The amount of used 

product was between 0.5 and 2.5 litre (AM 1.2 litre) and the paint contained between 0.4 and 
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3.2 % DEGBE (AM 2 %). The treated area during measurements was between 2 and 15 m2

(AM 6.4 m2). Exposure was mainly due to exposure on the hands. Results (given in DEGBE 

and product) are presented in table 4.19. 

Table 4.19: Dermal exposure rates during brushing ( Gijsbers et al, 2004) 

Hands 

exposure 

N W Range (mg) AM 

(mg) 

GM 

(mg) 

GSD 

(mg)

AMc

(μg/cm2/min)

GMc

(μg/cm2/min) 

GSDc

(μg/cm2/min) 

- DEGBE 

- product 

36 

24a

18 

13 

0.19-33.0 

11.3-733.3 

6.5 

170.5 

2.8 

98.4 

4 

3 

0.091 

2.8 

0.045 

1.7 

3.6 

2.9 

N, number of measurements; W, number of workers involved 

AM, arithmetic mean; GM, geometric mean; GSD, geometric standard deviation 

Nota a : for 12 of the measurements, exposure to product could not be calculated due to contamination of 

samples by other sources of DEGBE. 

Nota c : a surface area of 820 cm2 was assumed for the hands exposure

The 90th percentile of the measured exposure levels was approximately 400 mg product 

(unpublished data, derived from original data). 

No measurements for dermal exposure in spray painting of EGBE or other direct analogues 

(e.g. other glycol ethers) are available. A number of dermal exposure data sets is available for 

other spray applications. Exposure to a non-volatile pigment was measured in car painting. 

Dermal exposure to the hands, expressed as total formulation, was between 2 and 211 mg (n = 

30), with a median of 41 mg. Amount of product used was up to 1.5 kg in an average duration 

of 16 minutes (RISKOFDERM, 2002b ; Delgado et al., 2004). Exposure to another nonvolatile pigment was measured in marine anti-fouling painting. Dermal exposure to the hands, 

expressed as total formulation was between 286 and 27.000 mg (n = 24) for sampling periods 

of 1 to 3 hours. In this period 40 to 140 litres of paint were sprayed. Exposure was not only to 

workers actually doing spray painting, but also to auxiliary workers (so-called linesmen) 

assisting the spray painters. There was no difference in exposure between the two jobs. 

(RISKOFDERM, 2002c ; Hughson and Aitken, 2004). Spray application of a cleaning agent 

containing DEGBE was measured 12 times in the food industry. Duration of measurements 

ranged between 6 and 18 minutes. The in-use concentration of DEGBE was between 0.007 

and 1.1%. The total amount of diluted product used was between 16.5 and 97.2 L. Dermal 

exposure to the hands, expressed as total formulation, was between 37 and 1974 mg 

(RISKOFDERM, 2003a). The 90th percentiles of these three data sets were respectively 

approximately 100, 13,600 and 1000 mg per measurement period (unpublished data, derived 

from original data). Older data are reported in the TGD, where for spray painting on large 

surface areas a reasonable worst case value of 10 000 mg product per day (on 840 cm2

) is 

given (Appendix I.E). 

Modelling

SIDS (1996) retained continuous skin contact over the work period (8 hours) and a 1000 cm 2 

skin area exposed (a hand and a forearm). Intermittent contact seems more appropriate for 

tasks as brushing and rolling in this scenario. For spray application, extensive contact is 

assumed., For wide dispersive use, the EASE model estimates a dermal exposure in the range 

of 1-5 mg of product/cm2

/day for intermittent contact and 5-15 mg of product/cm2/day for 

extensive contact. The estimation is made from a formulation containing up to 20 % of EGBE 

(industrial paint) and a formulation containing up to 3 % of EGBE (decorative paint) and an 
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exposed skin surface area of 840 cm2 (two hands for consistency with other EU occupational 

risk assessments). This leads to estimated external dermal exposures of : 

- 168-840 mg/day for industrial painting (excluding spray application)- 840-2520 mg/day 

for industrial spray painting 

- 25-126 mg/day for decorative painting 

Assessment 

Skin contact due to manual transfer of liquids, spray application and brushing, rolling and 

cleaning is to be expected. In several of the available references, the importance of skin 

exposure is stressed. 

In the TNO study, cotton sample gloves can retain more material than skin leads to 

overestimation of exposure. The size of this possible overestimation is difficult to estimate. It 

can be expected that this overestimation is smaller when more viscous paints are used than 

when less viscous pure product is used. The fact that workers (except for breaks) may almost 

continuously do the same tasks suggests that the task based exposures should be extrapolated 

to a full work shift. Whereas extrapolation is clearly not valid for high (product) exposures, 

due to saturation of the surface of the skin, this is not so much of a problem with lower task 

exposure levels. Given the fact that painters spend part of their working period in preparation, 

moving between rooms, cleaning up the material and other non-painting tasks, the exposure 

period for painting can be estimated to be up to 6 hours. Based on the 90th percentile of the 

measured data (400 mg product in 2 hours), linear extrapolation to 6 hours of work and an 

assumed percentage of 20 % (industrial paint) or 3 % EGBE (decorative paint), the exposure 

value to be used in risk characterisation is estimated as 400x 3x 0.2 = 240 mg or 400x 3x 0.03 

= 36 mg EGBE per day for brushing and rolling of industrial or decorative paints. 

The exposure levels from industrial spray application apparently depend on the scale of 

application, as well as on control measures in use. Without further information, it is assumed 

that large scale application with limited exposure control can be done with paints containing 

up to 20% EGBE. The measured values over short periods cannot be extrapolated towards 

longer periods, because this would lead to oversaturation of the skin. Therefore, a reasonable 

worst case exposure level of 10,000 mg product per day is assumed, based on the levels 

mentioned in the TGD and the measurements by Hughson and Aitken (2004). This leads to an 

estimated exposure to EGBE of 2000 mg on 840 cm2

. Because EGBE is much more volatile 

than the measured substances, this may be an overestimation. Also, if less large scale tasks are 

done, the exposure levels may be substantially lower. These uncertainties should be taken into 

account in the evaluation of the MOS. 

Dermal exposure may be lower if suitable gloves are worn. 

Biomonitoring data

Three studies of biomonitoring have been reported for painting or coating activities. Results 

are presented in table 4.16. 

In the study performed by INRS (Vincent et al., 1996), the workers most exposed to EGBE 

were found in the area of cataphoresis. The authors note that cutaneous exposure was 

prevalent particularly in workers who cleaned car bodies with a rag dipped in EGBE before 

installing the electrode. Working conditions may have greatly improved since 1993. 
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In another French study (Delest and Desjeux, 1995), exposure of 54 house painters was also 

assessed by biological monitoring. Only 3 painters had BAA concentrations in urine higher 

than the detection limit (2 mg/g creatinine). The main reasons for such low figures were the 

application technique (brush or roller) and the low EGBE content in the products (<5 %). 

An assessment of skin penetration may be made from these biomonitoring studies with the 

same reasons, techniques and limitations that have been presented in scenario 2. Highest 

results are also obtained with peak value measurements, especially in this case from metal 

painting (63 mg/g creatinine and 0.3 ppm inhalation concentration), which gives a dermal 

equivalent of 96.2 mg/day, corresponding to a dermal load of 320 mg/day, assuming 30% 

absorption efficiency. As in scenario 2, a negative bias of 30% is also considered; the dermal 

load would then be re-assessed as ca. 430 mg/day. 

In the case of building painting (“decorative”; 13.2 mg/g creatinine and 0.2 ppm inhalation 

concentration), the calculated dermal equivalent is 16 mg/day, corresponding to a dermal load 

of 54 mg/day, assuming 30% absorption efficiency. If the same bias is also assumed, the 

dermal load would be assessed as ca. 70 mg/day. 

• Scenario 3-2 Printing 

EGBE is a solvent in a range of specialist inks particularly silk-screen inks used by 

professional trades. However there is a trend from solvent based inks to UV curing inks that 

contain no solvents. 

SIDS (1996) reports that inks contain approximately up to 20 % EGBE. In the enquiry 

recently performed in the ink formulating industry by CEPE (2002), the information collected 

is very limited. Only 6 answers were obtained in relation with the EGBE content in printing 

inks. They indicated content from traces up to 31 %. Screen ink seem to contain the highest 

concentration. Other recent data provided by one of the main producer of screen printing 

show that typical percentages range from 2 to 35 % (BP, 2002a). Taking into account all this 

data, typical maximum contents of 35 % EGBE in silk-screen inks and 20 % in others will be 

assumed in this assessment. 

Inhalation exposure

Measured data 

Monitoring data are reported in previous reports (SIDS, 1996 ; NICNAS, 1996 ; ATSDR 

1996), some of them are quite old and probably not representative of current working 

conditions. A compilation of these data is presented in table 4.20, completed with two further 

references (Auffarth et al., 1998 and Laitinen, 1998). Details have been added when available.
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Table 4.20: Atmospheric and biological monitoring during printing (8 hr-TWA inhalation 

exposure and BAA urinary concentration) 

Activity No/type 

of 

sampling 

Mean 

(ppm) 

Range 

(ppm) 

Comments / Postshift 

BAA urinary 

excretion (mg/g 

creatinine) 

Reference 

Printing press 

operators 3/p <0.2 <0.04-0.49 

2 %, cleaning 

printing press 

rollers 

Lewis and 

Thoburn, 

1981 

Label production /plate 

makers and pressmen 7/p 2 1-2 Apol, 1981 

Silk screening 6/p 3.4 1.1-5.3 Boiano, 1983 

Silk screening 

Spray printing 

p 

p 

 max 1.4 

max 0.75

Clapp et al., 

1984 

Silk screening 

 Silk screeners 

 Spray printers 

 Controls 

16/p 

5/p 

6/p 

6.8 

2.6 

0.3 

up to 45 % Baker et al.,

1985 

Silk screening 3/p 4 3-5 Apol, 1986 

Silk screening 108/p 0.64 Max 1.14 Half-shift 

sampling 

Veulemans et 

al., 1987b 

Printing (various) 25/a 0.8 0.3-3.5 Veulemans et 

al., 1987a 

Silk screening 

14/p 

8/a 

25 

63 

13-36 

23-169 

100 %, open spray 

troughs and wash 

table areas 

Kullman 

(NIOSH), 

1988 

Printing press 

operators 

1/p 8.3 Lee, 1988 

Silk-screening (cleanup and maintenance) 5/p 5.2 1.7-9.8 

10-50 %; cleaning 

with a 

hydrocarbonbased solvent 

Salisbury and 

Bennett, 1987 

Printing press 

operators 2/p <0.33 nd-0.53 

2 %; cleaning 

printing press 

rollers 

Kaiser, 1990 

General printing 9/p 0.64 0.4 - 0.8 9 urinary samples Sakai et al.,
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3.9 (1.3-9.9) 1993 

Tampography 84/p 0.2 <0.1-0.7 

48 urinary samples 

2.2 (<2-7.1) 

Vincent et al., 

1996 

Silk screening 295/p 0.2 <0.1-1.6 

154 urinary 

samples 

ndc

Vincent et al., 

1996 

Offset printing 39 Ndb 11 urinary samples 

2.2 (<2-3.8) 

Vincent et al., 

1996 

Printing press 

operation 

- semi automatic 

- 3/4 automatic process 

- fully automatic 

5/p 

14/p 

3/p 

0.5 

0.5 

0.5 

0.3-0.9 

0.3-0.9 

0.3-0.9 

Auffarth et 

al., 1998 

Silk screening 5/p 0.5 0.2-0.7 Auffarth et 

al., 1998 

p: personal sampling; a: area monitoring 

b : any concentration upper than 0.1 ppm was detected 

c: any concentration upper than 2 mg/l was detected 

More recent exposure measurements made in 2001 and provided by one of the main producer 

of screen printing inks in the EU have been presented by industry (BP, 2002a). Printers are 

exposed 1-8 hours per day but 2-3 hours is the typical length time when workers are exposed 

to solvents. Nearly all operations use LEV as well as general ventilation. EGBE was detected 

in 13 samples amongst 161. The results are presented in table 4.21. The figures represent 

TWA over 2-3 hours.

Table 4.21: Personal air measurements during screen printing (BP, 2002a) 

Activity 

No of results 

where EGBE 

detected 

Mean (ppm) Maximum 

(ppm) 

5-95 % 

percentile 

(ppm) 

Print shop 6 0.4 0.7 0.02-0.7 

Reclaim 2 0.9 1.7 

Other 5 <0.1 0.2 

Information in database 

In the HSE database (Brown et al., 1994), 66 personal exposure (8-hour TWA) have been 

collected (1986-1991) for screen printing. The range was 0-9.2 ppm (arithmetic mean 1.54 

ppm). 

Referring to the already cited French COLCHIC database (Vincent, 1999), 45 atmospheric 

personal samplings have been made between 1987 and 1998 in the printing industry, resulting 
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in an arithmetic mean concentration of 0.6 ppm (range 0.02-5.2 ppm; median 0.04 ppm; 95th

percentile 2.6 ppm). Results collected during the same period for printing activities in 

different industrial sectors are presented in table 4.22. 

Table 4.22: Personal exposure in printing activities made for measurements 60-480 minutes, years 1987-1998 

(Vincent, 1999). 

Activity No of 

results 

Mean 

(ppm) 

Range 

(ppm) 

Median 

(ppm) 

95th percentile 

(ppm) 

Screen printing 92 1.1 0.02-6.5 0.4 4 

Screen washing 9 0.4 0.04-1.6 - - 

Offset printing 13 0.3 0.02-1.2 0.04 1.2 

Cleaning in 

general 2 2.5 1.2-3.8 - - 

Flexography 20 0.08 0.04-0.1 0.1 0.1 

Rotogravure 7 0.04 0.02-0.1 - 

In the printing industry, it should be noted that exposure to EGBE has significantly decreased 

from 1987-92 to 1993-1998, with an arithmetic mean for personal samplings of 1.4 ppm 

(median 1.3 ppm; range 0.02-6.5 ppm; 68 samples) for the first period, and 0.3 ppm (median 

0.1 ppm; range 0.02-2.5 ppm; 79 samples) for the second (p<0.01) (Vincent and Jeandel, 

1999). 

Modelled data 

SIDS calculated the exposure during silk screening and general printing tasks using inks 

containing 50 % and 20 % of EGBE. Exposure estimates were 10 ppm and 2 ppm 

respectively. No information is available on the calculation method.

Exposure to vapours during printing is estimated by EASE to be in the range of 10-20 ppm for 

non dispersive use, direct handling with dilution ventilation. 

The model overestimates exposure levels, particularly because of non-consideration of the 

content of EGBE in the products. The estimates cannot be corrected for the partial vapour 

pressure because the composition of the formulations is not known. A simple approach based 

on a reduction of the exposure by a factor equivalent to the EGBE concentration in the 

mixture (35 % for screen inks and 20 % for others) would lead to exposure levels of: 

- 3.5-7 ppm for silk screening 

- 2-4 ppm for general printing. 

However the validity of these extrapolations is rather questionable. 
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Summary/statement of the exposure level 

Considering the above data, it seems reasonable to make distinctions between the different 

situations and uses. Mainly based on the data from COLCHIC, the reasonable worst-case 

exposures proposed would be the following: 

- Silk screening (including washing): 4 ppm (20 mg/m3) 

- General printing: 1 ppm (5 mg/m3) 

Dermal exposure

Measurements

In a study performed in Finland by Kuopio Regional Institute of Occupational Health 

(KRIOH)(RISKOFDERM, 2003b), a part of the EU RISKOFDERM project, potential hand 

exposure to EGBE was measured during silkscreening on 10 workers in three different 

enterprises. The measurements were made for 6 to 8 hours by giving protective gloves, which 

were collected after shift and analysed. Results (given in formulation) are presented in table 

4.23a. In the same study, measurements of actual hand exposure indicate that the use of 

gloves does lower hand exposure significantly. Hands seemed to be the most dominant 

potential dermal route exposure because EGBE could be found only on a few parts of the 

body parts of printers. 

Table 4.23a: Potential dermal exposure measurements during silkscreening (KRIOH, 2003) 

 1st measurement day 

(μg/cm2/h) 

2nd measurement day 

(μg/cm2/h) 

No of samples 6 10 

Average 5.328a 4.068b

90th % 15.684 12.937 

a : 3 results were below the limit of detection 

b : one result was below the limit of detection 

The range of the measured exposure values for the formulation was <0.04-85 mg and the 90th

percentile was 65 mg/day (unpublished data, from the original data). 

Modelling 

Dermal exposure may occur during mixing, application and cleaning activities. SIDS (1996) 

retained continuous skin contact over the work period (8 hours) and a 1000 cm2 skin area 

exposed (a hand and a forearm). Intermittent contact seems more appropriate for this scenario 

(as for painting). Assuming non dispersive use, direct handling and intermittent contact, the 

EASE model estimates a dermal exposure in the range of 0.1-1 mg of product/cm2

/day. The 

estimation is made from a formulation containing up to 35 % (screen inks) or 20 % (others) of 

EGBE and an exposed skin surface area of 840 cm2 (two hands for consistency with other EU 

occupational risk assessments). This leads to an estimated external dermal exposure of: 
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- 29-294 mg/day for silk screening 

- 17-168 mg/day for general printing. 

Assessment

For general printing, no relevant exposure data are available. The EASE estimate therefore 

has to be used for risk characterisation. For silk screen printing, a set of 16 potential full shift 

hand exposure data is available with a 90th percentile of 65 mg/day of product. The “product” 

in this case consisted of printing ink with up to 10% of EGBE and retarder (up to 100% 

EGBE) that was used by just two of the printers (as part of the total ink-system). Assuming 

that the concentration of EGBE in screen printing inks can be up to 35%, the reasonable worst 

case exposure level for EGBE in this process would be approximately 23 mg/day. Because the 

measured values are based on a more than 12 measurements and come from different 

workplaces, they can be considered sufficiently representative for use in risk characterisation. 

In conclusion, the following range are proposed for dermal exposure during printing: 

- 23 mg/day for silk screening 

- 168 mg/day for general printing. 

Dermal exposure may be much lower if suitable gloves are worn. 

Biomonitoring data

The results of two studies of biomonitoring performed in the printing industry are presented in 

table 4.20. 

In the study performed by Vincent et al. (1996), EGBE was not the main solvent to which the 

workers were exposed to. BAA was not detected in the silk screening printing workshops 

while its concentration was up to 7.1 mg/g creatinine when pads were used. 

In the study performed by Kuopio Regional Institute of Occupational Health 

(KRIOH)(RISKOFDERM, 2003b) and also described above in the paragraph “Dermal 

exposure – Measurements”, biological monitoring data were reported. Workers internal 

exposure was evaluated by urinary excretion of 2-butoxyacetic acid (BAA) in the first 

measurements day and in the second measurements day; in the first measurements, the 

inhalation exposure was excluded by using a respirator and in the second measurements, the 

dermal exposure was excluded by using protective overalls and gloves. The results are 

reported in the table 4.23b. 

Table 4.23b : Workers’internal exposures (Urany butoxyacetic acid (BAA) excretion, μmol/mol creatinine) 

 Urinary butoxyacetic acid (BAA) excretion, μmol/mol creatinine 

 1st measurements day 2nd measurements day 

 Before work shift After work shift Before work shift After work shift 

Average 3.2 3.8 3.5 18.4 

90th % 3.8 5.5 5.5 40.7 

Because of many results below the limit of detection, the correlations based on the results 

between the actual exposure and BAA excretion are highly inaccurate as it is mentioned in the 

report KRIOH (RISKOFDERM, 2003b). 
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If a tentative assessment of skin penetration is made in the same conditions that have been 

presented in scenario 2, biomonitoring data may in this case be entirely explained by 

inhalation exposure alone. Supposing also a negative bias, it is proposed to come back to the 

low end of the EASE assessment. 

• Scenario 3-3 - Cleaning 

The NICNAS report (1996) focuses on the use of EGBE in cleaning products in Australia 

because of the specific concern raised in this context : 

« Due to the large number of cleaning products containing EGBE, a large number of workers 

may be exposed to the chemical. The main groups of workers who handle cleaning products 

containing EGBE include: 

. carpet cleaners;

. contract cleaners; 

. food process workers; 

. hospital and nursing home workers; 

. hospitality industry workers; 

. householders; 

. laundry cleaners and workers; 

. mechanics; 

. metal workers; 

. school and office cleaners; and 

. window cleaners 

Exposure during cleaning is extremely variable, due to differences in frequency and duration 

of use, strength of solution used, method of application and precautions taken during use... 

The strength of solution used in the cleaning process is generally low as the product is usually 

diluted substantially before use; for example, most surface cleaners specify a dilution ratio in 

the range 1:3 to 1:100, depending on the application and the soil loading. A large proportion 

(68 %) of cleaning products contain less than 10 % EGBE (see table 4.24), so the final 

strength of solution is often less than 1%. A random survey of 20 general surface cleaning 

products containing <10 % EGBE indicated that the dilution ratio ranged from 1:1 to 1:250, 

with most ratios in the 1:5 to 1:100 range... 

Some products are sold as high level concentrates (>50 % EGBE) which must be diluted with 

large volumes of water before use. In some cases, products are diluted with hot water (up to 

80°C). 

Exposure during dilution 

Dilution is often carried out daily at the beginning of the shift. While the dilution procedure is 

usually of short duration, the potential exposure may be greater due to use of higher 

concentrations of EGBE and the possibility of splashing. Higher exposures may also occur if 
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the product is diluted with hot water as vapour concentrations may be higher and skin 

absorption facilitated. If dilution is carried out in a confined space or poorly ventilated area, 

exposure may be increased. 

Exposure during application of cleaning solution 

A number of different methods are used to apply the cleaning solution, for example, washing, 

wiping, mopping and spraying. Approximately half of the cleaning products were used in 

spray form, with a small number marketed in aerosol spray cans or trigger packs. This method 

of application will potentially increase both dermal and inhalation exposure as the 

atmospheric concentration of EGBE will be higher and dermal contact will be increased. The 

potential for exposure may also be increased where heat is applied during cleaning, for 

example, cleaning ovens and hot plates”. 

Table 4.24: Main uses of EGBE containing cleaners in Australia (NICNAS, 1996) 

Use Number % of total EGBE % 

 Min. Max. 

Surface cleaner 214 49 0.57 71 

Floor stripper 49 11 <1 30.5 

Glass/window cleaner 47 10 <1 40 

Carpet cleaner 40 9 <1 10-30 

Laundry detergent 15 4 <1.5 10-30 

Rust remover 11 3 <10 30-60 

Oven cleaner 11 3 <1 10-30 

Ink/resin remover 9 2 1 10-93 

Others 38 9 <10 94 

AISE (BP, 2002b) sent recently a questionnaire to their 10 member companies most likely to 

be using EGBE. Six (belonging to the group of the biggest companies) responded to the 

request. According to the data collected, the main uses of EGBE for cleaning are: 

- industrial and institutional cleaners such as professional laundry products 

- institutional kitchen/floor/window cleaners 

- strippers 

- other industrial cleaners for the food and beverage industry 

- industrial car/truck cleaners. 

The concentration of EGBE in these professional products is always below 12 %, and 

typically not higher than 8 %. 
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Taking into account the NICNAS data together with the information collected in European 

products registers (table 4.1 and 4.2), a maximum content of 50 % EGBE in professional 

cleaners with dilution 1:1 and a task duration of 2 hours will be assumed in this assessment. 

Inhalation exposure

Measured data 

1) Workplace measurements 

Monitoring data reported in NICNAS (1996) for cleaning activities are summarised in table 

4.25. 

Table 4.25: Measured atmospheric exposure (TWA) in cleaning activities. 

Activity 

Nb/type 

of 

sampling 

Mean 

(ppm) 

Range 

(ppm) EGBE % and comments Reference 

Window cleaning 

in hospitals (4 w) <0.2 Cleaner applied in spray, gloves worn 

Apol and 

Cone, 1983 

Cleaning in food 

plant (1 w) 1.6 

0.3 %, mechanical floor 

scrubbing, 95 min 

sampling, gloves, 

overalls, boots worn 

Apol and 

Johnson, 

1979 

School cleaning (4 w) <0.7 0.25 %, cleaner applied 

in liquid and spray form

Rhyder, 

1992 

Window cleaning 

Cleaning cars: 

garage A 

garage B 

garage C 

garage D 

Office cleaners1

: 

group A 

group B 

10/p(2 w) 

10/p(6 w) 

6/p (3 w) 

4/p (2 w) 

32/p(8 w) 

8/p (2 w) 

0.5 

0.84 

<0.1 

4.9 

0.32 

<0.3 

<0.1-1.2

<0.1-2.8

2.9-7.3 

0.3-0.73

14.4 %, 0.8-5 h 

exposure, no gloves 

worn 

21.2 %, 0.3-4 h 

exposure, no gloves 

worn 

5.7 %, 0.7-2 h exposure, 

no gloves worn 

21.2 %, 5.3 h exposure, 

short sleeves, gloves 

worn 

9.8 %, 15 min exposure, 

no gloves worn 

0.9 %, 15 min exposure, 

no gloves worn 

Vincent et 

al., 1993 

 p: personal sampling; a: area monitoring; w: workers. 

Note 1: for office cleaners, results are for half-day shift air samples. 

2) Experimental data 

A series of experiments was conducted in a room-size, controlled environment chamber to 

evaluate a user's inhalation and dermal exposure to constituents of a household spray cleaner 

dispensed from either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997). 
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Airborne concentrations and the amounts transferred to gloves worn by the product user were 

measured for EGBE at 5 % weight product concentration. The measured air exposure 

concentrations of EGBE were on average 5.7 ppm; air exposure concentrations resulting from 

a typical 20-minute usage were well below the OSHA OEL of 25 ppm. 

NICNAS estimation 

The inhalation exposure during cleaning activities was estimated by NICNAS (1996) for 

various EGBE concentration in products and taken up by SIDS (1996). The reasoning and the 

results are presented below. 

In the monitoring data available, TWA air concentrations up to 9.7 ppm were obtained for 

print machine operators using a cleaning solvent containing 10 – 50 % EGBE (Salisbury and 

Bennett, 1987) and up to 7.3 ppm for workers cleaning car windows with a 21.2 % solution 

applied as a spray (Vincent et al., 1993). Based on this data, the EGBE air concentration 

selected for worst-case estimates for a 30 % cleaning solution was 10 ppm TWA. 

For cleaning solutions containing <1% EGBE, the atmospheric concentrations were generally 

below the limit of detection; however, a personal monitoring reading of 1.6 ppm was obtained 

during floor scrubbing with a 0.3 % solution (Apol and Johnson, 1979). Consequently, an air 

concentration of 2 ppm was selected for 0.1 and 1 % cleaning solutions. 

Table 4.26: Exposure estimates for use of cleaning products (SIDS, 1996) 

% EGBE Max. exposure 

(ppm) 

0.1 2 

1.0 2 

10 4 

30 10 

Modelled data 

Several parameters should be chosen to correspond to the real variability of conditions of use 

of cleaning products: 

- wide dispersive use, low tendency to become airborne, direct handling would give 200-

300 ppm, 

- wide dispersive use, low tendency to become airborne, direct handling and dilution 

ventilation, would give an estimation of 100-140 ppm. 

Assuming the exposure would occur 2 hours a day, this leads to exposure levels of 50-75 ppm 

or 25-35 ppm, averaged on a shift duration. 

The model overestimates exposure levels, particularly because of non-consideration of the 

content of EGBE in the products. The estimates cannot be corrected for the partial vapour 

pressure because the composition of the formulations is not known. A simple approach based 

on a reduction of the exposure by a factor equivalent to the EGBE concentration in the 

mixture (25 % if the estimation is made for a formulation containing up to 50 % of EGBE 
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with dilution 1:1) would lead to exposure levels of 12.5-18.7 ppm or 1-8.7 ppm. However the 

validity of these estimates is rather questionable. 

Summary/statement of the exposure level 

Only limited measured exposure data are available and they are difficult to analyse because of 

the great variability of exposure conditions (in particular duration and content in the product). 

The highest exposure of 7.3 ppm measured for workers cleaning cars with a 21.2 % solution 

may be considered as a worst case for this scenario. Therefore it seems cautious to keep the 

NICNAS estimation of 10 ppm (49 mg/m3

). 

Dermal exposure

Measured data 

A series of experiments was conducted in a room-size, controlled environment chamber to 

evaluate a user's inhalation and dermal exposure to constituents of a household spray cleaner 

dispensed from either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997). 

Approximately 10 % of the amount of product used was transferred to the user's gloves 

(simulating dermal exposure), with the great majority found on the palms rather than the 

backs of the hands (88 % according to data presented). Using results presented, skin 

contamination can be evaluated (for both hands) at 0.22 mg/cm2/day for a 5 % solution. Note 

also that the right hand is roughly 4 times more contaminated than the left one (right-handed 

worker). 

In a study performed by TNO (RISKOFDERM, 2003a), a part of the RISKOFDERM project, 

potential hand exposure to an analogous but less volatile glycol ether DEGBE (2-(2-

butoxyethoxy)ethanol) was measured during spraying of a diluted cleaning agent (sampling 

duration between 6 and 18 min) and during wiping (car washing, sampling duration between 5 

and 12 min and cumulative duration of wiping per day 24-90 min). For spraying, the 

measurements were made using butyl rubber protective (recommended by the employers), the 

in-use concentration was between 0.0007 and 1.1 % of DEGBE. For wiping, hand exposure 

was evaluated by the hand wash method, the in-use concentration was between 0.04 and 0.82 

%. Exposure was mainly due to exposure on the hands. Results (given in product as well as 

recalculated to in-use product) are presented table 4.27. They show a substantially higher 

exposure rate during wiping activities. 

Table 4.27: Dermal exposure rates during spraying and wiping (RISKOFDERM, 2003b) 

Hands exposure Range (mg) AM (mg) GM (mg) GSD (mg) AM (μg/cm2

/min)* GM (μg/cm2/min)*

Spraying (n=12) 

- DEGBE 

- product 

36-6302a

37-1974 

1000a 

719 

438a

522 

3.73 

2.72 

0.15 

68.4 

0.04 

48.1 

Wiping (n=12) 

- DEGBE 

- product 

1.8-29.8 

1508-4861 

10 

3161 

6.8 

2985 

2.50 

1.45 

1.8 

535 

1.1 

499 

Nota a : these values are in μg 
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* Asurface area of 820 cm2 was assumed for the hands 

The 90th percentiles (expressed in mg of total diluted product) for the sampling durations were 

1000 mg for spray application and 4000 mg for car cleaning (unpublished data, derived from 

original data). 

Modelled data 

Dermal exposure is clearly predominant during cleaning activities. SIDS (1996) retained 

continuous skin contact over the work period (8 hours) and a 1000 cm2 skin surface area 

exposed (a hand and a forearm). Intermittent contact seems more appropriate for this scenario 

because the duration will generally be a part of the shift and continuous contact would 

probably overestimate the exposure. 

Assuming wide dispersive use, direct handling and intermittent contact, the EASE model 

estimates a dermal exposure in the range of 1-5 mg of product/cm2/day. The worst case 

estimation is made for a formulation containing up to 50 % of EGBE with dilution 1:1 and a 

420 cm2 skin surface area exposed (one hand). This leads to an estimated external dermal 

exposure of 105 - 525 mg/day for cleaning. 

Assessment 

The results of the TNO study during spraying and wiping indicate relatively high potential for 

dermal exposure. However interpretation is difficult due to the relative small data set of 

measurements and the fact that during spraying the use of sample gloves may lead to different 

exposure levels than direct contact to the skin. Furthermore, EGBE is much more volatile than 

DEGBE and this may lead to higher evaporation from the skin and lower effective exposure 

levels. Also, for tasks with cumulative exposure durations substantially longer than the 

sampling durations, extrapolation is difficult due to the effects of evaporation as well as the 

possibility of saturation of the skin with product, specifically for situations with direct contact 

and immersion, such as cleaning with a sponge or cloth. On the other hand, the kind of tasks 

performed with these products will often not be done for many hours per day. 

Although the measured data are difficult to interpret, they provide much more real exposure 

information than the EASE model, whose dermal exposure part is not based on real dermal 

exposure measurements. 

A rough estimator of reasonable worst case dermal exposure (to the hands) is therefore 

assumed to be the 90th percentile of the exposure levels during the measurement period. This 

appears to be reasonable, because the measurement periods were aimed at a covering one full 

“cycle” of the relevant activities and they were followed by periods without dermal exposure, 

when evaporation and other effects may lower the skin contamination levels before a new 

“cycle” will again contaminate the skin. Reasonable worst case exposure levels for hand 

exposure to diluted cleaning products containing up to 25% of EGBE are therefore expected 

to be: 

- spray application: 250 mg/day; 

- manual application including immersion of the hands into the solution: 1000 mg/day. 

The uncertainty in these values is rather large, because of the aspects mentioned earlier. This 

should be taken into account in the evaluation of the MOS. The estimate for manual 

application is substantially higher than that of EASE, while the estimate for spray application 
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is similar to that of EASE. However, the measured data are preferred. Dermal exposure may 

be much lower if suitable gloves are worn. 

Biological monitoring data

A study was conducted in France by INRS to evaluate the occupational exposure to EGBE of 

workers using window cleaning agents: 10 cleaning women working in a town hall and 13 

automobile cleaners working in 4 garages (Vincent et al., 1993). EGBE was detected in all the 

window cleaning agents (0.9 to 21.2 %). The results of exposures by job category are 

presented in table 4.28. 

Table 4.28: Characterisation of exposure by job category (Vincent et al., 1993) 

Air concentration of 

EGBE (ppm) 

Pre-shift urinary 

concentration of BAA 

(mg/g creatinine) 

End-shift urinary 

concentration of BAA 

(mg/g creatinine) 

Job 

category 

No of 

samples 

Mean Range No of 

samples

Mean Range No of 

samples 

Mean Range 

Cleaner 

of new 

cars 

15 2.33 <0.10-

7.33 14 17.9 <2-98.6 12 111.3 12.7-371 

Cleaner 

of used 

cars 

15 0.36 <0.10-

1.52 12 4.8 <2-33 11 6.3 <2-24.4

Office 

cleaner 32 0.32 <0.30-0.73 32 2.1 <2-4.6 32 2.1 2-3.3 

The authors noted that the high concentrations of urinary BAA relative to the EGBE TWA 

concentrations, indicated that dermal absorption was the predominant route of worker 

exposure. They concluded that for workers in these occupations not wearing protective 

gloves, environmental sampling probably underestimates the exposure, and that 

biomonitoring of urinary BAA appears to be the best method for estimating overall exposures. 

To indirectly assess skin exposure using these data as already proposed in the preceding 

scenarios, with the same limitations, it is seen that highest results are also obtained with peak 

value measurements in new car cleaning activity (272.4 mg/g creatinine and 7.3 ppm 

inhalation concentration), which gives a dermal equivalent of 240 mg/day, corresponding to a 

dermal load of 800 mg/day, assuming 30% absorption efficiency. For the same reasons 

already discussed in scenario 2, a negative bias is also assumed as 30%, which gives a dermal 

load re-assessed as ca. 1040 mg/day. 

Rettenmeier et al. (1993) reported biological monitoring data on car cleaners. Total BAA 

(free BAA and BAA glutamine conjugate) in 6 post-shift samples averaged 243 (range 67 to 

935) mg/g creatinine (assuming an average urinary creatinine concentration of 1.25g/l). No air 

monitoring was gathered in this study, which does not allow for an indirect evaluation of skin 

penetration. 

• Data from other uses (miscellaneous) 
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Exposure of 13 workers using cutting oils containing EGBE (1 % to 5 %) in a mechanical 

workshop was measured by biological monitoring. No atmospheric measurements were made 

in this case. Concentrations of BAA in post-shift urine varied from <2 to 8.3 mg /g creatinine. 

This result reflects a low exposure (Vincent et al., 1996). 

Exposure of 53 hairdressers working in 10 shops was evaluated. The composition of 43 

products was analysed, EGBE was detected in 8 products (0.5 to 5 %). No solvent was 

detected in the atmosphere and BAA in the post-shift urine was <2 mg/g creatinine (detection 

limit). This result was probably related to the very low quantities of products handled and the 

use of gloves (Vincent et al., 1996). 

Seven personal exposure measurements made during removal of asbestos-containing mastic 

result in EGBE concentrations in the range from 2 to 22 ppm with a mean of 12 ppm (Kelly, 

1993 quoted in ATSDR, 1998). 

4.1.1.2.4 Summary of occupational exposure 

Differences in assessments of skin exposure are striking. One could a priori consider that the 

work undertaken within the “RISKOFDERM” project should be given priority, since it is 

recent and has explored a diversity of real working conditions. In fact, this project has 

objectified the great difficulties of this exercise, leading Kromhout et al. (2004) to conclude: 

“For the time being, actual dermal exposure measurements and a better understanding of 

actual determinants of dermal exposure seem to be a necessity in order to evaluate dermal 

exposure hazards properly.” Important characteristics that may be retained are the extreme 

variability of skin exposure (e.g. “geometric mean levels of dermal exposure range over up to 

4 orders of magnitude between dermal exposure operation units when exposure is expressed 

in milligrams.”) and the predominance of hand exposure in very different tasks (e.g. Eriksson 

and Wiklund, 2004 ; Eriksson et al., 2004 ; Fransman et al., 2004 ; Gijsbers et al., 2004 ; Roff 

et al., 2004a and 2004b). 

In the present situation, skin exposure may also be indirectly assessed in real situations with 

EGBE itself (and not a parent compound with very different physico-chemical properties), so 

these data should also be considered as reliable although somewhat different. 

To take these 2 sources into account, it is proposed to take the available RISKOFDERM data 

as a basis and to adapt them to EGBE using biomonitoring data when applicable. This is done 

in the following table 4.29 (last column) where a factor of 4 has been applied to the 

biomonitoring data when already exposed reasons show that the RISKOFDERM results may 

not be directly used. 
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Table 4.29: Summary of proposed reasonable worst case exposures. 

Scenario

8-hour 

TWA 

inhalation 

(mg/m3

)

Dermal worst 

case derived 

from 

biomonitoring 

data (mg/day) 

External 

dermal 

(mg/day) 

Dermal worst 

case, EASE & 

RISKOFDERM 

& biomonit. 

Retained values 

for risk 

characterisation 

(mg/day) 

1 - Manufacture 12 No data 42b 42 

2 - Formulation 15.7 500 11,600c 2,000e

3 - Use of products 

3.1.1.1 - Coating/Painting – 

industrial: spraying 58.1

 

2,000c 2,000 

3.1.1.2 - Coating/Painting – 

industrial: other works

30.4 430 

(metal painting) 

240c 430 

3.1.2 - Coating/Paintingdecorative30.4 70 36c 70 

3.2.1 – Printing - silk screening 20 Negligible 23b 23 

3.2.2 – Printing - general printing 5 Negligibled

 168b 168 

3.3.1 – Cleaning - spraying 49 250c 250 

3.3.2 – Cleaning - wiping 49 1,040 1,000c 1,040 

Nota b : modelled data from EASE 

Nota c : data derived from measurements with a less volatile solvent (DEGBE) 

Nota d: in this case, due to a probable negative bias (see text), it is proposed to apply the low 

end of the EASE assessment. 

Nota e: see explanations in the § 4.1.1.2.2 – Assessment 

4.1.1.3 Consumer exposure 

4.1.1.3.1 Exposure from uses 

EGBE is used as a solvent in many products available for consumers as, paints, paints 

thinners and cleaning products. EGBE is no more used in cosmetics. 

Cleaning products which contain EGBE include general surface cleaners, floor strippers, 

window cleaners, carpet cleaners, spot cleaners, rust removers, oven and grease trap cleaners, 

laundry detergent, cars cleaners, bathrooms and toilets cleaners and disinfectants, ink and 

resin removers. 

Paints that contain EGBE are both waterborne paints and solvent-borne paints (CEPE enquiry, 

2002) including varnishes and products to preserve wood. 

An investigation was made by the journal "Que choisir" in 2001 showing that two paints 

contained 1 % of EGBE and that in cleaners for cars, the concentrations of EGBE ranged 

from 0.3 to 4 %. 
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Another source of exposure is linked to the stoppers of bottles used for perfusion in medicine 

which are made with natural or artificial rubbers. Chemicals used in rubber formulations can 

leach from rubber stoppers during sterilisation and enter the bloodstream of patients when the 

perfusion is administered. The concentrations of leached stoppers components found in 

parenteral solutions concentrations were measured by GC/MS after extraction by methylene 

chloride. Two parenteral solutions were tested containing 5 % dextrose and one containing 5 

% sodium bicarbonate. Results indicate a mean release of 68 μg/150 ml EGBE in dextrose 

solutions (respectively 70 and 65 μg/150 ml for each sample) and a release of 13 μg/500 ml 

EGBE in the sodium bicarbonate solution. The size of the sampling is too low to allow a good 

estimation of the real contamination of perfusion solutions by EGBE (Danielson, 1992). This 

scenario will not be used in the risk characterisation as it is of concern for a few people and it 

is not a frequent event to the single consumer. The consumer is exposed by this way only if 

two conditions are simultaneous: he needs a perfusion and the rubberstopper contains EGBE. 

By another way, the number of evaluations of the release of EGBE from rubberstoppers is too 

weak to allow a good estimation of the exposure. 

Scenario 1: Household surface cleaners

Two routes of exposures have to be considered during the use of household surface cleaners: 

inhalation and dermal exposure. 

Cleaning frequency and duration were assessed by a questionnaire applied to 342 subjects (75 

% of women), 18 to 80 years old (Kovacs et al., 1997). Subjects reported a longer duration for 

kitchen cleaning task that for the bathroom, with mean values of 37.7 and 30.7 mn 

respectively. (t = 3.67; p<0.0005). Depending on the frequency of cleaning in a week, the 

duration of exposure to household surface cleaners can be estimated to mean values of 98 and 

59 mn/week spent cleaning kitchen and bathroom respectively. When broken down by 

gender, the mean values for the kitchen are respectively 105 and 78 mn/week for women and 

men and, for the bathroom cleaning, 64 and 44 mn/week respectively. 

In the Technical Guidance Document, data from AISE (2002) are provided for the use of 

surface cleaners : the highest quantity of liquid surface cleaner used is 110 g/task when it is 

diluted in 5 l of wash water volume and the longest duration of exposure is 20 minutes. For 

sprays, the highest quantity of liquid used is 30 g/task during 10 minutes. 

Five tests were conducted in a 24.5 m3

, controlled-environment chamber to evaluate a user's 

inhalation and dermal exposure to constituents of a household spray cleaner dispensed from 

either a hand-trigger pump bottle or an aerosol can (Furtaw et al., 1997). The temperature of 

the room was 23 °C, the humidity was monitored but not controlled, outdoor air exchange rate 

was set at approximately 0.5 air changes per hour. The cleaning product contained 5 % 

EGBE. Subjects were asked to clean two "Formica" sheets (approximately 0.76 m x 1.83 m), 

one being placed horizontally in the room, the other being vertical on the wall. The cleaning 

process, which consisted on spraying the cleaning product on the "Formica" surface then 

wiping it with a cotton cloth continued approximately for 20 minutes. About 28 g of the 

product was used during the cleaning process. Five tests were performed. During the cleaning 

period, air from the breathing zone of the experimenter and air in a corner of the room were 

sampled on a charcoal tubes (type ORBOTM-32) and analysed on an automated gas 

chromatograph (Microsensor systems Inc. Model MSI-301). During the cleaning, the 

experimenter wore cotton gloves over PVC gloves. The purpose of the cotton gloves was to 

absorb cleaning solution that would have been in contact with the hands of the person if he 

had not been wearing gloves. The amount of EGBE transferred to the gloves is about 6.3 % ±
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3.8 % of the quantity used on the right hand and1.6% ± 3.0 % on the left one and a greater 

amount is found on the frontal area of the hand than on the back of the hand. The mass and 

size of particles emitted decay much faster than the air EGBE concentration in the room that 

suggests that EGBE is being reversibly absorbed and re-emitted from chamber surfaces. 

Median diameters of particles vary from 0.749 to 0.976 μm. The personal average ORBOTMtube result was 5.70 ppm. 

The interactions between EGBE and indoor materials were studied by placing the indoor 

material as a membrane between two air compartments. Both compartments are constantly 

flushed with air, the airflow containing EGBE leading to the primary cell, the other airflow 

with clean air entering the secondary cell. A permeation factor was defined as: PF(%) = CA x 

100/CB where CA is the outlet concentration of the secondary cell and CB the outlet 

concentration of the primary cell. Average inlet concentration of EGBE was 483.7 μg/m3 with 

a standard deviation of 2.2 μg/m3

. PF values were 88.1 % and 9.4 % for wallpapers, 70.4 % 

for acrylic wall-covering, 61.6 % for carpets. The capacity of retention of each material was 

also calculated. It varies from 5.3 to 33.3 % (mean value = 19.2 %; SD = 9.8 %) according to 

the category of wall paper (n=6), 3.1 % for the textile wall-covering (n=1), 82.9 % for the 

carpet (n=1), 37.4 for the chipboard (n=1), 12.1 for the PVC flooring (n=1) and 80.4 % for the 

gypsum board (n=1). All those materials can release EGBE after the use of cleaning products 

(Meininghaus and Knoppel, 1999). 

NICNAS (1996), using data available from monitoring of workers using window cleaners 

(n = 23) or surface cleaners (n = 19), estimates that a good worst-case estimate for 

concentrations in air during the use of cleaning solutions is: 

- 10 ppm (49 mg/m3

) for a 30 % cleaning solution 

- 2 ppm (9.8 mg/m3

) for a 0.1 or 1 % cleaning solution 

- 4 ppm (19.6 mg/m3) for a 10 % cleaning solution 

OECD (1997) estimated the exposure of consumers by cleaning products to 0.58 mg/kg/event 

assuming a respiratory rate of 0.8 m3/hour and a bodyweight of 60 kg for a 1 hour cleaning 

event. For dermal absorption, a skin absorption rate of 0.2 mg/cm2/hour and a skin surface of 

1,000 cm2

 were assumed. 

Zhu et al. (2001) made the determination of EGBE emissions from selected consumer 

products and made assessment of the inhalation exposure associated with cleaning tasks. Two 

all-purposes cleaners were tested and two spray glass cleaners. Concentrations of the products 

ranged from 0.744 % to 1.28 % for general-purposes cleaners, from 0.50 to 0.87 for glass 

cleaners. A standard room of volume 17.4 m3

 was used for the test. An average air exchange 

of 0.5 h-1 was applied to the standard room. It was assumed that for all-purpose spray-cleaners 

a mass of 76 g of product was applied on a surface of 4.5 m2

 for each task whereas a mass of 

17 g of spray glass cleaners was applied to a surface of 2.3 m2 for each task. The test included 

four various tasks for general-purposes cleaners: clean outside of cabinets during 0.87 hours, 

clean counters during 0.42 hours, clean bathroom or other tiled or ceramic walls during 0.57 

hours, clean outside of refrigerator and other appliances during 0.32 hours. Two tasks were 

assessed for glass cleaners: clean inside of windows during 2.12 hours, clean other glass 

surfaces like mirrors or tables during 0.40 hours. Samples of air in the chamber were collected 

every half hour or every hour or every 2 hours depending on the stage of the emission. An 

inhalation rate of 1.3 m3

/h and a mean bodyweight of 70.9 kg were used for exposure 

assessment. The daily exposure by task ranged from 0.013 to 0.063 mg/kg bw/d if only one of 

the tasks were performed in one day (mean value = 0.0325mg/kg bw/d; standard deviation = 
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0.017 mg/kg bw/d). If they were all performed on the same day, the sum of daily intake by 

inhalation is 0.186 mg/kg bw/d with the first product and 0.075 mg/kg bw/d with the second. 

In the case of glass cleaners, the daily exposure by task ranged from 0.001 to 0.004 mg/kg 

bw/d if only one of the tasks were performed in one day (mean value = 0.003 mg/kg bw/d; 

standard deviation = 0.001 mg/kg bw/d). If they were all performed on the same day, the sum 

of daily intake by inhalation is 0.006 mg/kg bw/d with the first product and 0.004 mg/kg bw/d 

with the second. 

Fortmann et al. (1999) performed tests to determine gas-phase and particulate emissions 

during application of a water-based cleaner with a hand-pump sprayer. Tests were performed 

in the bathroom of a test house and in an 18m3

 controlled-environment test-room. The waterbase cleaner which contained 50 mg/g of EGBE was applied to realistic surfaces (counter 

tops, glass, mirrors) with the manufacturer's hand-pump spray. During application, air 

samples were collected on Tenax for analysis of EGBE by gas chromatography. 

Concentrations of droplets were measured continuously with aerodynamic particle analysers. 

The average concentration of EGBE measured by collection on Tenax for a 10-minute period 

in a closed bathroom during and following application of 96 g of a 50/1 (v/v) dilution of the 

cleaner was 760 μg/m3. The mean particle droplet size during the application period was in 

the range of 1 to 2 μm. Particle concentrations increased during application with the sprayer 

but dropped quickly. 

Table 4.30: summary of studies about exposure by household cleaning products 

Study Route of 

exposure 

Concentration of 

EGBE in product

Duration of 

exposure 

number of 

tests 

Concentration Daily exposure 

Furtaw et 

al., 1997 

Inhalation 5 % 20 min n = 5 5.7 ppm (28 

mg/m3) in air 

Furtaw et 

al., 1997 

Skin 5 % 20 min n = 5 6.3 ± 3.0 % 

(right hand) 

1.6 ± 3.0 % (left 

hand) 

Meininghaus 

and 

Knoppel, 

1999 

Inhalation 0.48 mg/m3 in air 

30 % 10 ppm (49 

mg/m3

) in air 

10 % 4 ppm (19.6 

mg/m3

) in air 

NICNAS, 

1996 

Inhalation 

0.1 to 1 % 

n = 42 

2 ppm (9.8 

mg/m3

) in air 

OECD, 1997 Inhalation 

+ skin 

 1 hour modelled 

data 

 0.58 mg/kg/event 

Zhu, 2001 Inhalation 0.74 – 1.28 % 

0.5 – 0.87 % 

0.32-2.18 hr

0.4 – 2.52hr

n = 2 

n = 2 

2.8 – 62 mg/m3 0.013-0.063mg/kg/day 

0.001-0.004mg/kg/day 

Fortmann et 

al., 1999 

Inhalation 0.1 % 10 min n = 5 0.76 mg/m3 in air 
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The risk assessment will be based on data from NICNAS (1996) and AISE (2002) for 

inhalation exposure, assuming a cleaning product containing 10 % EGBE leading to an air 

concentration of 19.6 mg/m3 and a duration of exposure of 20 minutes (1/3 hour)for each 

event. The concentration of 10% will be chosen as a reasonable worst case as according to the 

Danish product register (2001) (cf table 4.1), products containing 10 % EGBE or less 

represents 76 % of the whole market of products containing EGBE (93 % contain 20 % or less 

EGBE). According to the french register SEPIA from INRS (2003), in 81 % of French 

cleaning products containing EGBE, its concentration is equal or less than 10 % (93% contain 

20 % or less EGBE). These data are furnished without any distinction between products sold 

to general consumers and these sold in the occupational field and generally, cleaning products 

sold in the occupational field are more concentrated in cleaning substances than those sold to 

consumers are. Data indicates that the great majority of cleaning products available to 

consumers contain less than 10% EGBE. It cannot be excluded that some products may be 

available with higher levels. However, anecdotal evidence suggests that such products are 

either used in small amounts (e.g. spot cleaners) or are diluted before use. Combined with 

other evidence to indicate a decline in use in this application, a figure of 10% is set as a 

reasonable worse case for the composition of EGBE in consumer cleaning products for the 

scenarios modelled. The worst case considered will be the use of undiluted product. 

For inhalation, based on a 20 m3

 respiratory volume a day for an adult weighing 60 kg, the 

exposure will be: 

20 x 19.6 x 1 

60 x 24 x 3 

= 0.09 mg/kg/d 

For skin contact, measured data from Furtaw et al. (1997) will be used to characterise each 

event : use of 28 g of undiluted product for a 20 minutes event (AISE, 2002) is considered as 

the worst case, transfer of 6.3 + 3.8 % of the quantity used on the right hand and of 1.6 + 3.0 

% to the left hand. For a 10 % EGBE product, the dermal exposure will be: 

0.1 x 28 x 1000 x (0.10 + 0.05) 

60 

= 7 mg/kg/d 

Scenario 2: Measurements in indoor air

Measurements of EGBE concentrations were performed in the indoor air of flats (Kirchner, 

2002). Preliminary results indicate that the 90th percentile is 3 μg/m3

 in the sleeping-room 

with a high value of 14 μg/m3

 in a flat. The 90th percentile is 3.5 μg/m3 in the kitchen with a 

high value of 24 μg/m3

 in a flat. Releases from building materials were also evaluated. 

Simulations were made by installing new carpets or new floor coverings or by applying paints 

on the walls of reference rooms. After 24 hours, the concentrations of EGBE in air ranged 

from below the detection limit to 3.8 μg/m3

 (mean value = 1.7 μg/m3; SD = 1.5 μg/m3) when 

release from carpets were tested. After 28 days, it ranged from non detectable to 23.3 μg/m3

(mean value = 6.1 μg/m3

; SD = 9.0 μg/m3). With other categories of floor coverings, it ranged 

from non detectable to 5.6 μg/m3 (mean value = 2.5 μg/m3; SD = 2.7 μg/m3) after 24 hours 

and from non detectable to 59.6 μg/m3 (mean value = 27.5 μg/m3; SD = 30.9 μg/m3) after 28 
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days. Release by paints applied on the wall were at non detectable values after 24 hours and 

reached 24 and 298 μg/m3 respectively for the two paints tested 28 days after application. 

The same type of measurements was performed in apartments in Italy: in five of them, EGBE 

was not detected, in a sixth, it was found at a concentration of 8 μg/m3

 (De Bortoli et al.,

1986). 

A study of the daily indoor concentrations for all indoor site types (n = 14) gave the following 

results: average air pollution by EGBE = 0.214 ppb (1 μg/m3

); median concentration = 0.075 

ppb (0.4 μg/m3); 25th percentile = 0.030 ppb (0.1 μg/m3); 75th percentile = 0.360 ppb (1.7 

μg/m3

) (Shah and Singh, 1988). 

According to data furnished by the FIPEC and SIGMAKALOM (Conseil supérieur d'hygiène 

publique de France (CSHPF), 2001), consumers should inhale 1 g of EGBE during a period of 

30 days. 

Table 4.31: summary of studies about exposure by indoor air 

Study Concentrations in air Number of 

measurements Mean values Reasonable Worst case exposure 

Bedroom : 3 μg/m3*

(highest value : 14 

μg/m3

 in 1 flat) 

Kitchen : 3.5 μg/m3

(highest value : 24 

μg/m3 in 1 flat)

n = 57 24 μg/m3

in 1 flat)

Release from carpets, 0-

28days : non detectable 

to 23.3 μg/m3

n = 6 

24 hours after : 

1.7 μg/m3 

28 days after : 

6.1 μg/m3

23.3 μg/m3

Release from new floor 

covering, 0-28days : 

non detectable to 59.6 

μg/m3

n = 5 

24 hours after : 

2.5 μg/m3 

28 days after : 

27.5 μg/m3

59.6 μg/m3

Kirchner 

(2002) 

Release from paints, 0-

28days : non detectable 

to 298 μg/m3

n = 2 298 μg/m3

De Bortoli et 

al. (1986) 

8 μg/m3 (Found in only 

1 out of 6 flats) 

surveillance in 

6 flats during 

1983 et the 

beginning of 

1984 

n = ? 

 8 μg/m3

Shah and 

Singh (1988) 1.7 μg/m3 ** n = 14 1 μg/m3

 1.7 μg/m3

CSHPF 

(2001) 1g/30 days modelled data by WPEM*** 1g/30 days 

* 90th percentile 

** 75th percentile 

*** WPEM = Wall Paint Exposure assessment developed by US EPA
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As a worst case approach, the scenario chosen will be: rooms with paints releasing 298 μg/m3

of EGBE and with a new covering floor releasing 59.6 μg/m3. For this scenario, the only 

exposure is by inhalation of indoor air. For inhalation; based on a 20 m3 respiratory volume a 

day for an adult weighing 60 kg and a 20 hours duration of exposure, the exposure will be: 

20 x (59 + 298) x 20

60 x 24 

= 0.1 mg/kg/d 

Scenario 3: Painting

Studies have been made to evaluate indoor concentrations of EGBE from paints. 

Norbäck et al (1995 and 1996) measured painters exposure to EGBE by sampling air in their 

breathing zone for one hour as they were applying various water-based paints: wall paints, 

ceiling paints, wood paints and wet room paints (N = 20 samples). All paints were applied by 

roller except one case of wall paint that was sprayed. Most of the measurements (90 %) were 

performed during renovation of old buildings. The average room temperature was 19.3 °C 

(17.5-20.5 °C) and the relative air humidity ranged from 34 % to 83 % (arithmetic mean value 

= 58 %). EGBE was detected during painting of walls and wet room painting. The arithmetic 

mean for EGBE is a concentration of 59.6 μg/m3

, the geometric mean is 8 μg/m3 (geometric 

standard deviation = 4.2 μg/m3

) with a highest value of 730 μg/m3. 

Hansen et al. (1987) measured volatiles emitted during the use of waterborne paints in one 

room during 3 days. 13 samplings were performed by adsorbing the organic vapours present 

in the ambient air.. The organic vapours were adsorbed on polymer in cartridges of stainless 

steel (Tenax AT, 60-80 mesh). The sampling time was 20 minutes corresponding to a 

sampling volume of 0.9 l. Samples were analysed by gas chromatography using a flameionisation detector. The content of EGBE in the paints ranged from 0 to 1.4 % w/w and the 

concentration in air of the workplaces ranged from 2 to 60 mg/m3 . 

OECD (1997) estimated the exposure of consumers by paints and surface coatings to 1.05 

mg/kg/event assuming a respiratory rate of 0.8 m3/hour and a bodyweight of 60 kg for a 6 

hour painting period. For dermal absorption, a skin absorption rate of 0.2 mg/cm2

/hour and a 

skin surface of 1,000 m2

 were assumed. 

EPA (2001) studied the emissions of EGBE from paints applied on two types of surfaces: 

stainless steel plate and gypsum board. The concentrations of EGBE in the paints ranged from 

4.68 to 5.20 mg/g (mean value = 4.98, n = 4). Peak concentrations measured in the air of the 

environment chambers were higher when the surface is a stainless steel plate (11.6 mg/m3

) 

than on gypsum board (5.14 mg/m3). 93 % of the EGBE is emitted during the 336-hour 

testing period when applied on stainless steel plate, 14 % when painting on gypsum board. 

The study was prolonged with gypsum board. After a rapid decrease in concentrations of 

EGBE in the environment chambers, levels of EGBE in the air remain at values near 0.01 

mg/m3 after 3 months. 

CEPE (2002) considers that a 3% concentration in paints can be considered as a reasonable 

worst case for decorative paints based on data gathered from their member companies (see 

table 4.14). 
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Table 4.32: Summary of studies about exposure by paints 

Study Average 

exposure 

Reasonable Worst 

case exposure 

number of 

measurements 

Comment 

Norback, 1995-1996 59.6 μg/m3 730 μg/m3 N = 20 

Hansen et al., 1987 2-60 mg/m3 N = 13 EGBE content in paint 

0-1.4 % 

OECD, 1997 1.05 

mg/kg/event 

 modelled data 6 hours painting period 

5.14 mg/m3

to 11.6 

mg/m3 

11.6 mg/m3 N = 4 2 weeks. Non adsorbent 

surface. Max EGBE 

content in paint 0.52 % 

EPA, 2001 

0.01 mg/m3

 0.01 mg/m3 value 3 months after 

painting 

CEPE, 2002 EGBE content in paint 3 

% 

The CONSEXPO model can also be used to estimate exposure. Another alternative model is 

the USEPA Wallpaint model. The parameters used in both models are shown in Appendix A. 

The calculated inhalation exposure results are : 

 CONSEXPO (worst case 

inhalation exposure) 

WPEM inhalation exposure 

Exposure per event (over 133 

minutes) 

377 mg/m3 144 mg/m3 instantaneous (8 hr 

TWA = 27 mg/m3

) 

Calculated external dose per 

event (60 kg/person) 

11.6 mg/kg/d Based on TWA : 3mg/kg/d 

Model prediction : 19.8 mg/kg/d 

To verify if this value can be considered as realistic, the same calculation was performed with 

a 1.5 % concentration of EGBE in paints to compare with the value obtained with measured 

data. 

A reasonable worst case inhalation exposure can be based on data from Hansen et al. (1987) 

for a 1.5% concentration of EGBE as the data were collected in real situations (renovation of 

old apartments in a building) and frequent samplings were performed during a 3 days work. 

Assuming a respiratory rate of 20 m3/day, a 6 hours event in one day and a mean bodyweight 

of 60 kg, the inhalation exposure by paints is: 

20 x 60 x 6 

60 x 24 

= 5 mg/kg/d 

Modelling the exposure according to the Technical Guidance Document, the inhalation 

exposure linked to the use of a 1.5% EGBE paint should be: 64.2 mg/kg/d. This shows that in 

this case, the modelling strongly overestimates the inhalation exposure (factor = 12.84). 

For CONSEXPO and WPEM models, calculations for a paint containing 1.5 % EGBE give 

the following results : 
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 CONSEXPO (worst case 

inhalation exposure) 

WPEM inhalation exposure 

Exposure per event (over 133 

minutes) 

188.5 mg/m3 72 mg/m3 instantaneous (8 hr 

TWA = 27 mg/m3

) 

Calculated external dose per 

event (60 kg/person) 

5.8 mg/kg/d Based on TWA : 1.5 mg/kg/d 

Model prediction : 9.9 mg/kg/d 

Comparison with measured 

data 

116 % of measured data 198 % of measured data 

As all models give an overestimation of consumer exposures, we have chosen to consider that 

a 3 % EGBE concentration in paint leads to a concentration of EGBE in air of 120 mg/m3 (60 

mg/m3 x 2 – 20 min TWA) and an external body burden exposure by inhalation of : 10 

mg/kg/d. 

This value will be used as the worst case for inhalation exposure in this scenario. 

For dermal exposure, the modelling proposed in the Technical Guidance document will be 

used. So the external exposure (Eed) of skin to EGBE from paints will be: 

C prod x TH der x AREA der x n Eed = D x BW 

where : 

C prod = concentration of EGBE in paint (kg/m3

) 

TH der = thickness of product layer on skin 

AREA der = area of contact between paint and skin 

n = number of events (n = 1) 

D = dilution. There is no dilution so D = 1 

BW = mean bodyweight for a consumer (60 kg) 

Assuming the concentration of EGBE in the paint is 3 % i.e. 30 kg/m3 (CEPE, 2002), the 

surface of the skin exposed is 840 cm2 (840 x 10 –4 m2

), the thickness of paint layer on the 

skin is 10-4 m, the external exposure of skin by paints will be: 

30 x 10-4 x 840 x 10-4 x 1 Ee = 1 x 60 = 4.2 mg/kg/d 

4.1.1.3.2 Summary of consumer exposure 

Table 4.33: Summary of proposed “reasonable worst-case” exposures in the main scenarios 

Scenario Inhalation 

(mg/kg/d)

Skin 

(mg/kg/d)

1 – Household surface cleaners 0.09 7 

2 – Indoor air 0.1 

3 – Painting 10 4.2 
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For risk characterisation, internal exposures will be estimated for each scenario. 

As a consumer is also exposed to indoor air when he paints, or uses household cleaners, we 

will also include in the risk characterisation, the scenarios: 

- household surface cleaners + indoor air 

- paints + indoor air 

Table 4.34: Summary of combined worst case scenarios 

Scenario Inhalation 

(mg/kg/d)

Skin 

(mg/kg/d)

Household surface cleaners + indoor air 0.19 7 

Paintings + indoor air 10.1 4.2 

4.1.1.4 Humans exposed via the environment 

The information relating to the estimation of the indirect exposure of humans via the 

environment are presented in table 1. The concentrations calculated in intake media (drinking 

water, fish, plant roots and leaves, milk, meat, air) and the subsequent estimation of human 

intakes via different routes are shown hereafter with the corresponding total daily intakes. 

Both local and regional levels are taken into consideration and the estimation of local 

environmental exposures has been performed for all scenarios listed in chapter 3.1.2.2. 

Concerning the production step, only the worst case has been reported. Calculations have 

been performed using default parameters in EUSES except for the use of an absorption factor 

via inhalation of 60% and a body weight of 60kg.
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Table 4.1: exposure assessment for human via the environment (concentrations in foodstuff, air and drinking water and connected dail

Conc. in drinking water (mg.L-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in wet fish (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in plant roots (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in plant leaves (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in milk (mg.kg-1 ww) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in (mg.kg-1 

Subsequdose (mg

Production (site-specific, worst case) 1.20.10-1 / 4.01.10-3 1.50.10-2 / 2.88.10-5 1.22.10-1 / 7.79.10-4 4.87.10-2 / 9.75.10-4 7.89.10-5 / 7.37.10-7 7.89.10-6 Paints IF

Paints IP

5.00.10-2 / 1.67.10-3

1.25.10-1 / 4.16.10-3

7.06.10-2 / 1.35.10-4

1.76.10-1 / 3.38.10-4

1.81.10-2 / 1.16.10-4

5.34.10-2 / 3.41.10-4

9.75.10-2 / 1.95.10-3

1.24 / 2.48.10-2

7.72.10-5 / 7.22.10-7

7.61.10-4 / 7.11.10-6

7.72.10-6 

7.61.10-5 

Paints IIF and IIIF

Paints IIP and IIIU

5.12.10-2 / 1.71.10-3

1.82.10-2 / 6.07.10-4

7.23.10-2 / 1.38.10-4

2.57.10-2 / 4.93.10-5

1.85.10-2 / 1.19.10-4

7.45.10-3 / 4.77.10-5

0.10 / 2.00.10-3

7.99.10-2 / 1.60.10-3

7.92.10-5 / 7.40.10-7

5.34.10-5 / 4.99.10-7

7.92.10-6 

5.34.10-6 

Paints IVF

Paints IVP

7.35.10-3 / 2.45.10-4

1.70.10-2 / 5.67.10-4

1.04.10-2 / 1.99.10-5

1.04.10-2 / 1.99.10-5

6.40.10-3 / 4.09.10-5

1.72.10-2 / 1.10.10-4

3.54.10-1 / 7.08.10-3

1.53 / 3.05.10-2

2.05.10-4 / 1.92.10-6

8.76.10-4 / 8.19.10-6

2.05.10-5 

8.76.10-5 

Paints VF

Paints VP

7.35.10-3 / 2.45.10-4

1.64.10-2 / 5.47.10-4

1.04.10-2 / 1.99.10-5

2.32.10-2 / 4.45.10-5

4.05.10-3 / 2.59.10-5

6.27.10-3 / 4.01.10-5

0.10 / 2.00.10-3

1.56.10-2 / 3.12.10-4

6.01.10-5 / 5.62.10-7

1.60.10-5 / 1.50.10-7

6.01.10-6 

1.60.10-6 

Paints VIF

Paints VIP

1.54.10-1 / 5.13.10-3

9.67.10-2 / 3.22.10-3

2.17.10-1 / 4.17.10-4

1.37.10-1 / 2.62.10-4

5.20.10-2 / 3.33.10-4

3.28.10-2 / 2.10.10-4

5.35.10-2 / 1.07.10-3

1.64.10-2 / 3.27.10-4

9.71.10-5 / 9.08.10-7

5.12.10-5 / 4.79.10-7

9.71.10-6 

5.12.10-6 

Paints VIIF

Paints VIIP

5.86.10-2 / 1.95.10-3

1.45.10-2 / 4.83.10-4

8.27.10-2 / 1.59.10-4

2.05.10-2 / 3.92.10-5

2.02.10-2 / 1.30.10-4

9.42.10-3 / 6.03.10-5

2.10.10-2 / 4.20.10-4

4.26.10-1 / 8.52.10-3

3.73.10-5 / 3.49.10-7

2.49.10-4 / 2.33.10-6

3.73.10-6 

2.49.10-5 

Paints VIIIF

Paints VIIIP

7.35.10-3 / 2.45.10-4

7.35.10-3 / 2.45.10-4

1.04.10-2 / 1.99.10-5

1.04.10-2 / 1.99.10-5

3.70.10-3 / 2.37.10-5

3.18.10-3 / 2.04.10-5

6.13.10-2 / 1.23.10-3

5.87.10-3 / 1.17.10-4

3.81.10-5 / 3.56.10-7

6.56.10-6 / 6.13.10-8

3.81.10-6 

6.56.10-7 

Paints IXF

Paints IXP

2.20.10-2 / 7.33.10-4

9.12.10-3 / 3.04.10-4

3.11.10-2 / 5.95.10-5

1.29.10-2 / 2.47.10-5

8.35.10-3 / 5.35.10-5

5.82.10-3 / 3.72.10-5

4.22.10-2 / 8.44.10-4

2.28.10-1 / 4.55.10-3

3.36.10-5 / 3.14.10-7

1.34.10-4 / 1.25.10-6

3.36.10-6 

1.34.10-5 

Detergents IF

Detergents IP

1.76.10-2 / 5.87.10-4

3.28.10-2 / 1.09.10-3

2.49.10-2 / 4.77.10-5

4.63.10-2 / 8.88.10-5

6.58.10-3 / 4.21.10-5

1.58.10-2 / 1.01.10-4

6.66.10-3 / 1.33.10-4

4.00.10-3 / 8.01.10-5

1.15.10-5 / 1.07.10-7

1.64.10-5 / 1.54.10-7

1.15.10-6 

1.64.10-6 

Detergents IIF 1.16.10-2 / 3.87.10-4 1.16.10-2 / 3.15.10-5 4.59.10-3 / 2.94.10-5 4.84.10-3 / 9.68.10-5 7.82.10-6 / 7.31.10-8 7.82.10-7 
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Conc. in drinking water (mg.L-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in wet fish (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in plant roots (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in plant leaves (mg.kg-1) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in milk (mg.kg-1 ww) / 

Subsequent daily dose (mg.kg-1.d-1) 

Conc. in (mg.kg-1 

Subsequdose (mg

Detergents IIU 1.67.10-2 / 5.56.10-4 2.35.10-2 / 4.51.10-5 5.69.10-3 / 3.64.10-5 3.63.10-3 / 7.27.10-5 9.32.10-6 / 8.72.10-8 9.32.10-7 Intermediates IP 2.29.10-2 / 7.63.10-4 3.17.10-2 / 6.08.10-5 2.31.10-2 / 1.48.10-4 4.83.10-3 / 9.66.10-5 1.25.10-5 / 1.17.10-7 1.25.10-6 Inks IF

Inks IP

9.36.10-2 / 3.12.10-3

8.75.10-3 / 2.92.10-4

1.32.10-1 / 2.53.10-4

1.24.10-2 / 2.37.10-5

3.19.10-2 / 2.04.10-4

8.73.10-3 / 5.58.10-5

3.29.10-2 / 6.59.10-4

5.56.10-1 / 1.11.10-2

5.93.10-5 / 5.54.10-7

3.21.10-4 / 3.00.10-6

5.93.10-6 

3.21.10-5 

Metal IF

Metal IP

6.27.10-2 / 2.09.10-3

1.18.10-2 / 3.92.10-4

8.85.10-2 / 1.70.10-4

1.62.10-2 / 3.11.10-5

2.18.10-2 / 1.39.10-4

1.19.10-2 / 7.60.10-5

4.06.10-2 / 8.12.10-4

7.99.10-1 / 1.60.10-2

5.03.10-5 / 4.70.10-7

4.61.10-4 / 4.31.10-6

5.03.10-6 

4.61.10-5 

Pharm IP 9.95.10-3 / 3.32.10-4 1.12.10-2 / 2.14.10-5 1.01.10-2 / 6.44.10-5 4.52.10-3 / 9.04.10-5 6.84.10-6 / 6.39.10-8 6.84.10-7 Elec IP 2.44.10-2 / 8.13.10-4 1.41.10-2 / 2.69.10-5 2.46.10-2 / 1.58.10-4 5.00.10-3 / 1.00.10-4 1.32.10-5 / 1.24.10-7 1.32.10-6 Leather IP 1.60.10-1 / 5.33.10-3 2.26.10-1 / 4.33.10-4 7.95.10-2 / 5.09.10-4 6.36.10-3 / 1.27.10-4 7.25.10-5 / 6.78.10-7 7.25.10-6 Adhesives IF

Adhesives IP

2.10.10-2 / 7.01.10-4

8.10.10-3 / 2.70.10-4

2.97.10-2 / 5.70.10-5

1.14.10-2 / 2.19.10-5

7.73.10-3 / 4.95.10-5

4.65.10-3 / 2.98.10-5

8.20.10-3 / 1.64.10-4

3.59.10-3 / 7.18.10-5

1.38.10-5 / 1.29.10-7

5.57.10-6 / 5.21.10-8

1.38.10-6 

5.57.10-7 

Agri IF

Agri IP

1.46.10-2 / 4.87.10-4

7.54.10-3 / 2.51.10-4

2.06.10-2 / 3.95.10-5

1.07.10-2 / 2.04.10-5

6.48.10-3 / 4.14.10-5

4.13.10-3 / 2.64.10-5

7.02.10-3 / 1.40.10-4

5.07.10-3 / 1.01.10-4

1.03.10-5 / 9.67.10-8

6.19.10-6 / 5.78.10-8

1.03.10-6 

6.19.10-7 

Oilfield IFOilfield IP

9.36.10-2 / 3.12.10-3

1.11.10-2 / 3.71.10-4

1.32.10-1 / 2.53.10-4

1.57.10-2 / 3.01.10-5

3.19.10-2 / 2.04.10-4

1.07.10-2 / 6.82.10-5

3.29.10-2 / 6.59.10-4

3.81.10-3 / 7.62.10-5

5.93.10-5 / 5.54.10-7

6.94.10-6 / 6.49.10-8

5.93.10-6 

6.94.10-7 

Cosmet IF and Fire IF Fire IP

Cosmet IU

3.49.10-2 / 1.16.10-3

7.78.10-3 / 2.59.10-48.70.10-3 / 2.90.10-4

4.93.10-2 / 9.44.10-5

1.10.10-2 / 2.11.10-51.23.10-2 / 2.35.10-5

1.23.10-2 / 7.90.10-5

4.02.10-3 / 2.57.10-53.53.10-3 / 2.26.10-5

1.29.10-2 / 2.58.10-4

3.57.10-3 / 7.14.10-53.55.10-3 / 7.10.10-5

2.25.10-5 / 2.10.10-7

5.42.10-6 / 5.07.10-85.28.10-6 / 5.44.10-8

2.25.10-6 

5.42.10-7 5.28.10-7 

Regional 7.35.10-3 / 2.41.10-4 1.04.10-2 / 1.96.10-5 1.47.10-3 / 1.11.10-5 3.48.10-3 / 4.05.10-5 5.19.10-6 / 4.06.10-8 5.19.10-7 
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The highest indirect exposure is estimated for the processing of can coating (Paints IVP) : 

3.73.10-2 mg.kg-1.day-1. It can also be noted that the highest exposures are to be expected 

through intake of drinking water and plants (leaves and roots). Moreover, based on the 

regional concentrations, the total daily intake for humans is 3.32.10-4 mg.kg-1.day-1. These two 

figures will be taken forward into the risk characterisation. 

4.1.2 Effects assessment: Hazard identification and dose (concentration)- 

response (effect) assessment 

4.1.2.1 Toxicokinetics, metabolism and distribution 

4.1.2.1.1 Studies in animals 

Inhalation 

Rat 

Sixteen male SD rats were exposed continuously (whole body) to EGBE (purity 99 %) by 

inhalation at doses of 20 ppm or 100 ppm for 1, 2, 3, 4, 6, 8, 10 or 12 days (Johanson, 1994). 

Urine was collected in 24-hr interval periods. After sacrifice, tissue samples of blood, muscle 

and liver were collected and analysed for EGBE and Butoxy Acetic Acid (BAA) content. 

No clinical signs of toxicity were noted during the duration of the study. In all the tissues 

examined, the EGBE concentration rapidly increased during the first three days and continue 

to increase slower during the remaining days of exposure. The table 4.35 shows the tissue 

concentrations reached in the 20 ppm exposure group. 

Table 4.35: Average tissues concentrations of EGBE and BAA following 20 ppm exposure 

Tissue Blood Liver Muscle Testis 

EGBE 

concentration 

10 – 20 μmol/l 10 μmol/l 10 μmol/l 5 μmol/l 

BAA concentration 30 – 40 μmol/l 15 – 20 μmol/l 10 μmol/l 10 μmol/l 

Following a 100 ppm exposure the tissues concentrations were approximately 5 times higher. 

(5 times higher in blood, 3.5 and 3.6 in muscle and testis respectively and 7.5 in the liver). 

The blood clearance of EGBE was estimated to 2.6 ± 1.3 l/h/kg bw whichever the dose 

administered. The kinetic profile of BAA was approximately the same as EGBE. The 

100ppm/20ppm concentration ratio were: 4.4 for blood, 4.7 for muscle, 2.3 for testis and 5.3 

for liver. The urinary excretion of BAA averaged 0.2 mmol/day in the 20 ppm group and 1.03 

mmol/day in the 100 ppm group. This correspond to 64 % of the calculated respiratory 

uptake. The renal clearance was 0.53 l/h/kg. 

[

14C]EGBE (purity 99 %) was administered to male F 344/N rats by inhalation (nose only – 

vapour form) for 6 hr at doses of 5, 50 or 450 ppm (Sabourin et al., 1992a). One group of 5 

rats was used to determine the fractional uptake of inhaled EGBE and the body burden of the 
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compound at the end of the exposure. Respiratory measurements were performed on this 

group. After termination, the entire carcass was digested. The 14C in these rats was used as a 

measure of the body burden. A second group of 4 rats was used to determine the paths of 

excretion of EGBE equivalents and the major metabolites excreted. Urine, faeces and exhaled 

breath were collected for 66 hr following the end of the 6 hr exposure. The rats were 

terminated and the carcass was digested and analysed for 14C. A third group of 30 rats was 

used for analysis of blood 14C and blood metabolite concentration at the 5 ppm exposure 

concentration only. At various time points and up to 24 hr following the start of exposure, 3 

rats were euthanized and bled by cardiac puncture. Blood samples were analysed for 

haematocrit as well as 14C associated with whole blood and plasma where 

14C associated 

with red blood 

cells 

= 

14C associated 

with whole 

blood 

- 

14C associated 

with plasma 

Plasma metabolites were also analysed. 

The respiratory rate and tidal volume were not significantly different form those of unexposed 

rats for any of the exposure. The amount of EGBE inhaled was proportioned to the exposure 

concentration in the 5 and 50 ppm exposures. A less than proportional amount of EGBE was 

inhaled at the high exposure concentration due to a lower minute volume. 

At all exposure concentrations, the majority of 14C was eliminated in the urine, less than 7 % 

of the parent compound was exhaled following the exposure. 10-20 % of the (14C)EGBE 

equivalent remained in the carcass up to 66 hr post exposure at all concentrations. 7 % of the 

excreted 14C was in the form of 14CO2 (see table 4.36). 

Table 4.36: Excretion of [14C]EGBE 

[

14C]EGBE equivalents as percentage of amount metabolised 

Exposure concentration in ppm (values in μmol) 

Route of excretion 5 50 450 

14CO2 during exposure 0.2 (0.008) 1.3 (0.6) 0.9 (3.5) 

14CO2 post exposure 7.6 (0.25) 7.2 (3.3) 5.9 (22.7) 

Urine 67.3 (2.3) 64 (31.4) 75.9 (296.8) 

Faeces 1.6 (0.06) 2.3 (1.1) 1.2 (4.6) 

Exhaled EGBE post exposure 3.3 (0.09) 5.1 (2.5) 2.3 (9) 

Carcass 19.8 (.068) 19.1 (9.4) 12.9 (50.6) 

Total 100 100 100 

Urinary 14C excreted during the first 41 hr represented more than 88 % of the total 14C 

excreted in urine. BAA was the major metabolite in urine at all exposure concentrations. EG 

and BEG were also found in lesser amounts (see table 4.37). As the exposure concentration 

increased, the proportion of 2 unidentified minor metabolites increased. At 5 ppm about 60 % 
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of the urinary 14C was excreted during the 6 hr exposure. Whereas at 450 ppm only 10 % was 

excreted during the exposure. The urinary metabolites profile varied with time following 

exposure : metabolism to Glucuronide conjugate of EGBE (BEG) was favoured during the 

exposure whereas metabolism to BAA and Ethylene Glycol (EG) was favoured at later time 

post exposure. This observation was more apparent at the high exposure concentration. 

Metabolism of EGBE leading to EG and BAA appear to be easily saturable. 

Table 4.37: Urinary metabolites of EGBE 

Percentage of retained dose excreted in urine by 41 hr 

Exposure concentration in ppm (values in μmol) 

Metabolite 5 50 450 

EG 16.1 (0.55) 13.4 (6.3) 7.9 (34) 

BAA 43.2 (1.47) 41.9 (19.9) 36.6 (143) 

BE glucuronide 3.4 (0.20) 6.7 (3.2) 10.4 (40) 

4 min unknown ND 10.5 (0.5) 5.3 (22) 

10 min unknown ND ND 1.5 (6) 

Others 0.5 (0.03) 0.7 (0.4) 2.2 (10) 

Analysis of whole blood as well as plasma indicated that the majority of the EGBE equivalent 

were in the plasma. 

During the first 2 hr of exposure about 20 % of the blood 14C was associated with the red 

blood cell fraction. However at the later time points the proportion of 14C in the cellular 

fraction declined and was undetectable following exposure. Bound 14C metabolites was 

greater after the exposure than during the exposure. 

In an inhalation study, F344 rats were exposed whole body for 6h per day, 5 days per week 

for 104 weeks, to EGBE (purity > 99 %) at concentrations of 31.2, 62.5 and 125 ppm (Dill et 

al., 1998). Post exposure blood samples were collected after 1 day, 2 weeks and 3, 6, 12 and 

18 months for EGBE and BAA determination. The post exposure time points depends of the 

day of collection. Post exposure urine samples were collected from animals after 2 weeks and 

3, 6, 12 and 18 months of exposure. This study was also performed on mice. 

The EGBE blood concentrations showed a rapid drop after the cessation of exposure. The 

elimination t1/2 for EGBE after 1 day of exposure was < 10 min, this t1/2 does not depend on 

the dose level. Increases in the initial blood concentration of EGBE and Area Under the Curve 

(AUC) were proportional to exposure concentration, supporting that elimination of EGBE 

from the blood follows linear kinetics. At the common concentration levels (62.5 and 125 

ppm), mice eliminated EGBE faster than rats, values of t1/2 and AUCEGBE were significantly 

lower in mice than in rats (see table 4.38). The rate of EGBE elimination from blood slowed 

with increasing days of exposure, this change was more obvious in rats than mice as their t1/2 

and AUC dramatically increased after 3 months of exposure. Male rats generally exhibited 

larger AUC than females, indicating a lower blood clearance of EGBE. k was not 

significantly different between sexes, implying that the volume of distribution, rather than the 

rate of elimination might be responsible for the sex-related difference in EGBE clearance. 
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Unlike EGBE, BAA was not rapidly cleared from the systemic circulation. The BAA 

concentrations in the blood did not start to decline until 20 to 80 min post exposure. It seems 

that BAA was eliminated from blood following saturable, nonlinear kinetics. The k was not 

constant, decreasing as EGBE exposure concentrations increased. As the rate of BAA 

production reflects the EGBE elimination, it seems that the nonlinear characteristics of BAA 

kinetics likely result from BAA distribution or elimination processes. When the number of 

exposure days increased, the rate of 2BAA elimination tended to decrease. Females 

eliminated BAA more slowly than males (smaller k, longer t1/2 and larger AUC). The 

maximum blood concentration was also higher in females at each exposure concentration. In 

mice, elimination of BAA was faster than rats when compared at common exposure 

concentrations. There was a clear difference in the urinary excretion of BAA between rats and 

mice (see table 4.39). Female rats excreted less BAA than males regardless of exposure 

concentration. Excretion of BAA tended to increase with time. 

Table 4.38: Comparison of EGBE elimination between rats and mice at the same dose level tested. 

C0 (μg/g) k T1/2 (min) AUC (μg/min/g) 

62.5 ppm 

 Rats Mice Rats Mice Rats Mice Rats Mice 

1d 1.86 1.59 1.29 1.35 0.0793 0.0809 0.229 0.185 8.74 8.56 3.03 3.75 23.5 19.6 5.6 7.3 

2w 3.06 2.24 1.14 1.54 0.0828 0.0839 0.161 0.199 8.37 8.26 4.31 3.49 36.9 26.8 7.1 7.7 

3m 0.54 0.47 1.30 1.3 0.0166 0.0248 0.126 0.199 41.66 27.9 5.51 3.49 32.3 18.9 10.3 6.5 

6m 1.31 0.69 1.48 0.9 0.0308 0.0230 0.128 0.098 22.51 30.20 5.41 7.08 42.5 29.9 11.6 9.2 

12m 1.08 0.97 1.17 1.54 0.0354 0.0439 0.091 0.129 19.6 15.79 7.63 5.38 30.5 22.0 12.9 12.0

125 ppm 

1d 4.27 3.11 3.78 3.18 0.0735 0.0736 0.171 0.139 9.43 9.42 4.06 4.99 58.1 42.3 22.1 22.9

2w 5.36 4.10 2.13 2.31 0.082 0.0852 0.126 0.147 8.45 8.13 5.5 4.72 65.3 48.1 16.9 15.8

3m 3.17 2.75 4.36 6.53 0.0379 0.0411 0.153 0.188 18.30 16.87 4.53 3.69 83.6 67 28.5 34.7

6m 3.58 3.58 2.43 2.39 0.0514 0.0589 0.084 0.082 13.47 11.76 8.29 8.44 69.6 60.7 29 29.1

12m 2.23 1.33 2.19 2.31 0.0347 0.0342 0.094 0.094 19.96 20.28 7.41 7.41 64.3 38.8 23.4 24.7
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Table 4.39: Comparison of BAA urinary excretion between rats and mice. 

 k T1/2 (min) AUC (μg/min/g) 

 Rats Mice Rats Mice Rats Mice 

62.5 ppm 

1d 0.0174 0.0109 0.0221 0.0224 39.9 63.7 31.3 31 5541 8228 1330 2808 

2w 0.0168 0.0088 0.0326 0.0304 41.2 78.4 21.3 22.8 4849 10605 797 1872 

3m 0.0168 0.0094 0.0217 0.0178 41.3 73.7 31.9 39 - - 3059 3872 

6m 0.0099 0.0090 0.0222 0.0225 70.3 76.8 31.2 30.8 - - 4592 7138 

12m 0.0139 0.0087 0.0134 0.0108 49.8 79.6 51.6 64.1 7279 8445 9026 13941 

18m 0.0067 0.0122 0.0144 0.0208 103.3 56.7 48.2 33.3 9351 8976 8158 7521 

125 ppm 

1d 0.011 0.0031 0.0241 0.0285 63.2 224.1 28.7 24.3 19010 33403 4475 8145 

2w 0.0116 0.0053 0.0263 0.0237 59.5 130.2 26.4 29.3 13597 37943 4640 10912 

3m 0.0059 0.0039 0.0248 0.0227 117.9 178.8 28 30.5 13554 29094 8295 11772 

6m 0.0035 0.0018 0.0124 0.0167 195.9 395.8 55.8 41.6 14142 26485 30151 18022 

12m 0.0037 0.0024 0.0124 0.0054 187 283.4 55.7 127.8 16649 25897 21094 40782 

18m 0.0023 0.0058 0.0143 0.0166 297.5 119.3 48.5 41.7 30768 25745 34787 21126 

Mouse 

In an inhalation study, B6C3F1 mice were exposed whole body for 6h per day, 5 days per 

week for 104 weeks, to EGBE (purity > 99 %) at concentrations of 62.5, 125 and 250 ppm 

(Dill et al., 1998). Post exposure blood samples were collected after 1 day, 2 weeks and 3, 6, 

12 and 18 months for EGBE and BAA determination. The post exposure time points depends 

of the day of collection. Post exposure urine samples were collected from animals after 2 

weeks and 3, 6, 12 and 18 months of exposure. Before the core study started, a separate set of 

mice was moved into the control chamber and designated as the “aged (naïve)” mice. At 18 

months into the chronic study, these mice (about 19 months old) were moved to the 125 ppm 

exposure chamber and exposed for 3 weeks. Blood was collected after 1 day and 3 weeks of 

exposure at post exposure time points of 10, 20, 40, 80, 180, 360, 720 and 1440 min. Post 

exposure urine samples were collected from the aged mice for 16 h after 2 weeks of exposure. 

This study was also performed in rats. 

The EGBE blood concentrations showed a rapid drop after the cessation of exposure. The 

elimination t1/2 for EGBE after 1 day of exposure was < 5 min, this t1/2 does not depend on the 

dose level. Increases in the initial blood concentration of EGBE and AUC were proportional 

to exposure concentration, supporting that elimination of EGBE from the blood follows linear 

kinetics. At the same concentration levels mice eliminated EGBE faster than rats. Values of 

t1/2 and AUC were significantly lower in mice at both exposure concentrations. The rate of 

EGBE elimination from blood slowed with increasing days of exposure , this change was 

more obvious in rats than mice as their t1/2 and AUC dramatically increased after 3 months of 

exposure. The kinetic parameters were not significantly different between male and female 
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mice. Unlike EGBE, BAA was not rapidly cleared from the systemic circulation. The BAA 

concentrations in the blood did not start to decline until about 40 min post exposure. It seems 

that BAA was eliminated from blood following saturable, non-linear kinetics. The k was not 

constant, decreasing as EGBE exposure concentrations increased. As the rate of BAA 

production reflects the EGBE elimination, it seems that the non-linear characteristics of BAA 

kinetics likely result from BAA distribution or elimination process. When the number of 

exposure days increased, the rate of 2BAA elimination tended to decrease. Elimination of 

BAA was faster than rats when compared at common exposure concentrations. No differences 

were seen between males and females. There was a clear difference in the urinary excretion of 

BAA between rats and mice. No sex differences in BAA excretion were seen regarding 

exposure concentration. Time-dependent changes in urinary excretion of BAA were not 

comparable between males and females as the trend appeared to change over time in the 

opposite direction (decreasing in males mice and increasing in females). 

Elimination of EGBE and BAA in aged mice : EGBE was rapidly cleared from systemic 

circulation and kinetic parameters were not significantly different from those of young mice. 

A slower terminal elimination phase was observed in aged mice suggesting that timedependent reduction in the rate of EGBE elimination in chronically exposed mice could be, in 

part, due to the age of animals. As with young mice, there was no sex difference in 

elimination kinetic. There was a great difference with young animals in blood concentration 

of BAA after 1 day of exposure. Aged mice eliminated BAA from blood more than 10 times 

slower than young mice. Consequently both t1/2 and AUC were greater in old mice compared 

to young animals after 1 day of exposure. This age-related difference mostly disappeared after 

about 3 weeks of exposure. 

In an inhalation study, female B6C3F1 mice were exposed whole body to 2-

butoxy[14C]ethanol (purity 97.6 %) at dose of 250 ppm for 6 hours (Green et al., 2000a). The 

mice (4 per time point) were terminated at 5 minutes, 24 and 48 hours post exposure. One 

animal for each time point was submitted to a whole body autoradiography, the remaining 3 

animals were submitted to an analysis of the free and bound radioactivity of the stomach and 

contents. 

The mice killed 5 min after the end of exposure contained high level of radioactivity without 

showing preferential labelling in any tissue or organ. The highest concentrations were found 

in liver, blood and nasal passages with lower concentrations in the gastro-intestinal. Similar 

concentration was observed in the glandular mucosa of the stomach but no radiolabelling 

were present in the forestomach. Very high levels of radiolabelling were present on the skin 

and fur near the hindquarters. 

At 24 hours post exposure, the highest concentrations of radioactivity were observed in the 

liver and buccal cavity. Similar high amount of radiolabelling was seen in the mucosa of the 

caecum, in the forestomach mucosa, the lower gastro-intestinal tract mucosa and the 

oesophagus. The glandular stomach was conspicuously different from the forestomach in 

having low, background levels of labelling. High levels of radiolabel were seen in the skin 

and fur particularly on the back and near the hind quarters. A lower level of labelling was 

observed in the salivary glands, thymus, kidney medulla, adrenal and spleen. Background 

labelling was seen in the rest of the internal organs. 

48 hours after exposure, high levels of radiolabelling was seen in the buccal cavity, the 

oesophagus, the forestomach and in the liver and the mucosa of the lower gastro-intestinal 

tract. The skin and fur, particularly near the hind quarters, were also heavily radiolabeled. 
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Low levels of labelling were observed in the duodenum, glandular stomach and the remainder 

of the internal organs. 

The analysis of stomach and contents showed that immediately after exposure, a greater level 

of radioactivity was present in the stomach and its contents than the two later time point and 

that most of this radioactivity was due to EGBE. At the 24 and 48-hour time point, more of 80 

% of the radioactivity present in the stomach tissues was covalently bound to protein. There 

was no significant difference between the glandular and forestomach. 

High concentrations of radioactivity were seen on the fur and skin of the animals in this study 

and according to the authors, this observation together with the presence of radioactivity in 

the buccal cavity, oesophagus and stomach contents suggests that EGBE is ingested during 

grooming (during exposure and post exposure) and mucous removal (muco-ciliary clearance) 

through the nasopharynx (during exposure). Although the whole body autoradiograms very 

clearly demonstrated highly selective labelling of the mucosa of the forestomach at the two 

later time points, the analysis of the stomachs failed to detect this, probably due to the high 

levels of background labelling in the remainder of the stomach tissues. 

In conclusion, the retention of radioactivity in the forestomach mucosa clearly indicates that 

the forestomach is a target organ following an inhalation exposure to EGBE. The high 

concentrations of radioactivity on the skin and fur and the presence of radioactivity in the 

buccal cavity and the oesophagus suggest that the radioactivity enters the forestomach as a 

result muco-ciliary clearance from the nasopharynx and grooming and ingestion of the 

material present on the skin and fur. 

A complementary study from Poet et al., 2003 identified both BAA and EGBE in the saliva of 

mice administered EGBE by both oral gavage and intraperitoneal routes. The author 

concluded that EGBE can distribute to the forestomach by multiple mechanisms: grooming of 

the fur, mucociliary clearance, saliva and from systemic blood circulation and that BAA can 

also distribute to forestomach tissues from saliva and blood circulation as well as being 

formed locally from EGBE. 

In this series of studies, Poet et al., 2003 also assessed the deposition of EGBE on fur after 

inhalation exposure: female mice were exposed either by whole-body or nose-only inhalation 

to a single 6-hour EGBE exposure of 250 ppm (vapour). At the end of each exposure, 5 mice 

were killed and immersed in hot water to collect the EGBE deposited on the fur. Additional 

groups of 5 mice were killed immediately after inhalation exposure for blood analysis. Two 

other groups of 5 mice were subjected to an 18-hour urine collection after the inhalation 

exposures. 

An average of 205 μg of EGBE was detected on the fur of mice exposed whole-body. For 

mice exposed nose-only an average of 170 μg was found. After corrections, the estimation of 

the authors was that whole body exposures averaged 25 % more EGBE on the fur than the 

corresponding nose-only exposure. 

At the end of each exposure, the mean concentrations of EGBE in blood were 3.0 and 3.9 

mg/l for whole-body exposure and nose-only exposure respectively. As the same time, the 

concentration of BAA in blood were 235 and 390 mg/l for whole-body exposure and noseonly exposure respectively. Low levels of EGBE were found in the 18-hour post exposure 

urine collection (about 68 μg) to either route. These concentration are supposed to come from 

the fur because EGBE is not expected to be excreted in the urine unconjugated. 

Concentrations of free BAA in the urine were about 2020 μg or 1780 μg for whole-body 

exposure and nose-only exposure respectively. 
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Summary inhalation route : 

Three studies performed with rats (two with F344 and one with SD rats) and 3 studies 

performed with B6C3F1 mice were reported. In one study, the distribution of EGBE and its 

metabolites was studied in relation with time. This study clearly showed that the liver and the 

forestomach were the main target organs. The great amount of radiolabelling found in the 

gastro-intestinal tract demonstrated that EGBE was ingested during grooming and by mucocilliary clearance from nasopharynx and not directly via inhalation route. However this 

mechanism cannot explain the great amounts found in the forestomach. EGBE metabolism 

leaded to the formation of a majority of BAA, EG and BEG and 2 metabolites unidentified in 

small quantities. BAA and EG formation followed a saturable mechanism and increased doses 

of EGBE leaded to a greater and greater formation of BEG compared to BAA and EG. Like 

the other routes of administration, elimination via urinary route was rapid and predominant. A 

small amount of the administrated dose was eliminated via inhalation route in the form of CO2

(less than 10 %). 

The blood half-life of EGBE was about 10 minutes in rats and 5 minutes in mice whatever the 

level of exposure. Unlike BAA which seemed to be eliminated by a saturable mechanism, 

elimination of EGBE followed a linear kinetic. When repeated doses of EGBE were 

administered, the rate of BAA elimination tended to decrease with increasing doses of EGBE. 

In rats, elimination of BAA varied with the sex of animals: females tended to eliminate BAA 

slower. This difference between the sex could be due to a difference in the renal excretion 

between males and females. This sex difference was not found in mice. A species difference 

was also reported, mice eliminating 2 fold faster than rats. An age difference was also 

described in mice, the older mice eliminating BAA 10 fold slower than the younger mice for 

an unique administration of EGBE. When exposures were repeated , the difference between 

aged and young mice tended the disappear. After 3 months of daily exposure, no difference 

was seen for the elimination of BAA when age is considered. 

Dermal 

In vivo studies

Rat

EGBE (99 % purity) was tested in a series of studies in rats for assessing its kinetic 

parameters via the dermal route (Bartnik et al., 1987). 

Cutaneous absorption in vivo: EGBE was applied to 6 males and 6 females Wistar rats (12 

cm2 on shaved skin) at a dose of 200 mg/kg. To avoid oral uptake, the site of application was 

kept covered with a perforated glass capsule. Urine was collected for 48 hours. After 

termination, treated skin area was dissected for the determination of the radioactivity. 

Percutaneous absorption was assessed by using the measurements of urinary excretion of 14C 

from 0 to 48 hr following cutaneous and subcutaneous administration of EGBE and 

expressing the results as total radioactivity (urine) following topical application / total 

radioactivity (urine) following subcutaneous application. 

In a similar experiment, blood levels were determined following cutaneous application of 

EGBE. EGBE was applied to twenty four female rats at a dose of 200 mg/kg (12 cm2

 on 

shaved skin). Animals were killed 0.5, 1, 2, 4, 6, 8, 16 and 24 hours after application of 

EGBE. EGBE and BAA were measured in blood. 
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In vitro absorption of EGBE was also studied (see in vitro section) 

Within 48 hr following topical application of EGBE, approximately 20 to 23 % of the applied 

radioactivity was found in the urine (95 % eliminated during the first 24 hr). A percutaneous 

absorption of approximately 25-29 % of the applied topical dose was determined. 

 The highest radioactivity in blood and plasma occurs at 2 hr following application. 

Measurement of BAA in the plasma presented the same qualitative course. Based on BAA 

levels in the plasma, it appears that the major part of the absorbed EGBE is metabolized to 

BAA. 

Male F344/N rats were exposed dermally (non occlusively) to EGBE (99.9 % purity) 

(Sabourin et al., 1992b) at dose levels of 122, 367 and 650 μmol/rat ). Parent compound, CO2, 

urine and faeces were collected for 72 hr. After 72 hr, rats were killed and the skin around the 

site of dosing was removed from the carcass. Digested tissues, urine and faeces were assayed 

for radioactivity. 

Analysis of urine from [14C]EGBE dosed animals indicated the presence of the parent 

compound. Some experiments demonstrated that 7 % of the volatilized [14C]EGBE was 

trapped in the water in the urine receptacle. It demonstrate that urine dripping down the sides 

of the metabolic cage can absorb significant quantities of the volatilized EGBE. 

At the mid dose, 3 rats per time point were killed at 0.5, 1, 2, 4, 8 and 16 hr after exposure. 

Blood was collected and haematocrit and radioactivity measurement made. Urinary and 

plasma metabolites were analyzed. 

The total recovery of the 14C was 74 to 90 %. 43 to 64 % of the dermally applied dose was 

trapped as volatile 14C. 20 to 25 % of the dermally applied dose were absorbed and 

metabolized. A small amount of the applied 14C (0.3-2 %) was still present at the site of 

application 72 hr following dosing. The majority of radioactivity was excreted in urine. Small 

amounts of 14C were found in faeces and exhaled CO2 on air. There was no significant effect 

of the dose on the excretion of 14C except for a slight increase in the proportion of 14CO2. 

At the end of the collection period 7-16 % of the absorbed radiolabel remained in the carcass. 

Urinary metabolites were analyzed up to the time at which < 10 % of the 14C excreted within 

72 hr remained. For EGBE this included urine collected up to 23 hr after exposure. BAA was 

the major urinary metabolite. Amounts of glucuronide conjugates were also detected. The 

metabolite profile was not significantly affected by the dose. Analysis of 14C in whole blood 

indicated that over 80 % of radioactivity was associated with the plasma. The maximum 

concentration of total plasma metabolites was reached at about 1 hr following dermal 

application of the mid-dose of radiolabelled EGBE, after which the concentration decreased 

with a half-life of about 4 hr. Plasma BAA, the major metabolite, was measured at 1-2 and 4 

hr post exposure and represented 53 to 75 % of plasma 14C. 

Guinea pig 

The percutaneous uptake rate of EGBE was studied in Guinea Pigs (Johanson and Fernström, 

1986d). Female Guinea-Pig (GP) were kept under anesthesia throughout the experiment. After 

Intra Venous (IV) bolus of 42 or 92 μmol/kg EGBE, blood was sampled after 5, 10, 20, 30, 

40, 50, 60, 75, 90, 105 and 120 min. At 150 min after the IV dosing, animals were exposed to 
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1 ml of undiluted EGBE percutaneously (3.14 cm2 of skin exposed) with occlusive conditions. 

Blood samples were collected at the same time intervals as the intravenous dose. 

The concentration of EGBE in blood after IV administration declined rapidly. Estimates of 

the total clearance (Cl) was 128 ml/min/kg bw corresponding to 2.7 ml/min/g of liver (0.8 

ml/min/g of liver in human and 2 ml/min/g of liver in perfused rat liver). After dermal 

administration, the blood concentration of EGBE rose rapidly and reach a plateau level during 

the later half of the exposure period (average 21 μmol/l). The average uptake rate was 

estimated to be 0.25 μmol/min/cm2. 

The influence of water on the percutaneous absorption of EGBE was studied in Guinea Pigs 

(Johanson and Fernström, 1988). Animals were kept under anesthesia for 6 hr and killed at the 

end of the experiment. GP were exposed for 2 hours to pure or aqueous EGBE concentrations 

of 80 %, 40 %, 20 %, 10 % and 5 %. After a recovery period of 2 hours a second 2-hour 

exposure to pure EGBE was performed. The skin uptake of diluted EGBE was compared with 

the skin uptake of undiluted EGBE, each animal serving as its own control. Arterial blood 

were collected at intervals of 10 to 30 min throughout the experiment and assayed for EGBE. 

The concentration of EGBE in blood increased with time after administration. After removal 

of the exposure source, concentration started to decreased immediately. During the 2nd hour of 

exposure to pure EGBE, the blood level appeared to approach a plateau level. The average 

concentration during the last hour was 4.6 μmol/l and the calculated skin uptake rate was 132 

nmol/min/cm2

. The absolute skin uptake rate and the average blood concentration of EGBE 

during the first exposure period increased as the percentage of EGBE in the applied solution 

increased up to a maximum of about 40 % and then decrease again. The relative skin uptake 

rate showed a similar pattern. The average relative uptake rate of EGBE was approximately 

the same from the 5, 10 and 20 % solution as from the undiluted EGBE, while it was twice as 

high from the 40 and 80 % solutions. In contrast, the concentration of EGBE in blood 

declined approximately twice as rapidly after the end of exposure to the 40 and 80 % solution 

(average t1/2: 28 min compared to 55 min for the other solutions). 

In vitro studies: 

Cutaneous absorption of EGBE in vitro was assessed by a modified method of Zesch and 

Schaefer (1973) (Bartnik et al., 1987). These studies also included an evaluation of cutaneous 

absorption of EGBE under semi occlusive and non-occlusive conditions. Skin of rats, pigs and 

humans was used. Before and at the end of each study, the skin was checked visually for 

integrity of the stratum corneum. For application of test materials, an area of 5 cm2 (animal 

skin) or 3 cm2

 (human skin) was demarcated on the skin samples. 30 microliters of each of the 

following test solutions was applied : EGBE (100 %), EGBE (3.5 %) in water, EGBE (10 %) 

in water. All test solutions were evaluated for absorption in rat skin (1, 6 and 16 hr semiocclusive, 1hr exposure non-occlusive) and pig skin (6 hr exposure semi-occlusive). 

Absorption of EGBE (3.5 % in water) were also assessed on both rat and pig skin under nonocclusive conditions (10, 30 and 60 min exposure). A comparative study involving samples of 

rat, pig and human skin was performed with EGBE (10 % in water) using an exposure period 

of 1hr under both semi-occlusive and non-occlusive conditions. The studies with 3.5 and 10 

% EGBE were repeated with 5 % 2-propanol (IPA) or 5 % alkyl sulfonate (LAS) added to the 

test solution. IPA nor LAS had any measurable effect on the dermal uptake of EGBE. 
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Under semi-occlusion in vitro, EGBE is readily absorbed and completely absorbed after 16 hr 

of exposure. Penetration depends on time as well as concentration. The penetration rate of 

pure EGBE is slower than from aqueous solutions, but more complete after 16 hr. A 

comparison of EGBE penetration at 6 hr under semi-occlusive conditions showed that for all 

the tested formulations, the penetration through the pig skin was 2 or 3 times less rapid than 

through rat skin. Application of EGBE on rat skin under non-occlusive conditions results in 

apparently great reduction in the absorption due to the volatility of the compound. Within 10 

min following application, a major proportion of the absorbed material had penetrated. In 

comparison with the pig skin, rat skin showed an approximate 2 fold higher absorption. Under 

semi-occlusive condition, the penetration rate of EGBE through human skin was comparable 

with that through pig skin (much slower than through rat skin). Under non-occlusive 

conditions, human skin exhibits the lowest percutaneous absorption (6.9 % of the applied 

dose). 

Percutaneous absorption of EGEE and EGBE was studied in humans and rats (Lockley et al., 

1999 – Abstract). 2 butoxy [1-14C] ethanol (1.41 mg/cm2

) was applied to rat dorsal skin and 

human breast skin in vitro. The results were compared with an in vivo study in which EGBE 

(9.22 mg/cm2

) was applied to the skin of male Wistar rats. Skin surface swab (unabsorbed 

material), tape strip (material in the stratum corneum), skin (material in the epidermis and 

remaining dermis) and absorbed dose (receptor fluids in vitro and mass balance cumulative 

dose in vivo) were analyzed up to 24 hr after application. Evaporated EGBE was trapped with 

carbon filters ( > 80% of the dose within 1 hr of application). 

Absorption of EGBE in vitro through rat skin most closely reflected penetration in vivo. 

Absorption through human skin in vitro was less than rat skin but reflected dermal absorption 

described in a previous study (Johanson et al., 1988). This study demonstrates that for EGBE 

in vitro studies are able to reflect in vivo conditions. 

Summary dermal route : 

Five studies were reported on EGBE kinetic properties via dermal route. Two performed with 

Wistar rat, one performed with F344 rats and two performed with Guinea pigs. A series of in 

vitro studies was also available, studying percutaneous uptake of EGBE in various species, at 

different concentrations in different solvents and with various patterns of application 

(occlusive or not). Under semi-occlusive conditions, dermal uptake of pure EGBE was 

between 20 and 30 % of the administrated dose. Dermal uptake of aqueous dilutions of 5, 10 

and 20 % EGBE was similar to that of the pure substance. Uptake was increased and was 

maximal for the 40 and 80 % aqueous solutions of EGBE. The rate of penetration was less for 

pure substance than for concentrated aqueous solutions. Some in vitro studies demonstrated 

that dermal uptake for pig skin was 2 or 3 times slower than rat skin. The results found with 

human skin were more or less equivalent to those seen with pig skin. 

If EGBE was administrated under non-occlusive conditions, the dermal uptake decreased 

dramatically (uptake < 10 %), mainly because of the volatility of EGBE. 

A plasma peak was obtained in about 2 hours after the beginning of the application and 

relatively constant until the cessation of exposure. 

The metabolism mainly lead to the formation of BAA and also to smaller quantities of BEG 

(Sabourin et al., 1992b). The half-life of metabolites in plasma was about 4 hours. The 

majority of metabolites was eliminated via urinary excretion and only a very small amount 
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were found in faeces. One part of administered EGBE was metabolised in CO2 and 

eliminated via respiration, this amount increasing with the increasing dose. 

Oral 

Rats

The metabolism and disposition of EGBE was studied in F344 rats (Ghanayem et al., 1987a). 

Male rats were given a single doses of radiolabeled EGBE by gavage of either 125 or 500 

mg/kg bw. Collection of urine, faeces and expired air was made during 48 hours after the 

administration. Biliary excretion of EGBE was also monitored. Determination of the 

radioactivity in each organ and in blood was performed at the end of the study. 

The amount of 14CO2 exhaled in the first 48 hours after dosing was approximately 18 and 10 

% of the administered doses of 125 and 500 mg/kg bw respectively. Exhaled Volatiles 

accounted for no more than 2 % of the administered doses. Faecal excretion of EGBE ranged 

between 2 and 3 % of the administrated doses. Urine was the major pathway of excretion and 

most of the radioactivity was excreted during the first 24 hours after dosing. A higher 

percentage (70 %) was found for the 125 mg/kg dose as compared to the 500 mg/kg dose (40 

%). Two major metabolites were detected by chromatography in the urine (Butoxy Acetic 

Acid (BAA) and the Glucuronide conjugate of EGBE (BEG). Three minor peaks 

corresponding to the Sulfate conjugate of EGBE (BES), EGBE and an unknown substance 

were also measured in urine. All 5 metabolites were observed in urine collected in the first 8 

hours after treatment with 125 mg/kg bw; EGBE and BES were not seen in later time periods, 

and EGBE and BES were not seen at any time in the urine of rats treated with 500 mg/kg bw. 

Analysis of urine collected between 8 and 24 hr after dosing revealed that approximately 90 

% of the EGBE derived radioactivity was BAA at both doses. While BAA was the only 

metabolite observed in the 24-48 hr urine of animals receiving 125 mg/kg bw, the ratio of 

BAA to BEG was approximately 3 to 1 in urine of rats receiving 500 mg/kg bw (see table 

4.40). 

Table 4.40: Effect of time and dose on the excretion of BE metabolites in urine 

Dose 

125 mg/kg 500 mg/kg 

8 hr 24 hr 48 hr 8 hr 24 hr 48 hr 

Metabolite 

X 3.0 <2 % ND <1 % <1 % <1 % 

BAA 74.4 93.5 100 76.4 89.9 75.3 

BEG 21.3 5.3 ND 23 9.2 23.8 

BES 2.7 ND ND ND ND ND 

BE <2 % ND ND ND ND ND 

Significant increases in bile flow were seen as early as 0.5 hr after EGBE administration (for 

the 500 mg/kg dose) that returned to normal after 4 hr. Biliary excretion of radioactivity 
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continued to increase in a manner parallel with the increase in bile flow. The cumulative 

excretion reached 8 % of the 500 mg/kg dose in 8 hr. BAA, BEG and EGBE were excreted in 

bile. EGBE was detected only during the first 2 hr after treatment. BEG was the major 

metabolite excreted in bile. 

Organs which had the most radiolabelling were forestomach, liver and kidneys. 

The modulation of EGBE metabolism after pyrazole (alcohol dehydrogenase inhibitor) or 

cyanamide (aldehyde dehydrogenase inhibitor) administration was studied in F344 rats 

(Ghanayem et al., 1987b). A single dose of 500 mg/kg of [14C]EGBE was administered to rats 

by gavage and another group received the same dose after a pre-treatment with 250 mg/kg 

Intra Peritoneal (IP) one hour before. Rats were placed immediately in metabolism cages for 

urine and faeces collections (8, 24 and 48 hr after dosing for urine and 24 and 48 hr after 

dosing for faeces). Expired volatiles and 14CO2 were collected over the 48 hr housing period. 

At the end of the 48 hr rats were sacrificed and tissues were collected. Biliary excretion and 

radioactivity after treatment or rats with radiolabeled EGBE of radiolabeled EGBE + pyrazole 

was determined. Qualitative and quantitative determination of metabolites in urine and in bile 

was performed. 

Approximately 11 % of the 500 mg/kg dose was exhaled as 14CO2 in 24 hr after dosing. 

However pre-treatment with pyrazole resulted in a significant decline in the percent of EGBE 

dose exhaled as 14CO2 . In contrast pyrazole significantly increased the urinary excretion of 

EGBE derived radioactivity. BAA was the major metabolite collected whatever the collection 

period (75 – 90 % of the total urine radioactivity). Most of the remaining was the glucuronide 

conjugate of EGBE. In contrast the major metabolite of pyrazole pre-treated rats was BEG (75 

– 85 % of the total radioactivity). Furthermore, 8 – 19 % of the radioactivity was the sulfate 

conjugate which was not detected in rats treated with EGBE only. The increase of biliary 

excretion in general was more important for pyrazole pre-treated than EGBE only animals. 

Moreover, the biliary excretion of EGBE derived radioactivity was higher in pre-treated rats 

(16 % vs 8 % for animals treated with only EGBE. A similar metabolic profile was obtained 

in bile compared to urine (BAA constituted 10, 21 and 46 % of the total radioactivity excreted 

in the bile fractions at 0 to 1, 2 to 4 and 6 to 8 hr after treatment. In contrast, no BAA was 

detected in any of the bile fractions collected from pyrazole pre-treated rats. In these rats 

approximately 12 % of the radioactivity was excreted in the first hr as unchanged EGBE with 

the remaining portion being BEG. In addition no metabolite other than BEG were detected in 

bile fractions collected at 2 to 4 and 6 to 8 hr after dosing with EGBE. With cyanamide pretreatment, the same results were obtained than with pyrazole. This study clearly demonstrates 

that metabolism of EGBE to BAA is mediated by alcohol and aldehyde dehydrogenases via 

the formation of BAL. 

The metabolism and disposition of orally administered EGBE (500 mg/kg) in young (4-5 

weeks old) or adult F344 rats (9-13 weeks old) was compared (Ghanayem et al., 1987c). 

There was a significant higher percentage of the EGBE exhaled as CO2, 48 hr after dosing, by 

young rats (approximately 10 and 22 % of the administered dose in adult and young rats 

respectively). Similarly, young rats excreted a significantly higher percentage of the 

administered dose in the urine than adult rats. BAA, BEG and BES were identified in the 

urine of EGBE treated rats. Young rats excreted significantly less BAA than adult rats. 

Further, young rats excreted more BEG in urine than adults. No BES was detected in the urine 

of either age group. An unknown metabolite was detected in the urine of both age groups. It 

constituted less than 1 % of the total radioactivity excreted in the urine of adult rats compared 
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to young rats. Lower levels of EGBE derived radioactivity were seen in all tissues of young 

animals compared to tissues of old animals. 

In a study performed by Medinsky et al., in 1990, EGBE was administrated via drinking 

water to male F344/N rats. In parallel 2 others rat groups were dosed with Ethylene Glycol 

Methyl Ether (EGME) and Ethylene Glycol Ethyl Ether (EGEE) for comparison with EGBE. 

Rat were allowed access for 24 hours to 2-butoxy[U-14C]ethanol (50 mCi/mmol) (purity 99.9 

%) at three doses: 290 ppm (237 μmol/kg bw), 860 ppm (401 μmol/kg bw) and 2590 ppm 

(1190 μmol/kg bw). Exhaled CO2, urine and faeces were collected during 72 hours from the 

beginning of the exposure. At the end of the 72-hour collection period, cages were washed to 

determine the amount of radioactivity remaining, the total amount of water consumed by each 

rat in the drinking water was measured and recorded. Rats were killed and carcasses analysed 

for total 14C. Urine was analysed for parent compound and metabolites. 

80 % of the radioactivity excreted appeared during the first 24-hour period. For metabolites, 

the major compound identified was BAA (50-60 % relatively constant for all doses), others 

compound identified were ethylene glycol, the glucuronide conjugate of EGBE and EGBE. 

No N-acetylglycine conjugate of BAA was identified in this study. 

Effects of multiple dosing of male Fischer 344 rats with EGBE on its own disposition and 

metabolism were studied (Ghanayem et al., 1992). This study was performed together with 

mechanistic studies on haematotoxicity (see § 4.1.2.2.3). Rats were treated with 125 mg/kg 

EGBE for 3 or 7 days by gavage followed by a single dose of 125 mg/kg dose of 14C EGBE 

on day 4 and 8 respectively. Rats were placed in metabolism cages and EGBE metabolism 

analysis were performed. 

No quantitative or qualitative alteration of EGBE metabolism and disposition were caused by 

repeat exposure compared to those observed in rats treated with a single dose. The ratio of 

BAA, the glucuronide and sulfate conjugates and parent EGBE excreted in the urine of rats 

treated for 4 or 8 days were relatively similar to those from rats treated with a single EGBE 

dose. Therefore, it is unlikely that tolerance development to the hemolytic effects of EGBE 

(seen in many studies) is caused by increased detoxification of EGBE or inhibition of EGBE 

metabolism to BAA. 

[

14C]EGBE (purity 96.3 %) was administered by gavage to male F344 rats (3 rats/dose) at 

doses of 10 and 125 mg/kg bw as a solution in water. An additional group of 3 rats was dosed 

with [14C]EGBE at 10 mg/kg in a corn oil vehicle (Dow, 1993). Samples of blood were taken 

from the rats administered EGBE in water at 1, 3, 6, 12 and 24 hr after dosing for 

determination of total 14C (plasma) and for analysis of EGBE and BAA (whole blood). Urine 

from each animal was collected at intervals of 0-12 and 12-24 hr after dosing and the 

metabolic profile assessed. Faeces were collected for 24 hr. Expired 14CO2 was collected 

throughout the study. Each animal was killed 24 hr after dosing. 

Haematological effects were seen in 2 rats of the 125 mg/kg bw dose group (the third rat was 

eliminated because of mis-dosing). 59 % of the dose was excreted in the urine during the first 

24 hr following administration of 10 mg/kg EGBE in either corn oil or in water. 10 % was 

excreted as 14CO2 in 24 hr. At 125 mg/kg bw the 2 rats eliminated 37.7 and 70.3 % of the dose 

in urine during the first 24 hr and 7.6 and 8.5 % of the dose as 14CO2. The maximum 

radioactivity plasma concentration was reached apparently just before the first blood sample 

was taken. Metabolite profiles in urine were similar to than of the Medinsky et al. (1990) 

study and the Ghanayem et al., 1987a study. Nearly 40 % of the 14C in urine was present as 

BAA during the first 12 hr following 10 mg/kg bw and 65 % after 125 mg/kg bw EGBE 
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dosing. An additional 15 % of the 14C in urine was present as the glucoronidase-labile 

conjugate of EGBE at 10 mg/kg bw and 10 % at 125 mg/kg bw. Ethylene Glycol (EG) was 

confirmed in this study as a metabolite of EGBE, but was present in small quantities. 

The potential of EGBE to form fatty acids conjugates was studied in F344 rats and in vitro

(Kaphalia et al., 1996). [Ethyl-1, 2-14C] BE (purity 99 %) was administered as a solution of 

water via oral route to 3 rats at a dose of 500 mg/kg bw. Three other rats served as controls 

and were given the same amount of water. The animals were killed 2 hr after exposure. The 

liver was excised and analyzed for radiolabeled lipids. 

Approximately 16 % of the total radioactivity detected in the liver was associated with lipids, 

mostly in the phospholipid fraction (about 85 % of the total lipids). 3 % radioactivity of the 

total lipids was detected in the ester fraction. 

The effects of co-administration of EGBE (99 % purity) with alcohols (Ethanol (EtOH), nPropanol (n-PrOH) and n-Butanol (n-BuOH)) was studied (Morel et al., 1996). Three sets of 

four groups of 10 male SD rats were used for the experiment in which all substances were 

administered orally by gavage (each set of groups differed from the others by the alcohol 

administered). In each set, the first group received distilled water and served as control. The 

second group was treated with only alcohol (10 or 30 mmol/kg) in distilled water, except for 

n-butanol (no vehicle), this group served as alcohol control group. The third group was dosed 

with EGBE at dose of 5 or 1 mmol/kg. The fourth group was given simultaneously alcohol 

and EGBE at the same doses than group 2 and 3 in distilled water (except for n-butanol: no 

vehicle). The haemolytic effect of EGBE (5 mmol/kg bw) was evaluated 4 hr after treatment 

by Red Blood Cell (RBC)-counting. Urinary concentration of BAA was determined on urine 

collected during 24 hr. 

Administration of EGBE alone (5 mmol/kg) resulted in a significant decrease on average 

RBC (26 %) accompanied by a large increase in the concentration of free plasma 

Haemoglobin (Hb). The co-administration of n-BuOH or n-PrOH (10 mmol/kg bw) partially 

reduced the haemolytic effect of EGBE. No effect was seen with co-administration of 10 

mmol/kg bw EtOH. At the dose of 30 mmol/kg bw, each of the three alcohols coadministrated with EGBE produced a complete protection against haemolytic effects. The 

alcohol control treatment did not have any effect on the haematological parameters. 

Urinary excretion of BAA in rats treated only with 1 mmol/kg bw EGBE was 0.083 mmol/24 

hr, which represents about 30 % of the ingested dose. The simultaneous administration of 

each of the three alcohols at a dose of 10 mmol/kg with EGBE did not modify the 24 hr 

urinary excretion of BAA, whereas at dose of 30 mmol/kg, decreases of 43, 33 and 31 % were 

observed for EtOH, PrOH and BuOH respectively. 

Mice

In a series of studies, the mechanism of EGBE (purity 99 %) accumulation in forestomach 

was assessed (Poet et al., 2003). Five experiment were conducted to examine various endpoints: 

- target tissue histology / forestomach irritation 

- tissue dosimetry and pharmacokinetics 

- fur deposition 
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- salivary excretion 

- retention in stomach content 

For tissue dosimetry and pharmacokinetics, groups of 30 female mice were administered 

EGBE (in saline solution) at doses of 50 or 250 mg/kg by IP injection or 250 mg/kg by oral 

gavage. Blood samples were taken and then the mice were killed 0.5, 1, 3, 6, 9, 12 and 24 

hours after dosing. Kidney, liver and stomach tissues were rapidly collected at each time 

point. The AUC and kinetic parameters for both EGBE and BAA were calculated. 

EGBE concentration in tissues paralleled the levels in blood regardless of dose or exposure 

route. For the 250 mg/kg dose to either route, concentration of EGBE were higher and 

persisted longer in forestomach than in blood or in other tissues. Regardless of the route, T1/2 

and AUC were higher in forestomach than in other tissues (Blood T1/2 could not be 

calculated because it was shorter than the earliest time point collected. The T1/2 for BAA was 

2.1 hours after gavage administration. 

By 24h, about 50 % of the total dose was eliminated in the urine (48 % for oral route) as 

EGBE, BAA or a conjugate. Following gavage administration of EGBE, BAA was the major 

urinary metabolite (38 % of the dose); there was also a small quantity (less than 0.2 %) of free 

EGBE, a conjugate of EGBE (up to 3 % - probably glucuronide).and a conjugate of BAA 

(between 0 and 7 %). 

Male and females B6C3F1 mice were administered EGBE (in distilled water) at dose of 600 

mg/kg (Deisinger and Boatman, 2004). Control animals were treated with distilled water. At 

5, 15, 45 and 90 min following the dose (5 min only for controls) animals were sacrificed and 

about 100 mg of blood, liver and forestomach were collected. Samples were analysed for 

EGBE, BAL and BAA by GC-MS. 

Five minutes after the administration, forestomach contained 50 fold more EGBE than blood 

or liver. Elimination from blood and liver samples was rapid (but still measurable at 90 min 

post dosing) whereas EGBE concentration in the forestomach decreased gradually and 

represent still 62 % and 31 % of the dose measured 5 minutes after administration in females 

and males respectively. Low concentrations of BAL were observed in all organs at maximum 

levels five minutes after administration. Forestomach levels were 10 fold higher than blood or 

liver concentrations. BAA concentrations increased till 45 and 90 min sample time. At all 

time points, BAA concentrations were lower in the forestomach than in blood or in the liver. 

No sex differences were detected for organ concentrations of EGBE and BAA but females 

seems to have higher BAL organ concentrations than males (at all time points). 

Summary oral route : 

EGBE kinetic properties via oral route were reported in up to 10 studies, 7 performed with 

F344 rats and one performed with SD rats. Two series of studies were also performed on mice 

These studies demonstrated the following results. 

Absorption of EGBE orally administered was rapid and essentially complete (assumed to be 

100 %). Target organs in which the most radiolabeled compound were found were: the 

forestomach, the liver and the kidneys. The major metabolite of EGBE is BAA which formed 

through a mechanism involving alcohol and aldehyde dehydrogenases which seems to be 

saturable. Simultaneous administration of EGBE and a primary alcohol (ethanol, n-propanol 

or n-butanol) in sufficient quantity inhibited BAA formation. The other metabolites were, in 
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order of magnitude: the glucuronide conjugate of EGBE (whose percentage increases relative 

to the dose at the expense of the BAA formation) and two minor metabolites, the sulphateconjugate of EGBE and ethylene glycol (which was not observed in all studies). Elimination 

is rapid and occurs mainly by urinary excretion. A small amount of metabolised EGBE is 

eliminated as CO2 in expired air (0 to 20 % for a high and low dose respectively). A small 

amount of unchanged EGBE (approximately 1 %) is also eliminated in expired air. In two 

studies, BAA, BEG and EGBE were found in bile. EGBE did not accumulate in tissues. The 

metabolic profile was not changed after repeated exposures compared to acute exposure. Agerelated difference was observed: young rats eliminating EGBE via CO2 and urine (with 

significantly less BAA and more BEG) to a greater extent than adult rats. 

Other routes 

Rat

Female SD rats (4 rats) were administered EGBE (purity unknown) via IP route at dose of 2.5 

mmol/kg EGBE concurrently with 20 mmol/kg EtOH (Romer et al., 1985). 

The degradation of EGBE was almost completely inhibited in combination with simultaneous 

EtOH treatment. 

Radiolabeled EGBE (purity 99 %) was administered subcutaneously to 3 male Wistar rats at 

the dose of 118 mg/kg. Faeces, urine and expired air were collected during the 72-hour period 

following EGBE administration. After termination the following organs and tissues were 

collected: liver, kidneys, spleen, fat, testes, thymus, sternum (including bone marrow) and 

blood (Bartnik et al., 1987). 

EGBE was metabolised and eliminated primarily in urine. Within 72 hr over 78 % of the 

radioactivity was found in the urine. A small amount (less than 1 %) was found in the faeces 

and 10 % was eliminated in the exhaled air as CO2. 1.6 % of the dose was found absorbed on 

activated charcoal indicating that the exhaled air contained only a small amount of the test 

compound or volatile metabolites. At the end of the experiment, 4.8 % of the radioactivity 

was found in the carcass. Spleen and thymus followed by the liver were the 3 major organs 

where the concentrations of radioactivity were highest. 

In a series of studies, EGBE was administered via intravenous route in order to study the 

effect of dose, age and inhibition of metabolism on the kinetic parameters of EGBE 

(Ghanayem et al., 1990a). Male Fisher 344 rats (young: 3-4 months old and old: 12-13 

months old), three to four animals per treatment group were given IP doses of either pyrasole 

(250 mg/2.5 ml of 0.9 % saline/kg – alcohol dehydrogenase inhibition), cyanamide (50 

mg/2.5 ml of 0.9 % saline/kg - aldehyde dehydrogenase inhibition) or probenecid (50 mg/2.5 

ml of 0.9 % saline alkalinised with NaHCO3/kg – renal tubular transport inhibition). A second 

dose of probenecid was administered 4 hr later. Twenty to 30 minutes after initial treatment, 

all groups received a single bolus dose of radiolabeled EGBE (31.25, 62.5 or 125 mg/kg) 

delivered IV. Blood samples were collected at 5, 15 and 30 min and at 1, 2, 4, 6 and 8 hours 

for analysis of radioactivity. EGBE and metabolites were determined in whole blood and in 

plasma. 

- Effect of dose and age: EGBE in blood is proportional to the administrated dose. At the 

same dose level, concentrations obtained in old animals were higher than in the young 

ones. The mean concentration of radioactivity in plasma was higher than in whole blood. 
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Only EGBE and BAA were found in the plasma. There was no significant effect of dose 

on T1/2 or Vd (Volume of distribution) of EGBE. Cmax, AUC and Cl of EGBE were 

affected by the level of administrated dose (increase of Cmax and AUC and decrease of Cl 

with increasing doses of EGBE). t1/2, Vd and Cl were not affected by the age of rats 

whereas Cmax and AUC were found to be age-dependent (increased in the older rats). For 

BAA, there was a significant increase in t1/2 in the older rats at the low and high dose but 

not in the middle. There is also a significant effect in AUC and Cmax, which tend to be 

higher in older animals than in young, this effect is more pronounced with high doses. 

- Effect of alcohol dehydrogenase inhibition by pyrazole: the radioactivity was higher in 

plasma than in whole blood. The total radioactivity was lower at all time point and at both 

dose levels than in rats treated with EGBE only. BE-glucuronide conjugate (BEG) was 

detected in the plasma (not in rats treated without pyrazole). Pyrazole resulted in 

significant increases in t1/2, AUC and Cl of EGBE at both dose levels with no effect on 

Vd. For BAA, there was a significant decrease in the values of AUC, Cmax and t1/2 in rats 

treated with pyrazole. 

- Effect of aldehyde dehydrogenase inhibition by cyanamide: the mean concentration of 

radioactivity was higher in whole blood than in plasma at all time points beyond the first 

30 minutes after EGBE administration. Furthermore, the mean concentration of total 

plasma radioactivity time profiles was higher at all time points in EGBE treated rats 

compared with that in rats treated with cyanamide + EGBE. Increases of t1/2, AUC, Vd 

and Cl of EGBE were seen in the cyanamide treatment group. No effect was seen on Cmax. 

The mean t1/2 of BAA was more than doubled but the difference was not statistically 

significant. A significant decrease of the Cmax and AUC of BAA was seen. 

- Effect of inhibition of renal tubular anion transport by probenecid : a consistent increase 

in the mean whole blood and plasma concentration of total radioactivity was seen. There 

were no significant changes in Cmax, AUC, t1/2, Vd or Cl of EGBE. In contrast, a 

significant increase in t1/2 of BAA (the increase being greater at the lower dose). 

Probenecid also significantly increased the AUC of BAA (also greater at the lower dose). 

Cmax of BAA was not significantly affected. 

In summary, this study has confirmed that BAA is the proximate hemolytic agent and that it is 

formed from EGBE by a metabolic pathway involving alcohol and aldehyde dehydrogenase. 

The renal organic acid transport may play a role in the clearance of BAA. The higher 

sensitivity of older rats compared to younger rats concerning haemotoxicity was due to a 

combination of factors: compromised renal clearance of BAA by the renal anion transport 

system in older rats, increased EGBE metabolism to BAA and diminished degradation of 

BAA to CO2 and greater sensitivity of erythrocytes in the older rats. 

Mouse 

Radiolabeled EGBE (purity 97.6 %) was injected intravenously to female B6C3F1 mice at 

dose of 10 mg/kg (Green, 2000b). Animals were terminated at 4, 24 and 48 hours after dosing 

(4 animals per sacrifice time). One animal for each sacrifice time was submitted to whole 

body autoradiography. The remaining three animals were submitted to an analysis of stomach 

and contents, the determination of radioactivity was made for forestomach, glandular stomach 

and stomach contents (free and bound radioactivity). 

Four hours after dosing, the highest concentrations of radiolabel were found in the liver, 

Harderian glands, salivary glands, nasal passages, oesophagus, buccal cavity and on the 
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surface of the feet. A lower concentration of radiolabel was seen in the stomach, Gastro 

Intestinal (GI) tract contents and mucosa, in the kidney cortex and associated with bones. 

Background levels of labelling were seen in the lung and the remaining internal organs. 

After 24 hours, the highest concentrations of radiolabel were observed in the liver, bone, 

Harderian glands, surfaces of the feet and buccal cavity. Slightly lower amounts were seen in 

the mucosa of the stomach, GI tract and the oesophagus. A lower level of labelling was 

observed in the salivary glands and kidney cortex. A much lower level of labelling was seen 

in the spleen, GI content and the kidney medulla. Background labelling was seen in the lung 

and the rest of internal organs. 

48 hours after dosing, radiolabel was observed in the liver, bone, buccal cavity and 

oesophagus. The mucosa of the forestomach and glandular stomach were also radiolabeled 

although there was no significant difference between them. Lower levels were observed in the 

salivary glands and GI tract mucosa. The lung and the remainder of the internal organs had 

background levels of labelling. 

The total radioactivity in the forestomach and glandular stomachs was similar over the 

duration of the study with the bulk of the radioactivity being present in the stomach walls: 80-

95 % of the radioactivity was bound to protein at the two later time points. 

In this study, the radioactivity in the stomach tissue appears to be derived from the systemic 

circulation and by ingestion from the buccal cavity. The origin of the latter is unknown, but 

according to the author, it may be derived from the salivary and Harderian glands which 

contained significant amounts of radioactivity. 

In a series of studies, the mechanism of EGBE (purity 99 %) accumulation in forestomach 

was assessed (Poet et al., 2003). Five experiment were conducted to examine various endpoints: 

- target tissue histology / forestomach irritation 

- tissue dosimetry and pharmacokinetics 

- fur deposition 

- salivary excretion 

- retention in stomach content 

- For Tissue dosimetry and pharmacokinetics, groups of 30 female mice were administered 

EGBE (in saline solution) at doses of 50 or 250 mg/kg by IP injection or 250 mg/kg by 

oral gavage. Animals were sacrificed 0.5, 1, 3, 6, 9, 12 and 24 hours after dosing. The 

AUC and kinetic parameters for both EGBE and BAA were calculated. 

EGBE concentration in tissues paralleled the levels in blood regardless of dose or 

exposure route. For the 250 mg/kg dose to either route, concentration of EGBE were 

higher and persisted longer in forestomach than in blood or in other tissues. For BAA, 

during the first three hours after IP administration the maximum concentration were found 

in blood, kidneys and liver. After 3 hours, forestomach concentration of BAA were higher 

than for other tissues. Unlike in the forestomach, BAA concentrations in the glandular 

stomach tissue were similar to other tissues. 

Regarding kinetic parameters, for EGBE both T1/2 and AUC were higher for gavage 

dosing than IP dosing. Regardless of the route, T1/2 and AUC were also higher in 
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forestomach than in other tissues (Blood T1/2 could not be calculated because it was 

shorter than the earliest time point collected. The T1/2 for BAA were 1h for IP injection. 

By 24h, about 50 % of the total dose was eliminated in the urine (54 % for IP) as EGBE, 

BAA or a conjugate for the 250 mg/kg administrations. A majority of BAA was found (50 

for the IP), a small quantity (less than 0.2 %) of free EGBE and of a conjugate of EGBE 

(up to 3 % - probably glucuronide) was detected. The last substance detected was a 

conjugate of BAA (between 0 and 7 %). 

- For saliva excretion assessment, groups of mice were administered EGBE at doses of 250 

mg/kg by IP injection or oral gavage. Salivation was induced by injection of pilocarpine a 

few minutes before saliva collection. Saliva was collected under anesthesia at various 

times after administration (up to 2.5 hr) by periods of 15 to 30 min (to obtain aliquots of 

saliva of 100-200 μl). Blood was collected at the midpoint of each saliva collection 

interval and at the end of saliva collection. Kinetic parameter of EGBE and BAA for 

blood and saliva were calculated. 

For EGBE, peak blood and saliva concentration were detected at 15 min and 7.5 min for 

blood and saliva respectively, regardless of the route. Concentration in blood and saliva 

were nearly identical at all time points and were below the level of detection after 1.3h. 

AUC and T1/2 in blood and saliva were similar. 

For BAA, the time curve was identical for blood and saliva, the values of saliva 

concentrations being 4 fold lower than the blood levels. The T1/2 was 2 fold higher in 

blood than in saliva (1.4 and 1.6 hr for IP and gavage respectively in blood). 

- For retention in stomach contents, a group of animal was treated with 250 mg/kg EGBE 

by IP route. After sacrifice (3, 6 and 9 hours after exposure) EGBA and BAA were 

quantified in stomach tissue and stomach contents. 

The concentration of EGBE was higher in the stomach content than that of the 

forestomach tissue. No EGBE was detected in either blood or glandular stomach tissue at 

any time point. The estimation of the T1/2 for EGBE in the content of the stomach was 

4.8h following IP injections, longest of all the tissues. 

Summary other routes : 

Two studies were performed via IV route (one in rats, the other in mice), two studies were 

performed via IP route and one via SC route in rats. Target organs evidenced in the previous 

described studies were confirmed in these studies: spleen, liver, thymus and stomach. 

However, a slight difference was seen in the distribution of the substance between the 

forestomach and the glandular stomach after an IV injection. The distribution to stomach 

comes from systemic circulation and also from ingestion of EGBE (which could come from 

salivary glands). 

In these studies, which are generally mechanistic studies, it was demonstrated that the BAA 

formation was caused by Alcohol Dehydrogenase and Aldehyde Dehydrogenase in the liver. 

This finding is validated by competitive inhibition studies performed with Ethanol. Inhibition 

of renal tubular anion transport caused a decrease in the renal excretion of BAA and therefore 

an increase of the EGBE toxicity. 
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4.1.2.1.2 Studies in humans 

Inhalation 

Seven human male volunteers were exposed to 20 ppm EGBE (0.85 mmol/m3) via inhalation 

route for a period of 2 hours during light physical exercise (50W) (Johanson et al., 1986a). 

Blood samples were collected during the exposure period and 3 hr afterwards and analysed 

for the determination of EGBE blood concentration. Urine was collected for a period of 24 

hours, the first sample was collected immediately before the volunteer entered the exposure 

chamber and thereafter sampling was made at 2-hour intervals for 6 hours. The samples were 

assayed for EGBE and BAA. 

No signs of adverse effects could be related to the exposure of EGBE and no effects were 

seen on electrocardiograms. The concentration of EGBE in blood rose rapidly reaching an 

apparent plateau level within 1-2 hours. The decay after exposure was rapid, with a biphasic 

slope in a semi-logarithmic plot (data are too limited to permit the calculation of more than 

one slope constant). EGBE could no longer be detected in the blood 2-4 hours after the end of 

exposure. The average elimination t1/2 was 40 min, the average plateau level in blood was 7.4 

μmol/l, the average blood clearance was 1.2 l/min and the steady-state volume of distribution 

was 54l. The total amount of EGBE excreted in urine was less than 0.03 % of the total uptake 

and the t1/2 of EGBE in urine was 1.36 hr. In urine, BAA reached a maximum concentration 

between 5 and 12 hours after the beginning of exposure. Elimination also reached a maximum 

between 2 and 10 hours after the beginning of the exposure. Great variations are observed 

between subjects. T1/2 for BAA in urine was calculated at 5.77 hours. 

According to the authors and on the basis of the very high in vitro blood/air partition ratio 

(above 1000, Johanson and Dynesius, 1988), one would expect the respiratory retention of 

EGBE to be limited by the alveolar ventilation and approximate 80 % at 50 W. However, the 

retention was about 57 % in the present study. The explanation may be that EGBE is partly 

adsorbed on the surface of the respiratory airways during inhalation and desorbed during 

exhalation. 

Five human volunteers were exposed to EGBE at 20 ppm for 2 hours during light physical 

exercise (50 W) (Johanson and Johnsson, 1991). Venous blood samples were collected and 

analysed for BAA before and immediately after exposure and also at 4 and 6hr after the 

beginning of exposure. Urine was collected every 2 hours and assessed for BAA content. 

After 2 hours of exposure BAA was found in blood. An average maximum concentration of 

45 μM BAA was reached after 2-4 hr. The decrease in BAA blood levels from 4 to 6 hr 

corresponds to an average half-time of 4.3 hr. The time profile in blood seems to be similar to 

that for urinary excretion, where the maximum occurred at about 5 hr and the half-time was 

estimated to 4 hr. The average clearance of BAA was estimated to be 23-39 ml/min and is 

only about 1/3 of the glomerular filtration rate of about 125 ml/min. The low renal clearance 

may be due to binding of BAA to blood proteins and absence or low efficiency in tubular 

secretion. The low pKa of BAA (3.5) indicates that more of 99 % of BAA present in urine is 

present in the ionised form and is not available for tubular re-absorption at normal urine pH. 

The average Vd of BAA was calculated to be 15 l. This value is approximately equal to the 

volume of extracellular water. 

Respiratory uptake of EGBE was investigated (concurrently with 9 other solvents) (Kumagai 

et al., 1999). The uptake of a chemical with high blood/air partition coefficient is predicted to 
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be high. But when a chemical is very water soluble, it can be partially absorbed in the mucus 

layer during inhalation and release during exhalation, leading to a low respiratory uptake. 

Four human volunteers were submitted to 25 ppm of EGBE (whole body in an inhalation 

chamber) for 10 minutes. The same people were also submitted to inhalation of 9 other 

substances in the same test conditions. Exhaled air was collected from 1 min before exposure 

to the end of the exposure period. 

The mean respiratory rate for each solvent was from 12.1 to 14 min-1. The mean tidal volume 

was from 470 to 530 ml, no differences were found among the solvents. The results obtained 

with the different solvents suggests that the wash in/wash out behaviour cannot completely 

explain the actual respiratory behaviour of these solvents. 

Summary human inhalation route: 

Three studies were reported in which human volunteers were exposed to EGBE by inhalation 

route. Kinetic parameters found in these studies were equivalent to those described in human 

studies earlier in the report for others routes of administration. Theoretical absorption 

(calculated) of EGBE via inhalation route was found to be 80 %. However, measurements 

performed showed a real absorption of 55 to 60 %. This difference is explained by a “wash in 

/ wash out” mechanism: due to its hydrophilic properties, EGBE is adsorbed to the surface of 

the respiratory tract during inspiration and it desorbed during exhalation leading to a decrease 

in the real uptake of substance. In the risk characterisation section, a 60 % of absorption for 

EGBE inhalation is used . 

Dermal 

In vitro

In an in vitro skin penetration test, the mean absorption rate of undiluted liquid EGBE (purity 

> 99 %) through human epidermis assessed (ICI, 1982a and 1982b). In this test, the 

penetration rate of EGBE was compared with 3 other glycol ethers (EGEE, EGME and 

Propylene Glycol Methyl Ether (PGME)) and 1 acetate (EGEEA). Disks of human abdominal 

skin were placed in diffusion chambers, the membrane integrity was assessed before the test 

by measurement of their permeability to tritiated water. The glycol ether absorption rate was 

measured for a period of 8 hours. After the test, the potential of the tested substance for 

impairing the epidermal diffusion barrier function was assessed by measuring the 

permeability to tritiated water again. A calculation of the damage ratio was made: 

Permeability constant after glycol ether contact / permeability constant before glycol ether 

contact. 

For EGBE the mean rate of penetration calculated in this study was 0.20 mg/cm2

/hr ( ±0.03 

SEM, n=8). EGBE did not produce large alteration in permeability in this study: damage ratio 

= 2.07. Human skin in vitro will always deteriorate over time and a ratio greater than 1 would 

be expected. The measured ratio indicates a marginal effect with little damage to the skin 

following prolonged exposure. 
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Another skin penetration study in vitro was performed with liquid EGBE (Eastman Kodak, 

1991). Human abdominal skin (stratum corneum) was exposed to EGBE in Franz-type 

diffusion cells. The integrity of each skin sample was determined before and after the test by 

measuring the permeability to tritiated water. 

The skin absorption results for EGBE were measured twice due to high variability observed in 

the first study. The mean absorption rates were: 0.857 mg/cm2/hour ± 0.282 in the first 

experiment and 1.52 mg/cm2

/hour ± 0.37 in the second experiment. The damage ratio was 

3.25 ± 3.33 in the first experiment and 5.14 ± 4.99 in the second experiment. Due to the high 

variability in each experiment, mean absorption rates were calculated separately for the 

undamaged skin (n=8) and the damaged ones (n=4), the results from the damaged skin 

specimens were about 3 times higher than the undamaged ones (3.39 mg/cm2

/hour vs 1.19 

mg/cm2/hour). When the results from the four cells showing the high damage ratio are 

excluded from the calculation of the overall mean result, the mean damage ratio is 1.66 ±

1.31. 

In a study performed by PMIC in 2001, in vitro percutaneous absorption of EGBE through 

human skin was assessed. Two different EGBE concentrations 10 and 5 % in an oxidative 

hair dye formulation was applied on human skin during a period of 30 minutes. For each 

experiment 33 mg of the different formulations (corresponding to 20 mg/cm2

) were applied on 

the skin surface (1.76 cm2). At the end of the 30 minutes of exposure, the surface of the skin 

was washed to eliminate the residual mixture. The diffusion of EGBE was monitored during 

the 24 hours following the applications. The receptor fluid was collected 2, 4, 6, 10, 21 and 24 

hours after the beginning of exposure. At the end of the 24 hr observation period, tissues 

(horny layer, epidermis and dermis) were analysed for EGBE remaining. This study was 

performed in compliance with GLP and following the recommendations of FDA, AAPS, 

COLIPA, SCCNFP and OECD for this kind of test. 

Results are expressed in μg equivalent of EGBE (μg/cm2) and in % of the applied dose for all 

the compartment analysed (see table 41). 

Table 4.41: Quantities of EGBE analysed in the different system compartments for the 2 tested concentrations 

(5 and 10 %) 

 EGBE 5 % EGBE 10 % 

μg/cm2 % of the applied dose μg/cm2 % of the applied dose

Filter (F) 29 ± 3 3.13 ± 0.34 53 ± 8 2.82 ± 0.38 

Washing (W) 

(30 min) 

639 ± 66 68.6 ± 7.5 1223 ± 178 65.2 ± 9.5 

Skin excess (F+W) 668 ± 68 71.7 ± 7.8 1275 ± 182 68.1 ± 9.8 

Epidermis (E) 

Including stratum 

corneum (SC) 

0.112 ±

0.065 

0.012 ± 0.007 0.283 ±

0.128 

0.015 ± 0.006 

Dermis (D) 0.023 ±

0.01 

0.002 ± 0.001 0.101 ±

0.051 

0.005 ± 0.003 

Receptor fluid (RF) 113 ± 54 12.1 ± 5.9 235 ± 138 12.5 ± 7.3 

Total skin +RF 

(SC+E+D+RF) 

113 ± 54 12.1 ± 5.9 235 ± 138 12.5 ± 7.3 

Total recovery (%) 84 ± 4 81 ± 4 
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These results showed that no accumulation of EGBE in skin occured. For these two 

concentrations, the percentage of absorption were similar (12.1 and 12.5 % for the 5 % and 10 

% concentrations). 

In vitro percutaneous absorption of liquid glycol ethers (including EGBE purity 98 %) was 

studied (Wilkinson and Williams, 2002). Human breast skin, full thickness or dermatomed 

(stratum corneum + upper dermis) was exposed to EGBE in aqueous solution at different 

concentrations (3 mg/ml or 6 mg/ml, 100 or 200 μl in the skin) or undiluted (10.5 μl). 

Percutaneous absorption was measured for 24 hours using flow through diffusion cells. Tissue 

culture medium was used as a receptor fluid with 2 % (w/v) Bovine Serum Albumin (BSA) or 

2-6 % (w/v) PolyEthylene Glycol 20 (PEG 20) added for some studies. 

In aqueous solution, a steady state flux of 544 ± 64 nmol.cm-2.h-1 (0.064 mg/cm2

/hr) was 

found with dermatomed human skin. Reducing the dose to 100 μl decreased the steady state 

flux of EGBE by about 55 %. Using full thickness skin increased the time to steady state (tau) 

and reduced the steady state flux. Absorption rates of undiluted EGBE in finite dose 

exceeded those measured with aqueous solutions, though the apparent permeability 

coefficient was higher with the aqueous doses. 

Modelling 

An alternative for dermal permeation estimation is the model SKINPERM (version 3.0), which 

provides an estimation of the amount of EGBE that penetrates the skin either from the vapour 

phase, the neat liquid or an aqueous solution. SKINPERM describes dermal exposure and 

absorption using a quantitative structure activity relationship (QSAR) which is based solely on the 

biological structure of the skin and the physico-chemical properties of the substance under 

investigation. SKINPERM 3.0 was validated with an extensive database of experimentally 

measured permeation coefficients and is considered valid for substances with log Kow values in 

the range from 0 to 4. The model estimates the permeation rate for EGBE to be 0.63 mg/cm2

/hr, 

which is consistent with the measured in vitro data. This model is used for EGBE only for 

comparative reasons as a lot of measured data is available.

In vivo

Five men, who have been involved in another study 2 years ago (Johanson et al., 1986a), were 

exposed to neat EGBE for 2 hr by placing four fingers of the left hand into a vessel filled with 

pure liquid solvent (Johanson et al., 1988). At regular intervals, thickness of the skin and the 

finger volume were measured. Blood and urine analysis were performed during a 24-hour 

period after the beginning of exposure. 

The skin of the exposed fingers was not irritated but appeared somewhat more rigid and less 

elastic after exposure and a wrinkled appearance. This effect reaches a maximum 2-4 hours 

after exposure and gradually disappeared. Concurrently, the volume of the fingers and the 

thickness of the skin decreased and then return to the normal. EGBE was detected in the blood 

of all subjects after exposure. Estimates of the skin uptake showed great variations between 

individuals (more than ten-fold variation): 7 to 96 nmol/min/cm2

 (0.05 - 0.63mg/cm2/hr) with 

a geometric mean of 20 nmol/min/cm2 (0.14mg/cm2/hr). The elimination t1/2 for EGBE in 

blood ranged from 0.6 to 4.8 hr (mean 1.3 hr). The excretion rate of BAA in urine increased 

during the first hours of exposure with a maximum 5 hours after the beginning of exposure. 

The cumulative excretion of BAA ranged from 2.5 to 39 % of the uptake of EGBE (with a 

mean of 17 %). 
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The percutaneous absorption of EGBE vapour was assessed in four male volunteers 

(Johanson and Boman, 1991). Subjects were exposed to EGBE by inhalation during 2 hours at 

concentration of 50 ppm (2 mmol/m3). The subjects were breathing chamber air while sitting 

outside the exposure chamber, mouth breathing was assured by the use of a nose clip. After 

the two hours, one hour of pause was made then a second period of exposure was performed. 

For this second exposure, subjects were sitting inside the exposure chamber (subjects were 

naked apart from a pair of shorts – the estimated surface area exposer being 16000 cm2) but 

breathing air was supplied from a tank of clean air. Heart rate was monitored during the entire 

day. Each volunteer participated in two experiments, one at normal (23° C, 29 % relative 

humidity) and one at raised temperature and humidity (33° C, 71 %). The two experiments 

were separated by two weeks or more. Capillary blood samples were collected during the 

experiments for the determination of EGBE. Based on earlier observations (Johanson et al., 

1986a), the relative respiratory uptake of EGBE was assumed to be 60 %. 

The EGBE blood concentration during the mouth exposure increased during the first hour and 

seemed to reach a steady state at about 3μM (1.8-4μM) during the 2nd hour. The mean 

respiratory uptake was 1.3 mmol or 11 μmol/min and the apparent blood clearance 3.8 l/min. 

For the percutaneous exposure, the concentration of EGBE in blood increased to about 9 μM 

during the second hour of exposure. In seven of the experiments the EGBE blood 

concentration was 2.4-5.5 times higher after skin exposure than after mouth exposure, in one 

it was lower. This difference was also reflected in the high percutaneous absorption of 31 

(8.6-48) μmol/min, or 2.5-5.9 times higher than the respiratory uptake. The half life of EGBE 

in blood after the skin exposure averaged 34 min (19-53 min). Only small and inconsistent 

differences in heart rate were found between the 2 routes of exposure, thus the observed 

differences in blood concentration and uptake rates of EGBE cannot be explained by 

differences in pulmonary ventilation. The concentration of EGBE in blood appeared to be 

slightly raised during mouth exposure to hot and humid, compared with normal chamber air. 

The increase may be partly explained by a raised pulmonary ventilation. The tendency 

towards higher blood concentrations in hot and humid conditions was more pronounced 

during the skin exposure. However, the differences between the two exposure conditions were 

not statistically significant. 

Six human volunteers were exposed arm only to 50 ppm (13C2)EGBE (purity 99.54 %) for 

2 hr (Corley et al., 1997). Blood samples were collected into the antecubital vein of the 

unexposed arm for the analysis of both EGBE and its major metabolite BAA. Finger 

prickblood samples were taken from the exposed arm only at the end of the 2 hr exposure to 

compare with the blood obtained in the antecubital vein. Blood samples were obtained before 

exposure and at 10, 20, 30, 40, 60 and 1.5, 2, 2.25, 2.5, 3, 3.5, 4, 8, 12, 16 and 24 hr after the 

initiation of the 2 hr exposure. Urine samples were collected before exposure and at 0.12 and 

12-24 hr intervals following the initiation of exposure for metabolite analysis. 

EGBE was not detected in blood samples from the unexposed arm until 30 minutes. By 1.5 hr 

EGBE was detectable in all 6 subjects. An apparent steady state was reached for EGBE 

between 1.5 and 2 hr. EGBE was rapidly cleared from the blood with an apparent elimination 

t1/2 of 0.66 hr (in 2 of the 6 subjects). 

As EGBE, BAA was not detected in blood samples from the unexposed arm until 30 minutes. 

BAA was detectable in all six subjects by 1 hr. Peak blood concentrations were reached 3-4 hr 

after the start of the 2 hr exposure. BAA was less rapidly cleared from the blood than EGBE, 

with an apparent elimination t1/2 of 3.27 hr. 
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The ratio of the finger prick blood sample taken at the end of the 2 hr exposure to the 

corresponding blood sample taken from the unexposed arm averaged nearly 1500/1 indicating 

a considerable influence by local absorption. 

No free EGBE was detected in any urine samples. Following hydrolysis to cleave acid-labile 

conjugates, EGBE was detectable in only 2 urine samples (0-12 hr). No EG nor glycolic acid 

were detected in urine. 

BAA was eliminated in urine during the first 12hr collection interval.2/3 of the total amount 

of BAA excreted in the urine was in the form of an acid-labile conjugate. In this study the 

skin permeability coefficient was estimated to be 3 cm/hr. 

Four volunteers (2 males and 2 females, aged 28-33) were exposed on 9 separate occasion, 

each occasion separated by at least 3 weeks (Jones et al., 2003). All exposure were 50 ppm 

EGBE for 2h. Two exposure were performed whole body and two skin only at 25° C, 40 % 

relative humidity and volunteers wearing shorts and T-shirts (baseline conditions). Other 

exposures were skin only and were performed with one parameter changed: high humidity (60 

% and 65 %), low and high temperature (20 and 30° C), minimal clothing (shorts (and bra) 

only and overalls (all-in-one boiler suit) and an industrial scenario (overalls and high humidity 

and temperature (30° C and 60 % relative humidity). Total BAA in urine was measured to 

assess the absorbed EGBE. Urine samples were collected before and after each exposure (0, 4, 

6, 8, 10, 12, 22, 26, 30 and 34h). To record any physiological changes in the volunteers under 

the different conditions, physiological monitoring equipment was used. The results obtained 

for skin only exposure are expressed as a percentage of the whole body measurement. 

The mean dermal absorption for baseline conditions contributes for 11 % of the total body 

burden. Low temperature did not affect significantly the dermal absorption whereas high 

temperature increased significantly the dermal absorption (14 %). Increasing humidity 

increased the dermal absorption but not significantly. Clothing has also a little effect on 

dermal absorption. In the industrial scenario skin absorption is significantly increased 

compared to baseline conditions. The maximum dermal absorption contributes for 42 % of the 

total body burden (mean 39 %). No significant differences in any of the physiological 

parameters were seen. 

This study shows that temperature is an important factor to take into account for assessing the 

percutaneous absorption of EGBE and also that the use of protective equipement under high 

temperature and relative humidity can lead to a high dermal absorption (higher than without 

protective equipment). 

Six male volunteers were dermally exposed to EGBE at various concentration: neat or 50%, 

90 % in water for 4 hours on the forearm (about 40 cm2

) (Jakasa et al., 2004). An inhalation 

exposure of EGBE vapour was also performed on each volunteer (93 ± 6.8 mg/m3 for 30 

minutes) and served as a reference dosage. Dermal absorption parameters were calculated 

during 24 hours after the beginning of exposures by measuring excretion of total BAA (free + 

conjugated) in urine and EGBE in blood. Blood samples were collected for 8 hours (16 

samples per experiment). Urine samples were collected every 4 hours during the 24 hour 

period. Each volunteer was exposed twice to a 50 % EGBE concentration (on each arm), once 

to the 90 % concentration and once to the pure EGBE. The period between two dermal 

exposures of the same site was at least 4 weeks. 

EGBE in blood was below the detection limits after exposure to pure EGBE. The average 

dermal flux and the apparent permeability coefficients were greater for the 50 % dilution than 

for the 90 % dilution (see table 4.42). The same results were obtained with urinary excretion 
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of BAA. The permeation rate reach a plateau between 1 and 2 hours after the beginning of 

exposure, indicating a steady state permeation. The maximum permeation rates are presented 

in the table 4.43 (apparent permeability coefficients). The uptake after dermal exposure was 

also compared with the uptake after inhalation exposure (see table 4.42) showing that 

significant amounts of EGBE are due to dermal exposure. Half life of BAA was calculated to 

be 3.4 hr (range 1.3 to 3.8 hr for inhalation experiment). From the inhalation study, it was 

calculated than 57 % of the inhaled EGBE was excreted as BAA in urine. 

Table 4.42: Kinetic parameters measured in the Jakasa study 

 50 % EGBE 90 % EGBE Neat EGBE 

Average dermal flux (mg/cm2/h) 

Based on blood data 0.92 +/- 0.34 0.74 +/- 0.25 ND 

Based on urine data 1.34 +/- 0.49 0.92 +/- 0.60 0.26+/- 0.17 

Apparent permeability coefficients (x10-

3

 cm/h) 

1.75 +/- 0.53 0.88 +/- 0.42 ND 

Estimated dermal uptake of EGBE(mg) 1,340 926 263 

Pulmonary uptake 346 

Intra and inter-individual variation of dermal fluxes were also studied with 50 % dilution. The 

results are reported in the table 4.43. 

Table 4.43: Intra and inter-individual variation of dermal fluxes after dermal exposure to 50 % EGBE in water. 

 Average dermal flux 

(mg/cm2/h) 

90 % confidence 

interval 

Intra individual 

CV (%) 

Inter individual 

CV (%) 

Blood data 0.92 +/- 0.34 0.63 – 1.19 16 35 

Urine data 1.34 +/- 0.49 0.94 – 1.74 20 34 

The results obtained in this study are in good agreement with animal data and in vitro data on 

human skin. It also demonstrate that demal absoption of EGBE is important when compared 

with inhalation exposure. 

Summary human dermal route: 

Five in vivo studies on volunteers and four in vitro studies are available for this end point. The 

four in vitro studies, measuring the rate of absorption of liquid EGBE through human skin, 

gave results that varied by a factor of 25 (0.064 mg/cm2

/hr for the lowest and 1.66 mg/cm2/hr 

for the highest). In vitro, rate of absorption is highly dependent on the concentration of the 

aqueous solution of EGBE used. In vivo, in one study, an estimation of the skin penetration 

gave results in the following range: 7 to 96 nmol/min/cm2 (0.008 mg/cm2

/hr to 0.0114 

mg/cm2/hr) for pure liquid EGBE. Another study performed with liquid EGBE has showed 

that greater absorption was found with EGBE 50 % in water than neat EGBE (dermal flux 

1.34 mg/cm2

/h and 0.26 mg/cm2/h respectively). This is consistent with data available in vitro

and in animals. This is also critical for risk characterization because percentage of absorption 
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taken into account in each scenario can be different if exposure is exposure is to EGBE pure 

or EGBE in formulations.

EGBE half life was approximately 1 hour in all studies whereas BAA half life was about 5 

hours. If temperature and humidity conditions increased, the percutaneous uptake increased 

also (in contrast with the inhalation uptake which remained constant). 

In one in vivo study, percutaneous absorption of vapour EGBE was assessed. Depending on 

the external conditions during exposure, the internal dose of EGBE due to percutaneous 

absorption varies between 11 % and 39 %. The percentage of 11 % was found for “normal” 

conditions of use (temperature, humidity) and 39 % for the worst case of industrial use (high 

temperature, high humidity and overalls wearing). This worst case percentage is use in the 

risk assessment section to estimated the internal dose of EGBE due to dermal absorption of 

vapour EGBE. 

Based on the PbPk modelling it is estimated that for a worst-case exposure to EGBE vapour 

(100 % of the body exposed and no cloths), percutaneous absorption would account for 15-27 

% of the internal dose of EGBE (Corley et al., 1997). 

Oral 

In a suicide attempt, a 50-year woman ingested 250-500 ml of a window cleaner containing 

12 % of EGBE, corresponding to about 0.5 to 1 g/kg bw (Rambourg-Schepens et al., 1988). 

On admission to the intensive care unit, the patient was comatose. Biologically she presented 

with metabolic acidosis, hypokalaemia, a rise in serum creatinine level and a markedly 

increased urinary excretion of oxalate crystals. 

In another suicide attempt, quantitative analysis of blood BAA was performed (Gualtieri 

et al., 2003). A high maximum blood concentration was found (4.86 mmol/L). Symptoms 

recorded were metabolic acidosis and hepatic biochemical disorders. Si sign of haemolytic 

anaemia were described. 

4.1.2.1.3 Other data 

In vitro studies have been conducted to determine the relative degree to which some glycol 

ethers act as substrate for alcohol dehydrogenase (ADH) (Dow Chemicals, 1983). Four 

concentrations of EGBE (purity 98.55 %) were used to define the kinetic constants. Each 

concentration was run in duplicate. Experiments were conducted at both pH 8.8 and 7.4 in 

order to determine if pH was a critical factor. 

Assays conducted at pH 7.4 resulted in data sets which were not suitable for plotting. For 

EGBE, Vmax was 4.06 μM/min, Km was 1.18x10-3 M with a correlation coefficient of 0.98. 

Kinetic of EGBE was studied in a perfused rat liver system with and without ethanol 

(Johanson et al., 1986b). The liver of a Sprague-Dawley rat was isolated and perfused with 

EGBE (99 % purity). Concentration ranging from 0.057 to 2.7 mM of EGBE were achieved 

and submitted to the perfusion system for periods of 10 min. Two samples from the perfusion 

medium and from the perfusate were collected during the last 4 minutes and were analysed for 

EGBE concentration determination. Preliminary studies indicated that the half life of the 

adaptation of the liver to new concentrations of EGBE was approximately 1 min. Hence, 6 

min was regarded sufficient to reach a plateau level. At the end of 3 experiments, the effect of 
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EtOH was studied while maintaining the concentration of EGBE at 0.45 mM. The liver was 

then perfused with a medium containing 17.1 mM EtOH during 20 min, followed by an equal 

period of time of perfusion with EtOH free medium. 

The elimination data fit well to the Michaelis-Menten equation. The estimates of the apparent 

maximum elimination rate of EGBE range between 0.59 and 1.3 μmol/min/g. The estimate of 

the Km vary from 0.19 to 0.4 mM. Maximum clearance ranged from 2.7 to 3.1 ml/min/g. In 

the presence of EtOH, the extraction ratio of EGBE decreased from 0.44 to 0.11. When EtOH 

is withdraw, the liver returned nearly to its previous elimination capacity in approximately 10 

min. This finding supports the hypothesis that EGBE is mainly metabolised by ADH in the rat 

liver. 

Testicular and hepatic capacities to metabolise EGBE by alcohol dehydrogenase (ALD) was 

assessed (Moslen et al., 1995). These comparisons were done with SD rats (highest testicular 

capacity to metabolise EGME) and with Syrian Golden Hamster (no detectable testicular 

capacity to metabolise EGME). 

Testicular and hepatic activities of ALD for EGBE was greater in hamster than in rat. 

Comparing with other glycol ethers (GE), testicular and hepatic activities of ALD for EGBE 

were the less important in rats whereas testicular ALD in hamster was greater for EGBE than 

for EGME or EGEE. In hamster liver, the ALD activity was less than those of EGME and 

EGEE. 

In a series of in vitro studies the metabolites of some glycol ethers, EGME, EGEE and EGBE 

were determined in human and rat hepatocyte cultures (Green et al., 1996). Adult male Fisher 

344 hepatocytes and human hepatocytes were incubated with EGBE (purity unknown) at 

doses of 0.02 – 0.2 – 2 and 10 mM for 4 hr. 

Over 90 % of the radioactivity was detected in 3 peaks: EGBE – BAA and EG. BAA was the 

major metabolite formed in both species. More than 90 % of the 0.2 mM EGBE was rapidly 

converted to BAA by rats hepatocytes (40 % for human hepatocytes). The percentage of 

EGBE that was converted to BAA after 4 hr of incubation with rat hepatocytes was similar at 

0.02 – 0.2 and 2 mM. In human hepatocyte the percentage of EGBE that was metabolised to 

BAA decreased between 0.2 and 2 mM. The percentage of EGBE metabolised to EG was 

greatest at the lowest substrate concentration for both human and rats suggesting that this 

pathway became saturated at relatively low glycol ether concentration. Another minor 

metabolite of EGBE was identified in this study: glucuronide conjugate of EGBE. 

Vmax values were 15–20 fold greater in rat hepatocyte than in human hepatocytes (741 

nmol/h/106 hepatocytes in rat and 113 in human). The Km value was similar for rats and 

humans (1 mM). 

The potential of EGBE to form fatty acids conjugates was studied in F344 rats and in vitro

(Kaphalia et al., 1996). Fatty acid ethyl synthase (FAEES) was extracted from rat liver 

microsomes. Different quantities of this enzyme were incubated with 2 μmol of [1-14C] oleic 

acid and 750 μmol EGBE at 37 ° C for 2 hr. A control reaction mixture was also incubated 

without enzyme. After extraction radioactivity was measured. 

Incubation of [1-14C] oleic acid and EGBE in the presence of the FAEES results in the 

formation of 18:1 fatty acid 2-butoxyethyl ester (not detected in the mixture incubated without 

enzyme). 
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The role of liver alcohol dehydrogenase isoenzymes in the oxidation of glycol ethers in rats 

was explored (Aasmoe et al., 1998). Wistar rats were killed to prepare liver homogenate. 

ADH activity was assayed by gel electrophoresis after addition of EGBE at the following 

concentrations: 0.05-20 mM. Km and Vmax were calculated. Activities were expressed as 

specific activity (the rate of NADH formation/min/mg protein). This assay was also 

performed concurrently with Methanol (MeOH), Ethanol (EtOH),BuOH, EGME, EGEE, 

Ethylene Glycol Propyl Ether (EGPE) and Ethylene Glycol Hexyl Ether (EGHE). 

After electrophoresis, the liver homogenate revealed one enzymatically active zone on 

staining with MeOH, EtOH, BuOH, EGME, EGEE, EGBE and EGPE (this pattern was 

identical for male and females). This enzymatic form is probably identical with the ADH-3. 

The intensity of the ADH-3 increased with increasing chain length of the alcohol or glycol 

ether. The ADH-2 isoenzyme was only active with medium and long chain aliphatic alcohols 

(not with glycol ethers). The same ADH isoenzyme was active in males and females, however 

the activity of ADH is sex-dependent, with higher enzyme activity in females than in males. 

This experiment also showed that this difference was a result of higher amount of ADH in 

females rather than of sex-specific isoenzymes. 

The substrate specificity (Vmax/Km) decrease in the following order: EGBE > EtOH > EGEE 

> EGME. The Km value for EGBE was approximately 10-fold lower than the other 2 glycol 

ethers, while the Vmax values were almost the same. Consequently, at low and identical 

glycol ether concentrations, the biotransformation of EGBE would be more efficient than that 

of the other two glycol ethers. At the same time, the low Km value for EGBE compared to 

EGME and EGEE indicates that metabolism of EGBE via ADH will be saturated at lower 

substrate concentrations than those of EGME and EGEE. 

A PbPk model was developed with the results obtained from various studies (Johanson, 

1986c). In his study, Johanson tried to extrapolate the in vitro rat data to man in vivo. 

Concentration-time curves are generated by computer simulation. The outcome of the 

simulation is compared with the results from the experimental exposure in male volunteers. 

The model used is designed for simulation of organic solvent kinetics in general. The organs 

are lumped into compartments according to blood flow and fat content. Muscles and skin 

form a separate compartment due to their wide variation in blood flow at different work loads. 

It is assumed that solvent uptake occurs only in the lungs and elimination only in the liver. It 

is further assumed that the solvent instantaneously distribute homogenously in each 

compartment, and as a consequence the solvent retained in the respiratory airways 

immediately reaches the arterial blood. The model structure is depicted in the figure 4.44: 

Figure 4.44: First PBPK developed for EGBE 

All partition ratios between blood and other compartments are set to 1. Strict flow limitation, 

i.e. diffusion equilibrium between end-capillary blood and tissue is assumed for all 

compartments. The relative pulmonary uptake of EGBE was set to 0.6. Volumes and blood 

flows to the different organ groups were computed from literature data. 

There is a good agreement between simulated and experimental blood concentration curves, 

indicating that the assumptions made have a certain degree of validity. Increased physical 

activity increased the blood concentration of EGBE due to increased pulmonary uptake rate. 

Similarly, the co-exposure to EtOH caused elevated blood concentration of EGBE due to a 

decrease in the elimination rate. The simulation also suggested that venous blood samples are 

useful, if not slightly more useful, as arterial or capillary blood sample in the biological 
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monitoring of exposure to EGBE. In any case, blood samples should be collected during the 

exposure phase because of the rapid decay of EGBE. The risk of accumulation of 

unmetabolized solvent in the body appeared low. According to the model, non-linearities 

because of saturated elimination occur at concentrations well above 100 ppm, even in 

combination with physical exercise and ethanol, thus linear kinetics of EGBE may be 

expected at ordinary occupational inhalation exposure. 

The model was improved by Shyr et al., 1993. Improvements consisted in the modelisation of 

the metabolite formation by three routes of exposure (oral, dermal, inhalation). 

In order to improve the PbPk model described by Johanson in 1986, a series of Good 

Laboratory Practice (GLP) studies were performed (Dow, 1993 – Corley et al., 1994). The 

objectives were to : 

- expand the PbPk model to include additional exposure routes (oral gavage, drinking 

water, IV infusion and dermal), 

- determine the relative solubilities of EGBE and BAA in rat blood, rat tissues and human 

blood for use in estimating partition coefficients 

- incorporate the disposition of BAA into the model 

- add allometric scaling factors for rat and human physiological parameters 

- add competing pathways for the metabolism of EGBE into the model. 

- develop a method for analysing trace levels of both EGBE and BAA in blood 

- validate the new analytical method by administering 14C-labeled BE to rats and analysing 

blood for BE and BAA in urine for BE and major metabolites. 

- confirm the presence of the metabolite, EG, in urine by mass spectroscopy 

- validate the new PbPk model with data developed as a part of these studies and relevant 

data from the literature. 

The model used was significantly different from that of Johanson (see figure 4.45): 
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Figure 4.45: Improvement of the PbPk model (from Dow, 1993) 

Allometric-scalable physiological and biochemical parameters were used in place of standard 

values for a 70 kg man to normalise these parameters to the actual body weights reported in 

several human studies. The rat was added to expand the database for model validation and to 

assist interspecies comparisons. Additional routes of exposure were added for validation of 

the model simulations with data from published studies. A second model was linked to the 

EGBE model specifically to track the disposition of BAA. The kidney was added to the BAA 

model since it is an organ of elimination for BAA. 

Nose only inhalation, IV infusion, oral gavage and drinking water simulations were modelled 

according to Andersen et al., 1987. Dermal exposure to EGBE were modelled according to 

Mc Dougal et al (1986 and 1990) for neat and aqueous solutions and according to Johanson 

and Boman, 1991 for vapour absorptions. 

Physiological constants were taken from the literature (Anderson, 1987 ; Fiserova-Bergerova, 

1983 ; Johanson, 1986c). The effect of exercise on the ventilation rates and blood flows were 

adapted from Astrand (1983) and Johanson (1986c). 
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Partition coefficients were available only for EGBE in human blood/air and saliva/air 

(Johanson and Dynesius, 1988). Partition coefficients for EGBE and BAA in the blood, liver, 

kidney, skin, fat, muscle, stomach, small intestine, caecum and lung tissues of male F344 rats 

and the blood of humans were determined experimentally. 

After calculation of the partition coefficients the following assumptions were made for the 

PbPk model: 

- the partition coefficients for EGBE and BAA for the rapidly perfused compartments were 

assumed to be equal to that of the liver 

- the partition coefficients for the slowly perfused compartments were assumed to be equal 

to that of the muscle 

- the average of the stomach, small intestine and caecum partition coefficients was used to 

represent GI tract 

- the skin/air partition coefficient for dermal uptake of EGBE vapours was assumed to be 

equal to the blood/air partition coefficient 

- the skin/liquid partition coefficient for dermal dosing of neat EGBE and solutions of 

EGBE was assumed to be equal to the measured skin/saline partition coefficient. 

Biochemical constants : the metabolism of EGBE and BAA was assumed to occur only in the 

liver through a single metabolic pathway (alcohol / aldehyde dehydrogenase) following 

Michaelis-Menten kinetics. Other minor metabolic pathways have been reported by various 

authors, since there was insufficient kinetic data available to differentiate the parameters 

associates with each of these other pathways, a second saturable pathway was included in the 

model to simulate the disappearance of EGBE from the liver by all other pathways combined. 

Parameters for this pathway were estimated (based on Medinsky et al., 1990 studies). 

The model also assumed that BAA was bound to proteins in blood and was eliminated by a 

saturable process in the kidney. A model similar to that of Russel et al., 1987 was used for 

describing protein binding and saturable elimination of BAA. 

Cmax and AUC of the concentration for BAA in blood were used as an estimates of the 

“delivered dose” or “internal dose surrogate” for high dose/low dose, dose/route and rat to 

human extrapolation. 

A metabolism study was conducted to assist in validation of model parameters studied (see § 

4.1.2.1.1.1). These results with other data sets were compared with model predictions. 

Data are generally in good agreement with the model except for dose levels which cause 

toxicity. The model has no systematic provision for correcting for potential diminished renal 

excretion and/or liver metabolism that arise secondary to the haemolytic activity of BAA and 

will therefore tend to overpredict the amounts of BAA excreted by the kidneys in urine. 

Nevertheless the model satisfactorily predicts concentrations of BAA in blood and amount of 

BAA eliminated in the urine at dose levels that do not cause toxicity. 

The PbPk model of Corley et al.,1994 served as a background to develop a new model to take 

into account chronic inhalation and female rat and mice parameters. (Lee et al., 1998). The 2-

year toxicokinetic studies utilized in the model development were conducted as a part of the 

2-year inhalation toxicity and carcinogenicity studies of EGBE (NTP, 1998) and are described 

previously in this report (Dill et al., 1998). 
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Compared to the previous model, the muscle compartment was included in the poorly 

perfused compartment, a kidney compartment was added for EGBE and spleen was added as 

a separate compartment. The same assumptions than before were made. It was also assumed 

that partition coefficients of EGBE and BAA in male rats were representatives of those for 

other species regardless of the sex. Some adjustments have been made due to chronic 

modifications induced by EGBE: 

- cardiac output start to diminish after 6 month exposure (down to 2/3 of the estimate used 

for young animals by the end of the 18 months of exposure). 

- volume of the fat compartment starts to increase after 6 month of exposure (up to 3 fold at 

the end of the 18 months of exposure). 

- Total plasma protein binding sites in mice start to increase after 6 months of exposure (up 

to 2 fold at the end of the study) 

- The maximum rate of EGBE metabolism to BAA for a male rat starts to increase after 12 

months of exposure (2 fold at the end of exposure). 

- The maximum rate of 2BAA renal elimination starts to decrease after 6 months of 

exposure (down to 3⁄4 of the estimate for young animals at the end of exposure). 

Figure 4.46: Last PbPk model (Lee et al., 1998) 

In human via inhalation and dermal route, the study of the PbPk model demonstrated that it is 

not possible to reach a BAA blood concentration that might be able to cause even a slight 

haemolysis in humans (Corley, 1996). This is based on the assumption that a concentration of 

8mM of BAA in blood is needed in human to be able to develop sign of haemolysis (only 0.5 

mM in rats) and was calculated for theoretical exposure of 6 hr regardless of the concentration 

(even up to the saturated concentration of 1160 ppm EGBE) or inclusion of the dermal 

absorption of vapors. 
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Recently, the model was improved to include key studies that independently determined sex, 

age and species differences in partition coefficients, metabolism and renal clearance (Corley 

et al., 2005). 

Summary other data : 

In vitro studies have showed that EGBE transformation in BAA depended on ADH (isotype 

3). This enzyme is more active in females than in males. Moreover, it has been demonstrated 

than metabolic rate in rat was 10 to 20 fold higher than in human. 

A PbPk model has progressed with experimental data collected in humans and in animals. 

This model included at the present time various exposure route (inhalation, oral, dermal, IV), 

the differences between humans and animals, the kinetic parameters of the main metabolite 

BAA and allow also the modelling of repeated exposures. Recent studies have given results 

which are consistent with the current PbPk model. 

4.1.2.1.4 Summary of toxicokinetics, metabolism and distribution 

Oral administration of EGBE leads to a quite complete absorption of the substance. Via 

inhalation route, a “wash in / wash out” mechanism limits the absorption to 55 – 60 % of the 

administrated concentration. Via dermal route, uptake of liquid EGBE depends on the 

administration mode, the species and the concentration of EGBE in the final product. An 

occlusive administration will be responsible of a great percutaneous uptake whereas a nonocclusive administration will minimize absorption due to the volatility of EGBE. 

Rat skin seems to be readily permeable compared to pig or human skin (2 or 3 fold more). 

Percutaneous uptake also depends on the EGBE concentration in the tested product : for 40 

and 80 % aqueous solutions of EGBE, absorption was demonstrated to be maximum. In two 

rat studies, dermal absorption of liquid EGBE was estimated to be between 20 and 30 %. 

In human, dermal studies with liquid EGBE give penetration uptakes which vary by a factor 

of 10 between different subjects exposed to EGBE with the same experimental conditions. A 

percentage of absorption of about 12 % was calculated in one study using EGBE at 5 and 10 

% in water. 

Dermal penetration of vapour EGBE was studied in humans. Depending on the experimental 

conditions, the percentage of the absorbed substance due to dermal uptake varies between 11 

and 39 % of the total body burden. 

EGBE reaches a maximum blood concentration rapidly after exposure whichever the route of 

exposure. EGBE is rapidly metabolised (with a plasmatic half life of about an hour). 

Target organs are the liver, kidneys, thymus and stomach, in particular forestomach in the rat 

whichever the route of administration (oral and inhalation route, no data for dermal route). 

The main metabolism pathway leads to the formation of BAA via Alcohol dehydrogenase and 

Aldehyde dehydrogenase in a saturable mechanism. With increasing doses of EGBE, the 

formation of glucuronide conjugate of EGBE or BAA is enhanced. Minor metabolites of 

EGBE are also reported depending on the species used (see figure 4.47).
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Figure 4.47: Metabolism of EGBE (Patty, 2001) 

Materials identified with an asterisk (*) have been identified in either rodents or humans. 

**: BAA-Blu = N butoxyacetyl glutamine conjugate 

Elimination is rapid and mainly via urinary route (80 to 90 % of the metabolites). The 

plasmatic half-life of metabolites is about 4 hours. A small amount is eliminated as CO2 by 

the respiration (10 to 20 %). Normal renal excretion is conditioned by physiological state of 

the kidneys: females excreted less rapidly BAA than males and aged animals have a trend to 

eliminate metabolites with more difficulties than young animals. Any renal injury will 

enhance BAA toxicity by increasing its blood persistence. 

However if renal integrity is respected, a repeated administration of EGBE lead to an 

adaptation of the metabolism. In this case elimination of BAA occurred more rapidly. This 

mechanism of extra hepatic adaptation is also described for action of EGBE on red blood 

cells, especially on erythrocyte deformability (see § 4.1.2.2.3). 

EGBE BAA * 

EGBE glucuronide * 

BES (sulfate- conjugate) * 

EG * 

BAA -– 

Glu ** 

Humans * 

CO2 * 

URINE

Mixed 

function 

oxydase 

Alcohol and 

aldehyde 

dehydrogenase 

Butyraldehyde 

Butyric acid 

CO2 * 

 + glycine Glycine conjugate 

oxalic acid 
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For risk characterisation, if absorption factors are needed, the following values will be taken : 

3⁄4 Oral route: 100 % absorption 

3⁄4 Inhalation route: 60 % absorption 

3⁄4 Demal route: distinction should be made between dermal absorption of EGBE vapour 

(in the atmosphere) and dermal absorption of liquid EGBE. For these two cases, the 

absorption factors should be discussed on a case by case basis because a lot of factors 

can modify these factors (temperature and humidity, clothing, concentration of EGBE 

if diluted, duration of the exposure...). 

Overall, dermal absorption of EGBE vapour will contribute for 27 % of the total 

EGBE body burden in normal uses and 39 % if extreme conditions are expected. 

Dermal absorption of liquid EGBE will vary in function of the duration of exposure 

and the concentration of EGBE in the formulation, internal doses will be calculated on 

a case by case basis using the dermal flux calculated in the Jakasa study. Where 

required, a permeation rate of 0.63mg/cm2

/hr will be used. This is the upper figure 

from the in vivo data and is consistent with the in vitro data. 

It is considered that the PBPK model for EGBE is sufficiently well developed to justify its 

used to derive animal to man toxicokinetic extrapolation factors for the inhalation route. 

These factors are based on the toxokinetics of BAA since this is the metabolite that causes the 

critical toxic effects. A detailed description of how the model is used is given in the General 

Aspects introduction to the risk characterisation. Where required, the half life of BAA will be 

assumed to be 5 hours. 

4.1.2.2 Acute toxicity 

4.1.2.2.1 Studies in animals 

Inhalation 

Rats

Carpenter et al. 1956 and the report of the Mellon Institute of Industrial Research (1952) 

reported some experiments concerning acute toxicity of EGBE in various animals. For rats via 

inhalation route, a LT50 of 9 hours was reported for a concentration of 1300 ppm. At 800 

ppm, mortality was 3/6 rats for a 8-hr exposure period and 0/6 rats for a 4-hr exposure period. 

At 500 ppm no rats out of 6 were dead for a 8 hr exposure period and only one out of 6 for a 4 

hr exposure period. When older rats were exposed to 375 ppm (age not reported) 11 out of 13 

and 23 out of 23 died following a 7 hr exposure period. 

Saturated vapour of EGBE caused no mortality among 6 adult female rats exposed for 8 hr 

and observed for a 14-day observation period. Clinical symptoms were limited to bloody 

urine and poor coordination. Concentration was measured to be 475 ppm. 

Earlier results (1940 and 1943 cited in Carpenter et al., 1956) with saturated vapour give the 

following results: 4 out of 6 rats died following a 9 hr exposure, 2 out of 6 rats died after a 4 

hr exposure and no rats died after a 2 hr exposure. 
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In a poorly reported study (Gage, 1970), rats (4 males and 4 females) were exposed whole 

body to EGBE (purity unknown) at an unique concentration of 2400 ppm (13 mg/l) during 5 

hr. At this concentration test substance was in aerosol form. 

All rats died within 2 days. Clinical symptoms before death were a comatose state and 

haematuria. Blood Hb was measured 35 to 50 % of the normal. 

Groups of Fisher 344 rats (6/sex/dose) were exposed whole body to EGBE at doses of 867, 

523 or 202 ppm (purity 99.4 %) for 4 hr (Bushy Run Research Center, 1980a). EC50 was 

calculated at the end of the 14-day observation period. Clinical observations and gross 

macroscopy was recorded during and at the end of the study. 

In rats exposed to 867 ppm, loss of coordination was observed concurrently with laboured 

breathing and red fluid discharge around the urogenital region. All animals were dead 2 days 

after exposure. In rats exposed to 523 ppm, coordination loss was also observed and red fluid 

discharge around the urogenital region. 2 males and 3 females died during the 14-day 

observation period. At the end of the study, the tail of 2 males and 2 females appeared 

necrotic. In rats exposed to 202 ppm, except a slight amount of dried red material observed on 

the tail the day following exposure, no other sign of toxicity was observed. Body weight of 

the 523 and 202 ppm groups were depressed 1 day after exposure but a normal body weight 

was recovered by 4 days. Animals that died had enlarged and discoloured kidneys with 

urinary bladder filled with red stained urine. 

In this study, a LC50 of 486 ppm was calculated for males and 450 ppm for females. 

In a validation study for the OECD method 403 (ring test), 8 chemicals including EGBE were 

tested (Shell Chemicals, 1982). Wistar rats (3/sex/group) were exposed whole body to 

saturated vapour concentration (theoretically 617 ppm) for a variable period of time. The 

maximum exposure time was 7 hr, if deaths occurred during either the exposure period or the 

observation period, exposures were repeated for shorter intervals (eg. 3 hr, 1 hr, 30 min...). 

For Butyl oxitol (EGBE – purity 99 %) durations of exposure were 7 hr, 3 hr and 1 hr. 

Measurements of the atmospheric concentrations in the exposure chamber gave values 

between 750 and 910 ppm. 

During exposure animals exhibited signs of lethargy. For rats exposed during 7 hr, mortality 

was 1/3 males and 3/3 females at the end of the observation period. Clinical signs were 

necrosis of the end of tails, blood in urine and paleness of eyes and feet. For the 3 hr exposure 

group mortality was 0/3 males and 1/3 females. Except for necrosis of the tail, the same 

symptoms than the 7 hr group were observed. For the 1 hr group, no mortality was observed, 

symptoms were limited to the presence of blood in urine and paleness eyes and feet. Recovery 

was full 2 days after exposures. 

The results of other laboratories participating at the ring test were published (Klimisch et al., 

1988). The nominal concentration at which animals were exposed varied from 3.3 mg/l to 4.2 

mg/l (mean 3.7 mg/l). The LT0 (time for which at least one death were found) was 3 hr for 5 

laboratories and 1 hr for only one laboratory. 

Mice

In a poorly reported inhalation study in mice,, the effects of EGBE and other glycol ethers 

was studied (Werner et al., 1943a). Swiss mice were exposed in groups of 16 for 7 hr periods. 
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At 1210 ppm all animals died within 32 hr. At 920 ppm 11/16 animals died within 32 hr. At 

770 ppm 14/16 animals died within 1 week after exposure. At 670 and 560 ppm, 2/16 animals 

died in 32 hr and 1 week respectively. No animals died with exposure to 390 ppm of EGBE. 

Dyspnoea was the major toxic symptom described moreover when doses were near the lethal 

level, severe haemoglobinuria was seen. Moderate to marked follicular phagocytosis and 

congestion of the cavernous veins were frequent findings in spleens of exposed animals. Focal 

necrosis and lymphoid hyperplasia was also seen in 3 surviving animals. Interstitial nephritis 

and typical pictures of bronchopneumonia was seen in a few animals. 

Guinea pigs

Carpenter et al., 1956 reported some experiments concerning acute toxicity of EGBE in 

various animals. A LT50 of 7 hr was reported for guinea pigs for an exposure of 1300 ppm 

(Union Carbide Corp., 1943). Guinea pig mortality was 1/6 in a 4 hr exposure (Mellon 

Institute of Industrial Research, 1952). 

In a GLP study, EGBE (99.87 % purity) was administrated via inhalation route (whole body) 

to Guinea Pigs for a single hr of exposure (Bushy Run Research Center, 1994). Target 

concentration for the exposure was the highest attainable EGBE concentration (saturated 

vapour concentration) at 20°C and 740 mm Hg: 791 ppm. Study design was identical to 

OECD guideline 403 except for the use of GP and for the during of the exposure (1hr). 

Real concentration obtained were 633 and 691 ppm for the females and the males 

respectively. 

No death nor clinical signs were seen during the exposure or during the 14-day post-exposure 

period. No changes in body weight or bodyweight gain were observed. Gross findings at 

necropsy were observed in 4/5 males and 3/5 females, all of these findings were considered to 

be incidental. 

For this study, it can be concluded that LC50 in GP in greater than 633 ppm in females and 

greater than 691 ppm in males. 

Summary inhalation route: 

Table 4.48: Summary inhalation route 

Lethal Concentration 

LC50 (mg/kg) 

Mortality and treatment related 

effects in function of exposure time 

Validity 

* 

Reference 

Rats inhalation route 

 Exposure to 800 ppm during 8 hr 

caused 50 % mortality whereas an 

exposure time of 4 hr did not cause 

ant death (6 animals in each group) 

Exposure to 500 ppm (2.45 mg/l) for 

8 hr – did not cause any mortality 

(0/6) wherease a tratment period of 

4hr caused one death (out of 6) 

If Older rats are exposed to 375 ppm: 

2 Mellon Institute of 

Industrial Research, 

1952 

Carpenter et al., 1956
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11/13 and 23/23 died after 7 hr of 

exposure 

LC50: 486 ppm (2.38 

mg/l) for males and 

450 ppm for females. 

4hr exposure 

Laboured breathing, loss of 

coordination, tail necrosis, renal 

toxicity, haemolysis. 

Mortality from 523 ppm (2.56 mg/l) 

2 Bushy Run Research 

Center, 1980a 

 Saturated vapours (617 ppm – 3 mg/l) 

and exposure time variable. 

Mortality 4/6 for 7 hr exposure, 1/6 

for 3 hr exposure and no mortality for 

1 hr exposure. 

Lethargy, necrosis of the tail and 

haemolysis. 

2 Shell Chemicals, 

1982 

 2400 ppm (11.76 mg/l) aerosol for 5 

hr exposure. All rats died within 2 

days. Comatose state and haemolysis.

3 Gage, 1970 

Mice Inhalation route 

 Mortality from 670 ppm (3.28 mg/l) 

and 560 ppm (2.74 mg/l) for 

exposure of 32 hr and 1 week 

respectively. No dead for 390 ppm 

(1.91 mg/l). 

Dyspnea and haemolysis. 

3 Werner et al., 1943a 

Guinea Pigs Inhalation route 

 LT50: 7 hrs at 1300 ppm (6.37 mg/l) 2 Carpenter et al., 1956

> 633 ppm (3.1 mg/l) 

(females) 

> 691 ppm (3.39 

mg/l) (males) 

1 hr exposure. 

No effects 

1 Bushy Run Research 

Center, 1994. 

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed 

according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment 

possible. 

In a good quality study performed on rats, LC50 was calculated to be comprised between 450 

and 486 ppm (for a 4 hour exposure (Bushy Run Research Center, 1980a). Other studies give 

results quite consistent with this one. In these studies, females and old animals were more 

sensitive than males or young animals. 
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Clinical symptoms and pathology were: lethargy, ataxia, laboured breathing, loss of 

coordination, haemolysis and tail necrosis. Renal injuries were commonly seen during 

pathological examinations. 

According to the data available, a classification R20 is needed. 

Dermal 

Rats

EGBE (purity unknown) was applied dermally (occlusive) on rats for 4 hr (Mellon Institute of 

Industrial Research, 1961). Animals were observed during 14 days after application. 

The calculated LD50 was 2275 mg/kg (1680 – 3079 mg/kg). 

The dermal toxicity of EGBE (purity unknown) was assessed in rats in a GLP study 

(Safepharm laboratories, 1993a) according to OECD guideline 402. SD rats (5/sex) were 

given a single 24 hr, semi-occluded dermal application of undiluted test substance and then 

observed for 14 days. 

No deaths were reported. No signs of systemic toxicity or irritation were noted during the 

study. All rats gained body weight as expected during the study. No abnormalities were noted 

at necropsy. The LD50 in this study was greater than 2000 mg/kg for both males and females 

rats. 

The dermal toxicity of EGBE (purity unknown) was assessed in rats in a GLP study 

(Safepharm laboratories, 1993b) according to OECD guideline 402. SD rats (5/sex) were 

given a single 24 hr, occluded dermal application of undiluted test substance. Animals were 

observed for 14 days following application. 

One female was found dead 2 days after dosing. Comment signs of systemic toxicity noted 

were ataxia, pallor of the extremities, emaciation, lethargy, decreased respiratory rate, 

laboured respiration and tiptoe gait with incidents of ptosis and red/brown staining around the 

eyes. A full recovery was seen before the end of the study. No signs of irritation were noted 

during the study. Surviving animals showed expected gain in bodyweight during the study 

except for one female which showed bodyweight loss during the firs week. No abnormalities 

were noted at necropsy in surviving animals. Female that died during the study showed 

haemorrhagic lungs, dark liver, dark kidneys, sloughing of the non-glandular epithelium of 

the stomach and haemorrhage of the small and large intestines. The LD50 in this study was 

greater than 2000 mg/kg for both males and females rats. 

Guinea pigs

Results of studies were reported briefly (Mellon Institute of Industrial Research, 1952). 

Dermal LD50 was found to be 6411 mg/kg (5400 to 7765 mg/kg). 

LD50 in rabbits and in guinea pigs were compared (Roudabush et al., 1965). Groups of 

Hartley guinea pigs and white rabbits (4 animals / dose) were dosed (at least 3 doses) with 

EGBE (purity unknown) in intact skin for GP and rabbits and also in abraded skin for GP 

only. 
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The LD50 values were614 mg/kg in rabbits and 208 and 271 mg/kg for intact skin and 

abraded skin respectively in guinea pigs. 

A comparative percutaneous toxicity study of 10 industrial solvents (including EGBE) was 

performed in guinea pig (Wahlberg and Boman, 1979). A fixed volume of EGBE (purity > 99 

%) was administrated occlusively to 20 guinea pigs for a period of time which allow the test 

substance to be totally absorbed (between 5 and 7 days). Animals were then observed for 35 

days for clinical symptoms and were weighted once daily except on week end. For 

comparison the same amount of EGBE was injected IP and the animals were followed in the 

same manner as the epicutaneously exposed guinea pigs. The applied volumes of EGBE were 

450 and 1800 mg. 

No deaths were seen for the 450 mg dose. For the high dose, 5 animals died on day 3 

cumulative death reached 11 on day 4 and finally 13 on day 7 and after. No changes on 

bodyweight compared to controls were seen for the 450 mg dose. With IP injection of 1800 

mg, 100 % mortality was reached in 6 hours, and with 450 mg dose, 70 % of the mortality 

was reached in 48 hr. 

In a GLP study performed according to the OECD guideline 402, Hartley guinea pigs were 

exposed to 2000 mg/kg of EGBE (purity 99.8 %) via dermal route (Eastman Kodak, 1994b). 

The test substance undiluted was applied under occlusive conditions to the guinea pigs (5 

/sex) for a test period of 24 hr. 

No death was reported. All animals appeared clinically normal during the 14 day post 

exposure period. No effects were seen on body weight, bodyweight gain and no treatment 

related changes were noted at necropsy. Based on this assay, the dermal LD50 of EGBE can 

be considered greater than 2000 mg/kg. 

Rabbits 

New Zealand White rabbits were dosed with EGBE (purity unknown) via dermal route 

(Mellon Institute of Industrial Research, 1952 ; Carpenter et al., 1956). Test material was 

applied occlusively during 24 hr at different doses. LD50 was calculated after a 14-day 

observation period. 

Extreme congestion of the kidneys, bloody urine, pale liver and engorged spleen were noted 

in dead animals. The LD50 calculated was 560 mg/kg (480 to 640 mg/kg). 

In the same report, previous studies were reported: in 1947, 10 rabbits were doses with 505 

mg/kg undiluted EGBE and all died within 48 hr. Bloody urine was a common finding. In 

1948, 505 mg/kg was applied to 6 rabbits and 3 out of 6 survived but had bloody urine. 

Similarly in 1951, 4 out of 6 rabbits died when dosed with 505 mg/kg EGBE. 

Ten New Zealand White rabbits were exposed dermally to Polysolv EB (EGBE – purity 

unknown) at dose of 2000 mg/kg during 24 hr (MB research laboratories, 1976). Epidermal 

abrasions were made every 2-3 cm over the exposed area (sufficiently deep to penetrate the 

stratum corneum but not deep enough to produce bleeding). The wrapping was occlusive. 

After the 24 hr exposure period, animals were observed for 14 days. 

At 24 hr, all rabbits exhibited lacrimation, bloody urine, flaccid muscle tone and anorexia. No 

spontaneous movement were noted. All animals died during the second day of observation. It 

can be concluded that LD50 is lesser than 2000 mg/kg. 
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In order to estimate more precisely the value of the LD50, another study was performed with 

the same experimental conditions except for the dose level chosen and the number of animals. 

Four doses were applied to three groups of four NZ white rabbits: 250, 500 and 1000 mg/kg. 

In this second study, mortality was seen at 500 mg/kg (1/4) and 1000 mg/kg (4/4). No toxic 

signs were reported for the 250 mg/kg group, for 500 and 1000 mg/kg group blood was found 

in urine and for the 1000 mg/kg group animals were flaccid. In died animals, necropsy 

findings were blood in urine and sometimes liver and renal injuries. The calculated LD50 

based on this study was 580 mg/kg (0.31 – 0.85). 

The percutaneous toxicity of butyl cellosolve was determined on rabbit skin (Duprat and 

Gradiski, 1978). Groups of 6 New Zealand White rabbits were exposed to undiluted EGBE 

(purity > 99.5 %) at doses of 72, 90, 108, 135, 180 and 225 mg/kg for a 8 hr period. Animals 

were observed daily over 15 days. Macroscopic and microscopic examination were performed 

on dead animals and those surviving on the 15th day. 

At the end of the exposure period, the total amounts of EGBE applied were absorbed. 

Mortality was 2/6 animals for 72 and 90 mg/kg, 4/6 animals for 108 mg/kg, 5/6 animals for 

135 and 180 mg/kg and 6/6 animals for 225 mg/kg. Death occurred between day 1 and day 8. 

Clinical signs were prostration, hypothermia and hemoglobinuria especially in the groups 

receiving the highest doses. Early death were considered to be caused by narcosis or failure of 

respiration and delayed death were probably due to renal impairment. Lungs appeared 

modified. Histopathological investigation in dead animals showed congestion of the liver, 

necrotic foci with mesenchymatous reactions and inconstant steatosis, passive congestion of 

the spleen, enlarged kidney with haemoglobinemic nephrosis, cutaneous lesions including 

necrosis. There were no evident dose-effect relationship since the findings were exhibited at 

all dose levels. In surviving animals no effects were seen for 90 and 72 mg/kg dose. Other 

animals showed signs of renal toxicity and skin irritation. 

In this study, the calculated LD50 was 100 mg/kg bw. 

In a briefly reported study, groups of male New Zealand rabbits were exposed occlusively to 

EGBE during 24 hr at concentrations of 900 and 450 mg/kg (Bushy Run Research Center, 

1980b). Mortality was 4/4 animals for the high dose and 1/4 for the low dose. Signs of 

erythema and necrosis were seen at the administration point. In dead animals necropsy 

showed orange red lung and liver, dark spleen, dark red kidneys, orange peritonea and 

intestine, blood in urine. In this study LD50 was 569 mg/kg (348 – 931 mg/kg). 

Nine glycol ethers including EGBE were tested for determination of acute dermal LD50 

(Eastman Kodak, 1981b). EGBE (purity > 99.5 %) was administrated dermally to groups of 

New Zealand White rabbits (5 animals/group) at doses of 153, 307, 614 and 1239 mg/kg. 

After 24 hr of occluded exposure, the wrap was removed and animals were observed during 

14 days for clinical and irritation signs. Body weights, gross pathology were also checked. 

Clinical signs were anorexia, depression, cyanosis and ataxia for the lowest doses. Salivation, 

nasal discharge, iritis, depression, laboured breathing and prostration were observed at higher 

doses. No gross pathological effects were seen at the 153 and 307 mg/kg doses. At the two 

highest doses, however, adverse and dose-related effects on kidneys were recorded. Reddish 

coloured fluid was observed in the urinary bladder of animals. Some effects were also seen in 

liver and in thymus. Calculated LD50 was 435 mg/kg (range 330 to 578 mg/kg). 

A GLP study was performed to assess the acute dermal toxicity of EGBE (purity unknown) in 

the New Zealand White rabbit using a method described in the OECD guideline 402 
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(Safepharm laboratories, 1994a). 2 groups of 10 animals (5 males and 5 females) were given a 

single 24 hr semi-occluded dermal application to intact skin at dose levels of 1000 and 2000 

mg/kg. Animals were observed for 14 days after the day of treatment and were killed for gross 

pathological examination. 

There were no death for the 1000 mg/kg dose. For the high dose, one female was found dead 

2 days after dosing and 2 animals (1 male and 1 female) were killed in extremis one day after 

dosing. No signs of toxicity were seen for the low dose, for the high dose, common signs of 

systemic toxicity related to EGBE administration were reported: lethargy, red stained urine, 

laboured breathing, hunched posture and isolated incidents of loss of righting reflexes, 

hypothermia, ataxia and diarrhoea. Surviving animals recovered before the end of the 

observation period. Very slight to well-defined erythema with or without very slight or slight 

oedema was noted at treatment sites of all animals and at all doses, in high dose animals, 

some signs of necrosis were seen in 5 animals. All surviving animals except one female in the 

low dose group and one male in the high dose group showed expected gain in bodyweight 

during the study. For surviving animals, no abnormalities were reported at necropsy (1000 

and 2000 mg/kg), only the animals dead in the high dose group showed signs of hepatic and 

renal toxicity and haemorrhage of the gastric mucosa, of the non glandular epithelium of the 

stomach, of the small and large intestine and red fluid present in the bladder. 

In this study, it can be conclude that LD50 is greater than 2000 mg/kg. 

A GLP study was performed to assess the acute dermal toxicity of EGBE (purity unknown) in 

New Zealand White rabbit using a method described in the OECD guideline 402 (Safepharm 

laboratories, 1994b). 3 groups of 10 animals (5 males and 5 females) were given a single 24 

hr occluded dermal application to intact skin at dose levels of 500, 707 and 1000 mg/kg. 

Animals were observed for 14 days after the day of treatment and were killed for gross 

pathological examination. 

One female treated with 500 mg/kg and 3 animals treated with 1000 mg/kg were killed in 

extremis. Common signs of toxicity noted in all groups were ataxia, hunched posture, 

lethargy, laboured breathing, diuresis, red-coloured urine and skin and eyes pale yellow in 

appearance. Yellow coloured eyes was commonly noted in animals treated with 707 or 1000 

mg/kg. Isolated incidents of toxicity noted in animals treated with 1000 mg/kg were 

diarrhoea, hypothermia and loss of righting reflexes. Common signs of skin irritation noted 

were very slight to well defined erythema, very slight to severe oedema, scattered areas of 

grey/green-coloured dermal necrosis, desquamation, slight haemorrhage of dermal capillaries, 

small superficial scabs. Animals that died or were killed in extremis during the study showed 

haemorrhagic lungs, dark or pale liver, dark kidneys and red liquid present in the bladder. In 

dead animals, slight haemorrhage of the gastric mucosa and haemorrhage of the large and/or 

small intestines. No abnormalities were noted at necropsy of animals that were killed at the 

end of the study. The calculation of LD50 gives an overall value of 841 mg/kg, LD50 for 

males only was 1060 mg/kg and LD50 for females only was 667 mg/kg. 
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Summary acute toxicity dermal route

Table 4.49: Summary acute toxicity via dermal route. 

LD50 (mg/kg) Experimental 

conditions 

Effects Validity * Reference 

Rat dermal route 

2275 mg/kg 4 hours occlusive 2 Mellon Institute 

of Industrial 

Research, 1961. 

> 2000 mg/kg 24 hours, semi 

occlusive 

No irritation, no sign of 

toxicity 

1 Safepharm 

laboratories, 

1993a 

> 2000 mg/kg 24 hours, 

occlusive 

Ataxia, pallor of 

extremities, lethargy, 

laboured breathing. No 

signs of irritation. 

1 Safepharm 

laboratories, 

1993b 

Guinea-pigs Dermal route 

6411 mg/kg 2 Mellon Institute 

of Industrial 

Research, 1952 

208 mg/kg 

(intact skin) 

271 mg/kg 

(abraded skin) 

 2 Roudabush et al., 

1965 

450 – 1800 

mg/kg 

 Mortality at 1800 

mg/kg only (13/20 

animals) 

2 Wahlberg and 

Boman, 1979 

> 2000 mg/kg 24 hours, 

occlusive 

no irritation, no signs of 

toxicity 

1 Eastman Kodak, 

1994b 

Rabbits Dermal route 

560 mg/kg 

505 mg/kg 

24 hours 

occlusive 

Haemolysis, renal – 

hepatic – splenic 

toxicity 

2 Mellon Institute 

of Industrial 

Research, 1952 

Carpenter et al., 

1956 

580 mg/kg 24 hours Anorexia, depression, 2 MB Research 
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occlusive, dermal 

abrasion 

cyanosis, ataxia, 

laboured breathing, 

renal – hepatic and 

thymic toxicity. 

Mortality from 500 

mg/kg. 

Laboratories, 

1976 

100 mg/kg 8 hours Haemolysis, narcosis, 

laboured breathing, skin 

irritation. Liver, splenic 

and kidney toxicity. 

2 Duprat and 

Gradiski, 1978 

569 mg/kg 24 hours 

occlusively 

Mortality from 450 

mg/kg. 

2 Bushy Run 

Research Center, 

1980 b 

435 mg/kg 24 hours 

occlusively 

Anorexia, depression, 

cyanosis, ataxia, 

laboured breathing, 

renal – hepatic and 

thymic toxicity. 

2 Eastman Kodak, 

1981b 

> 2000 mg/kg 24 hours, semi 

occlusive 

Lethargy, haemolysis, 

ataxia, signs of 

irritation mild to severe 

at high doses (necrosis). 

Hepatic and renal 

toxicity. 

1 Safepharm 

laboratories, 

1994a 

841 mg/kg 

1060 mg/kg for 

males 

667 mg/kg for 

females 

24 hours, 

occlusively 

Mortality from 72 

mg/kg. Signs of 

irritation mild to severe 

at high doses (necrosis). 

Very slight systemic 

effects. 

1 Safepharm 

laboratories, 

1994a 

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed 

according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment 

possible. 

In rats, three studies showed LD50 greater than 2000 mg/kg. In the most recent studies, 

performed according to the same experimental protocol except for occlusion (one occlusive 

and the other 1⁄2 occlusive) animals exhibited clinical signs only when exposed to EGBE under 

complete occlusion. Clinical signs were the same than those seen with inhalation 

administration: mainly haemolysis, lethargy, ataxia, hepatic and renal toxicity. No signs of 

irritation were noted in any of the studies. 

In guinea-pigs, great variations were seen depending on the studies. LD50 ranged from 208 

mg/kg to 6411 mg/kg. Only one recent study were performed according to guidelines. This 

study gave a LD50 of greater than 2000 mg/kg. For this study, no adverse effects were 

described (local or systemic). Very few details are available about the local or systemic 

toxicity for the other studies. 
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In rabbits, except for one study (Duprat and Gradiski, 1978), results were quite consistent. 

When EGBE was applied occlusively, calculated LD50 for a 24-hour application ranged from 

435 to 841 mg/kg in 6 studies. When applied semi-occlusively, LD50 was greater than 2000 

mg/kg, showing the importance of evaporation. Common systemic signs of toxicity usually 

seen with EGBE were described: ataxia, laboured breathing, depression, cyanosis, renal – 

hepatic, thymic and splenic toxicity. Local signs of irritation were seen in some studies, mild 

irritation for lower doses and sometimes severe irritation , even necrosis for the higher doses. 

Overall, for dermal toxicity, great differences were seen between the tested species and the 

mode of occlusion. When EGBE was administered non-occlusively or semi-occlusively the 

LD50 was much higher than when administered occlusively. As for acute oral toxicity rabbit 

seems to be the most sensitive species with LD50 of about 500 mg/kg when administered 

occlusively whereas other species exhibited LD50 greater than 2000 mg/kg in the same 

experimental conditions. Clinical signs were the same than those observed via oral route. 

Local signs of irritation were noted, mild for low doses and sometimes necrosis for the high 

doses. By dermal route, depending on the application (occlusive or not) the LD50 was 500 

mg/kg or > 2000 mg/kg respectively. 

According to the data available, a classification R21 is needed. 

Oral 

Rat studies

Oral acute toxicity of 69 glycols and glycols derivatives was assessed (Smyth et al., 1941). 

The substance was administrated diluted in water via oral route. Ten Wistar male rats were 

used for each dosage and enough dosages were administrated to include those at which all 

died. Observation period after dosing was 14 days. The maximum tested concentration was 2 

%. 

Most deaths occurred within the first 2 days after dosing Lethal Dose: LD50 was 1260 mg/kg 

(1120 to 1420). 

In the publication of Carpenter et al., 1956 and Mellon Institute of Industrial Research, 1952, 

some LD50 results were reported. These tests were performed according to a standard 

procedure (exceptions were specified) on non fasten rats (5-6 weeks old) with dose levels 

differing by a factor of 1.26 or 2 in a geometric series. A 14-day post exposure observation 

period was made. Wistar rats were used until 1942, then Sherman rat from 1942 to 1952 and 

Carworth – Wistar from 1952. 

Symptoms were sluggishness, ruffling of coats, prostration, and narcosis. Autopsies on dead 

rats revealed congested of haemorrhaged lungs, mottled livers, severely congested kidneys 

and haemoglobinuria. 

Results are summarised in the table 4.50: 
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Table 4.50: Summary of the LD50 reported in Carpenter et al., 1956 

Sex Weight (g) Concentration 

EGBE in Water (%) 

LD50 and range (mg/kg) Year 

M 90-120 10 1480 (1150 to 1910) 1938 

M 90-120 10 2600 (2300 to 2900) 1951 

F 90-120 10 2300 (1900 to 2800) 1951 

M 90-120 5 2800 (2400 to 3300) 1952 

F 90-120 5 1600 (1200 to 2100) 1952 

M 90-120 5 1900 (1300 to 2600) 1953 

F 90-120 5 1600 (1400 to 1900) 1954 

M 30-60 5 3000 (2600 to 3400) 1954 

F 30-60 5 2300 (2100 to 2600) 1954 

M 335-460 5 560 1954 

F 260-320 5 530 (380 to 750) 1954 

M 90-120 5 2400 (2100 to 2700) 1954 

F 90-120 Undiluted 2800 (2200 to 3700) 1954 

M 90-120 5 2100 (1400 to 3100) 1955 

In a range-finding test performed on rats (poorly described), EGBE (purity unknown) was 

administered orally in a water vehicle to groups of 5 animals at doses of 252, 500 and 1000 

mg/kg (Dow, 1959). 

Mortality was 3/5 animals at 500 mg/kg and 2/2 at 1000 mg/kg. From the lower dose, animals 

appeared sick and drowsy. Signs of haematotoxicity appeared at the dose of 500 mg/kg 

(haematuria). From this study a LD50 of 470 mg/kg can be calculated. 

EGBE (purity unknown) was administered by oral route to groups of 10 female rats at doses 

of 1000 – 1600 – 2000 – 2500 and 4000 mg/kg in a 10 % solution in corn oil. Rats were 

fasted 12 hours before administration. Post exposure observation period was 7 days (Hoechst, 

1966). 

Mortality occurred from 1600 mg/kg bw dose and higher and between 1h30 and 24 hrs after 

treatment. In this study the calculated LD50 was 1950 mg/kg. 

Male Wistar rats were administrated Polysolv EB (EGBE – purity unknown) orally by 

intubation ( MB research laboratories, 1976). Doses of 670, 1310, 2560 and 5000 mg/kg were 

used. Animals were observed for mortality, toxicity for 14 days and the LD50 calculated at 

the end of this period. 

No mortality was found for the 670 mg/kg group, 3/10 animals died in the 1310 mg/kg group, 

9/10 animals died in the 2560 mg/kg group and 10/10 animals died in the 5000 mg/kg group. 

Signs of toxicity were lethargy in all groups and ataxia in the high dose group. At necropsy, 

signs of liver and kidney toxicity were seen in the 1310 mg/kg group. Blood in bladder was 
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seen in all animals of the 2 highest dose groups. The LD50 calculated in this study was 1590 

mg/kg. 

Male Wistar rats (5/group) were dosed with EGBE at doses of 9030- 4515 - 2257 - and 1128 

mg/kg (Bushy Run Research Center, 1980b). Animals were observed during 14 days before 

calculating the LD50. 

All animals died at doses of 9030 and 4515 mg/kg during or the day after administration. At 

2257 mg/kg two out of 5 animals died on day 5 after administration. No deaths were seen at 

1128 mg/kg dose. 

For the 2 high doses, clinical symptoms were difficult breathing, sluggish and bloody 

salivation. For the 2257 mg/kg dose, animals were sluggish and had an unsteady gait. Nothing 

remarkable was seen on animals of the lowest dose. On dead animals, necropsy revealed dark 

livers, distented stomach, filled with liquids and gaz. Red kidneys, intestine containing blood, 

red adrenals were also reported. Nothing was seen in survivors. In this study, calculated LD50 

was 2420 mg/kg (1670 - 3504 mg/kg). 

EGBE undiluted (purity unknown) was administered orally to groups of females CDF rats 

(3/group) at doses of 130, 250, 500, 1000 and 2000 mg/kg (Dow, 1981). 

Mortality was seen at 200 mg/kg (2 out of 3animals). Clinical signs were lethargy and 

respiratory difficulties at 2000 mg/kg and necrosis of the tails in all surviving rats of the 1000 

and 2000 mg/kg dosed groups. For this study, the LD50 was between 1000 and 2000 mg/kg.

 

The acute oral LD50 was determined in fasted and fed rats (5/group) for 9 chemicals 

including EGBE (purity > 99.5 %) (Eastman Kodak, 1981a). Male CD/BR rats were given 

EGBE undiluted by gavage (five different doses progressing by a factor of 2). 

The LD50 calculated for fasted or fed rats was 1746 mg/kg. For both groups (fed and fasted) 

clinical signs were: inactivity, laboured breathing, anorexia, tremors and death. Haematuria 

was seen at high dose. In dead animals, blood was found in stomach and intestines. 

Some LD50 studies were reported without details concerning the method used (Tyler, 1984). 

A LD 50 of 620 mg/kg was reported in rats (Rowe and Wolf, 1982). 

Mouse 

Carpenter et al., 1956 reported some experiments concerning acute toxicity of EGBE in 

various animals. A LD50 of 1230 mg/kg was reported for mice via oral route. 

The acute oral LD50 was determined in fasted and fed mice (5/group) for 9 chemicals 

including EGBE (purity > 99.5 %) (Eastman Kodak, 1981a). Male CD1 mice were given 

EGBE undiluted by gavage (five different doses progressing by a factor of 2). 

The LD50 calculated for fasted mice was 1519 mg/kg, whereas LD50 for fed mice was 2005 

mg/kg. For both groups (fed and fasted) clinical signs were: inactivity, laboured breathing, 

anorexia, tremors and death. Haematuria was seen at high dose. In dead animals, blood was 

found in stomach and intestines. 

Some LD50 studies were reported without details concerning the method used (Tyler, 1984). 

Two LD 50 of 1170-1700 mg/kg and 1000-1600 were reported (Rowe and Wolf, 1982 and 

Saparmamedov, 1974 respectively). 
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Rabbit 

Carpenter et al., 1956 reported some experiments concerning acute toxicity of EGBE in 

various animals. A LD50 of 320 to 370 mg/kg was reported for rabbits via oral route. 

Guinea-pig 

In a GLP study (performed according to OECD GL 401), guinea pig were given orally EGBE 

(purity 99.8 %) in a distilled water vehicle at dose of 500, 1000 and 2000 mg/kg (Eastman 

Kodak, 1994a ). 

Mortality was 3/5 in males and 5/5 in females at the top dose. At 1000 mg/kg, the mortality 

was 1/5 for males and females. Clinical signs were slight to severe weakness, sialorrhea and 

staining of the hair of the face and/or abdomen for the 2000 mg/kg dose. At 1000 mg/kg 

animals evidenced weakness and prostration the day of administration. Only a slight weakness 

was noted for the 500 mg/kg group. Sign of necrosis and minor haemorrhage in the gastric 

mucosa was seen after termination in animals doses with 1000 and 2000 mg/kg. For this 

study, the LD50 was calculated to be 1414 mg/kg. 

Carpenter et al., 1956 reported some experiments concerning acute toxicity of EGBE in 

various animals. A LD50 of 1200 mg/kg was reported for guinea-pigs via oral route. 

Summary oral route:

Table 4.51: Summary of LD50 values after oral route administration 

LD50 (mg/kg) Toxicological effects Validity * Reference 

Rats Oral route 

1260 mg/kg 3 Smith, 1941 

560-2800 mg/kg for 

males 

530 and 2300 mg/kg 

for females 

Narcosis - prostration 2 Mellon Institute of 

Industrial Research, 

1952 

Carpenter et al., 1956 

470 mg/kg Mortality from 500 mg/kg. 

Haematuria. 

3 Dow, 1959 

1950 mg/kg Mortality from 1600 mg/kg 2 Hoechst, 1966 

1590 mg/kg Lethargy, laboured breathing, 

haemolysis, liver and kidney 

toxicity. 

Mortality from 1310 mg/kg 

2 MB Research Labs, 

1976 

2420 mg/kg Laboured breathing, sluggish 

and bloody salivation. 

Haemolysis, dark liver and red 

2 Bushy Run Research 

Center, 1980 b 
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kidneys. 

1000-2000 mg/kg Lethargy, laboured breathing, 

necrosis of the tail. 

Mortality from 2000 mg/kg. 

2 Dow, 1981 

1746 mg/kg 

(fasted or fed) 

Inactivity, laboured breathing, 

anorexia, tremors, haemolysis. 

2 Eastman Kodak, 

1981a 

620 mg/kg 3 Rowe and Wolf, 1982

Mice Oral route 

1230 mg/kg 2 Carpenter et al., 1956 

1170 – 1700 mg/kg 

1000 – 1600 mg/kg 

 3 Rowe and Wolf, 1982 

Saparmamedov, 1974 

1519 mg/kg (fasted) 

2005 mg/kg (fed) 

Laboured breathing, anorexia, 

tremors, haemolysis. 

2 Eastman Kodak, 

1981a 

Rabbits Oral route 

320 – 370 mg/kg 2 Carpenter et al., 1956 

Guinea pig oral route 

1200 mg/kg 2 Carpenter et al., 1956 

1414 mg/kg Weakness, prostration, necrosis 

and haemorrhage of gastric 

mucosa. 

Mortality from 1000 mg/kg 

1 Eastman Kodak, 

1994a 

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed 

according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment 

possible. 

In rats, numerous studies has been performed to assess the LD50 via oral route. Results varies 

a lot between 470 mg/kg and 2600 mg/kg. Recent studies (performed according to well 

defined experimental methods) have given results between 1000 and 2600 mg/kg. Clinical 

signs noted were: lethargy, laboured breathing, ataxia. For pathology, haemolysis was seen in 

the majority of the studies, sometimes accompanied with renal and hepatic lesions (certainly a 

consequence of haemolysis). 

In mice, available studies exhibited LD50 ranging from about 1500 to 2000 mg/kg (these 

results are more consistent than those obtained in rat studies). Clinical symptoms similar than 

those of the rat studies were seen. 

One study was performed in rabbits showing a LD50 ranging from 320 to 370 mg/kg. This 

value seems very low compared to other studies on other species via oral route. The rabbit can 

be considered to be the most sensitive species concerning acute oral toxicity of EGBE. 
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Considering that this study is an old study, and that only the result is available (no details on 

purity or experimental conditions), it is difficult to take this value into account for the risk 

characterisation or classification purposes. 

Two studies are available in Guinea-pigs. The LD50 calculated were 1414 and 1200 mg/kg. 

The same clinical signs and pathology than other species tested were seen in these studies. 

Necrosis and haemorrhage of the gastric mucosa was also seen. 

According to the data available, a classification R22 is needed. 

Other routes 

Rats 

Various results concerning IV administration of EGBE were reported (Mellon Institute of 

Industrial Research, 1952 ; Carpenter et al., 1956): LD50 in female rats (170-230 g) when 

administrated as 3 % dilution was 380 mg/kg (290 – 500 mg/kg) – LD50 in female rats (90-

120 g) when administered undiluted was 340 mg/kg (300 to 380 mg/kg). 

In a non-GLP study, unique IP injection of 2 different EGBE (n butyl oxitol ® 98-99 purity 

and Dowanol EB ® > 99 % purity) was performed on female SD rats (4/group). Doses of 200, 

252, 316, 398 were tested for both substances with an additional dose of 500 mg/kg for 

Dowanol EB ® (Dow, 1972). 

For both substances, mortality was seen from 252 mg/kg. In all dose groups and with both 

substances blood was seen in urine. A LD50 of 252 mg/kg was calculated for n butyl oxitol ®

and a LD50 of 317 mg/kg was calculated for Dowanol EB ®. 

Ten female SD rats were injected IV with 0.034 ml EGBE/kg (Freundt and Helm, 1986). 

Twenty four hour urine samples were collected during the next 4 days. The following 

parameters of kidney function were examined in the urine: volume, osmotic pressure (by 

means of freezing point depression), haematuria (using a semi-quantitative test), albumin, 

Lactate DeHydrogenase (LDH). Gel filtration of the urine was carried out before enzyme and 

albumin analyses. 

After treatment, the following parameters were found to be changed significantly: the albumin 

level and the LDH activity was augmented only on the 2nd day, and the GAL activity was 

decreased only in the 24-h urine samples from the 4th day. The urinary LAP activity, the 

volume, and the osmotic pressure of the urine remained unchanged. EGBE caused a 

microhaematuria (approximately 50 erythrocytes/μl) in 2 (of 10) rats on the 1st dose day after 

its application. 

SD rats were injected IV with 0.034 ml/kg EGBE (purity unknown) (Freundt et al., 1993 – 

abstract). The activities of lactate dehydrogenase (LDH), leucine aminopeptidase (LAP) and 

beta-galactosidase (GAL), the concentrations of albumin and creatinine, the volume, the 

specific gravity and the pH, leucocytes, erythrocytes, nitrite, total protein, ketone, bilirubin 

and urobilinogen were analysed in the 24 hr urine. 

A significant increase of albumin and LDH activity was seen in the female rats at the 2nd day 

after administration. All other parameters remained normal. Because these changes were 

small and transient, the authors concluded that EGBE have a slight nephrotoxic potential.
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Mice

SC injection of 0.5 cc/kg to white mice produced severe toxicity (Von Oettingen and Jirouch, 

1931). For 0.25 cc/kg no mortality was seen and one out of two animals died with 0.5 cc/kg 

dose. The only animal treated with 0.75 cc/kg died. Due to the small number of animals 

treated no LD50 was calculated. 

One study concerning IV administration of EGBE to mice was reported (Carpenter et al., 

1956): LD50 in male and female mice when administrated as 3 % dilution was 1130 mg/kg. 

Acute toxicity was assessed in vitro and in vivo in mice (Tanii et al., 1992). Male ddY mice 

(4 animals/dose – 4 doses) were given IP 2000 mg CCl4 in olive oil or olive oil only 24 hr 

before dosing with EGBE. CCl4 is known to inactivate the hepatic microsomal 

monooxygenase system and may influence the detoxication of EGBE. It also inhibits hepatic 

non-microsomal enzymes including ADH. Animals were observed 1 week and after 

termination the LD50 was calculated. Concurrently a cytotoxicity test was performed to 

determine ED50. 

LD50 without CCl4 was 6.77 mmol/kg whereas it was equal to 4.99 mmol/kg with CCl4. As 

CCl4 at the dose used appears to inhibit all hepatic enzyme activities (microsomial and non 

microsomial), the authors concluded that it is unlikely that the metabolite contribute to the 

acute toxicity of EGBE. 

There was no significant correlation between ED50 for the different cells used in the 

cytotoxicity studies and LD50. A correlation was found between LD50 and logP for the 

majority of the tested substances. No quantitative relationship was described. 

Rabbits

The LD50 for male rabbits by IV route of undiluted EGBE is 0.14 ml/kg (Mellon Institute of 

Industrial Research, 1952). Death occurred precipitally after 0.2 ml/kg while after 0.1 ml/kg 

the rabbits were prostrated and rapidly recovered. At the 0.05 ml/kg dosage level, the only 

symptoms were increased rate of respiration. 

In the same report, various results concerning IV administration of EGBE were reported: 3 % 

EGBE in 0.75 % NaCl solution was not haemolytic to rabbit red blood cells. LD50 of a 3 % 

EGBE solution was 500 mg/kg (380 – 650 mg/kg). 

Table 4.52: Summary other routes 

LD50 (mg/kg) Experimental 

conditions 

Effects Validity * Reference 

Rats 

380 mg/kg IV route in 3 % 

water dilution 

 2 Mellon Institute 

of Industrial 

Research, 1952 

317 mg/kg IP route. Mortality from 252 

mg/kg. Haemolysis 

2 Dow, 1972 

 IV route Increase of LDH 

activity and decrease of 

3 Freundt and 

Helm, 1986 
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0.034 ml/kg GAL activity. 

Microhaematuria 

 IV route 

0.034 ml/kg 

Increase of albumin and 

LDH activity. 

3 Freundt and 

Helm, 1986 

Mice 

About 0.5 cc/kg SC 

0.25, 0.5 and 

0.75 cc/kg 

0.5 cc/kg produced 

severe toxicity (1 death 

out of 2 treated). 0.75 

cc/kg: death of the only 

treated animal. 

3 Von Oettingen 

and Jirouch, 

1931 

1130 mg/kg IV, 3 % dilution. 3 Carpenter et al., 

1956 

6.77 mmol/kg 

without CCl4 

4.99 mmol/kg 

with CCl4 

IP 3 Tanii et al., 1992 

Rabbits 

500 mg/kg IV route 2 Mellon Institute 

of Industrial 

Research, 1952 

* 1: valid (performed according to GL and GLP) – 2: valid with restriction (not performed 

according GL and/or not GLP but scientifically acceptable – 3: not valid or not assessment 

possible. 

Some studies performed via IV, IP and SC routes in various species gave different results. 

These studies are not suitable for RA because these routes of administration are not relevant 

with exposure scenarios. However, some of them give valuable data about the mode of action 

of EGBE for renal toxicity and valuable information about hepatic metabolism. 

4.1.2.2.2 Data from human exposure 

Inhalation 

2 males and 1 female were exposed to 200 ppm two 4-hr periods separated by 30 minutes 

lunch (Mellon Institute of Industrial Research, 1955). Erythrocyte fragility test, blood 

pressure and pulse-rate were determined the exposure day (3 measure before - during lunch 

and after exposure - for fragility test, one measure was also made during exposure). Urine 

analysis for glucose and albumin were conducted the following morning, and the BAA levels 

were determined in 24-hour urine samples first collected at the end of the exposure day. 

One male and the female excreted considerable amount of BAA in the two 4 hrs periods 

following exposure but the other male excreted only trace amount of this metabolite for the 

same period. Clinical symptom (subjective) were: immediate irritation of the nose and throat, 
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followed by ocular irritation and disturbed taste was experienced by all three subjects. The 

female subject, who excreted the largest amount of BAA, reacted most adversely to the 

exposure, and acquired a headache which lasted about 24 hr. Unlike rats, no effects were seen 

in the fragility test in human tested. 

Another study was performed on human with lower doses: 2 females and 1 male and the male 

of the precedent study whom had only traces of BAA in urine were exposed to 100 ppm 

EGBE in the same conditions that the precedent study. 

The only objective finding of significance in the exposed subjects was the urinary excretion of 

BAA. Other measured parameters did not shown significant differences. Even one subject 

who had not excreted significant quantities of the metabolite after the 200 ppm exposure, did 

eliminate 75.5 mg in 24 hours. The urinary BAA levels of the other subjects were similar to 

that found at the 200 ppm exposure. Surprisingly, the subjective response of the humans 

exposed to 100 ppm EGBE was at least as great, if not greater, than that elicited at the 200 

ppm exposure level. The subject whom participated at the former experiment reported that the 

EGBE odor was not as strong as at the 200 ppm level, although he did have a slight cold at the 

time of exposure. The two female subjects, apparently experienced greater distress during and 

after exposure than did the males. One of them vomited after 7 hours of exposure and several 

times during the day after while the other contracted a headache the following day. The 

former subject stated that high air temperatures often cause emesis, and was convinced that 

the present attack was caused by the relatively high chamber temperature (28.5° in the 

afternoon), rather than the solvent vapours. The other subject also though that the chamber 

temperature caused her headache. 

Oral 

In a suicide attempt, a 50-year woman ingested 250-500 ml of a window cleaner containing 

12 % of EGBE, corresponding to about 0.5 to 1 g/kg bw (Rambourg-Schepens et al., 1988). 

On admission to the intensive care unit, the patient was comatose. Biochimically she 

presented with metabolic acidosis, hypokalaemia, a rise in serum creatinine level and a 

markedly increased urinary excretion of oxalate crystals. 

In another case of attempted suicide (Gijsenbergh et al., 1989), a 23-year-old woman had 

ingested about 500 ml of a mixture containing EGBE (12.7 %) and of EtOH (3.2 %) (about 57 

g of EGBE corresponding to a dose of about 1g/kg bw). 

Upon admission, she was in a comatose state, had breathing difficulties was in metabolic 

acidosis and had a concentration of EGBE in blood of 432 mg/l. 2 hr after admission, the 

blood concentration of EGBE was 304 mg/l . A fall in Hb from 11.9 g/dl on admission to 8.9 

g/dl on the second day, together with the appearance of haematuria was noticed. The half-life 

of EGBE in the blood was 210 min (greater than the commonly values described in the 

literature probably due to the presence of EtOH in the mixture). BAA concentration in the 

urine was maximum 4 hr after dialysis treatment was initiated. 

A case of acute poisoning with EGBE was reported (Bauer et al., 1992). A 53-year-old man 

ingested intentionally 500 ml of a mixture containing 9.1 % of EGBE (45.5 g – about 750 

mg/kg bw) and 2.5 % EtOH. 

Clinic signs were coma, tachycardia, metabolic acidosis and hypoxemias, pulmonary oedema 

and Adult Respiratory Distress Syndrome (ARDS). By 36 hr after admission, the subject 
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showed signs of non haemolytic hypochromic anaemia with thrombopenia. No signs of 

haemolytic anaemia were noticed. 

Some cases of children poisoning which have occurred in the Pittsburgh Poison Center were 

summarized in a publication (Dean and Krenzelok, 1992). The ingested products contained 

EGBE in concentrations ranging from 0.5 % to 9.9 %. The children poisoned ages ranged 

from 7 months to 9 years. The quantities ranged from 5 to 300 mL - 24 children were studied 

- 2 of them were treated by gastric gavage followed by a 24 hr hospitalisation. The other 

children were treated at home with simple dilution. Common symptoms seen in acute animal 

dosing (haemolysis, CNS depression, metabolic acidosis and renal compromise) did not 

occur. 

A 18-year-old male consumed between 360 and 480 ml of a glass cleaner containing 22 % 

EGBE (maximum 95 g of EGBE – about 1,25 g/kg bw) (Gualtieri et al., 1995 –and Gualtieri 

et al., 2003). 

He was hospitalised with metabolic acidosis and hepatic biochemical abnormalities (Serum 

Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT) 

and hepatic bilirubin). After treatment with EtOH and haemodialysis, the patient returned 

home without sequelae. Nine days following his initial discharge, he was again admitted after 

ingestion of 480 ml of the same cleaner. The second hospitalisation was absent of any 

significant metabolic or hepatic abnormalities. Haematological and renal abnormalities were 

absent in both hospitalisations. Quantitative analysis for BAA has given a maximum 

concentration of 4.86 mmol/L approximately 16 hours after ingestion. 

A 19 year old man ingested about 20-30 ounces of a product containing 25-35 % EGBE 

(maximum 336 g of EGBE – about 4.5 g/kg bw) (Burkhart and Donovan, 1998). The product 

had a pH of 13 and was therefore considered as an alkaline corrosive. The patient developed 

characteristic features of coma, acidosis and haematuria which were previously reported 

following EGBE ingestion. After treatment, the patient still exhibited neurologic sequelae 

(difficulties with fine motor skills and stop speaking). However the patient medical history 

was significant for mental retardation, depression, hyperactivity and he was treated with 

various strong psychiatric medecine stopped after the ingestion. This weakens the direct 

relationship between EGBE and the sequelaes reported. 

A case report of a massive ingestion of a mixture containing 10-30 % of EGBE and 10-40 % 

of isopropanol was reported (McKinney et al., 2000). A 51-year-old woman ingested 8 

ounces of this mixture (corresponding to an estimate dose of 24-72g of EGBE corresponding 

to a range of 0.4 – 1.2 g/kg bw) in a suicide attempt. 

Clinical symptoms were metabolic acidosis and mental status depression. She was treated 

with EtOH therapy and recovered without apparent sequelae. No signs of haematotoxicity 

were noticed. 
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Table 4.53: Summary human acute toxicity data 

Estimation of 

absorbed dose 

Patient history Patient pathology Reference 

Between 0.5 and 1 

mg/kg bw 

50-year woman. 

Suicide attempt with 

glass cleaner. 

Coma, metabolic 

acidosis, 

hypokaliemia, 

increase in serum 

creatinine level and 

urinary excretion of 

oxalate crystals 

Rambourg-Schepens 

et al., 1988. 

About 1 g/kg bw 23-year woman. 

Suicide attempt with 

mixture containing 

EGBE. 

Coma, breathing 

difficulties and 

metabolic acidosis. 

Haematuria and 

decreased Hb for 2 

days. 

Gijsenbergh et al., 

1989. 

About 750 mg/kg bw 53-year man. Suicide 

attempt with mixture 

containing EGBE. 

Coma, tachycardia, 

metabolic acidosis, 

hypoxemia, 

pulmonary oedema 

and ARDS. Non 

haemolytic anaemia 

with thrombopenia. 

Bauer et al., 1992. 

About 1.25 g/kg bw – 

2 times separated by 9 

days 

18-year man. 

Ingestion of a glass 

cleaner. 

Metabolic acidosis 

and hepatic 

biochemical 

disorders. 

Nothing after the 

second ingestion. 

Gualtieri et al., 1995, 

Gualtieri et al., 2003. 

About 4.5 g/kg bw. 19-year man. 

Ingestion of a mixture 

containing EGBE. 

Coma, acidosis and 

haematuria. 

Burkhart and 

Donovan, 1998 

Between 0.4 and 1.2 

g/kg bw 

51-year woman. 

Ingestion of a mixture 

containing EGBE. 

Metabolic acidosis 

and mental status 

depression. 

McKinney et al., 

2000 

According to this data, a LOAEL of 400 mg/kg bw can be taken into account for acute 

toxicity by oral route in humans. It should be noted that this is a worst case estimation derived 

from the McKinney paper in which the possible range of exposure was between 0.4 and 

1.2 g/kg bw. 
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Summary human data:

Acute human toxicity data comes from children accidental ingestion or adult suicide attempts 

made with mixtures containing EGBE and from one study on human volunteers by inhalation. 

For oral route case reports, ingested doses are difficult to evaluate because of the lack of data 

concerning the body weight of all patients and the exact ingested dose, but a semi-quantitative 

estimation of the ingested doses was made for each case (see table 4.7). The range of doses 

which lead to clinical symptoms varies between 0.5 and 4.5 g/kg bw. In all cases, patients 

exhibited SNC depression and metabolic acidosis. Signs of haemolysis were seen in some 

cases but this finding was not systematic (this showed that human is much more resistant to 

haemolysis than rodents). After a first acute ingestion, a second administrator some days later 

did not exhibit the same symptoms, this finding was also seen with animals in some studies. 

In these cases, EGBE was ingested together with other substances (ethanol and/or unknown 

substances) that could have some influence on the symptoms seen. Between 0.5 and 1.5 g/kg 

bw the patients totally recovered after treatment. According to this data, a LOAEL of 400 

mg/kg bw can be taken into account for acute toxicity by oral route in humans in the risk 

characterisation section. If a risk characterisation by dermal and inhalation route is needed for 

acute effects, kinetic data is sufficient to extrapolate this oral LOAEL to inhalation and 

dermal LOAEL. Human data is preferred for risk characterisation, especially for EGBE 

because of its haematotoxicity more marked in animals than in humans. It should be noted 

that this is a worst case estimation derived from the McKinney paper in which the possible 

range of exposure was between 0.4 and 1.2 g/kg bw. 

By inhalation route, symptoms reported by the three volunteers were signs of irritation (throat 

and ocular) and headache. These symptoms did not seem to be dose-related. 

Summary acute toxicity 

For the inhalation route, the 4 hour LC50 in rats, which are susceptible to haemolysis (see next 

section), was of the region of 450ppm (2214mg/m3). Higher values are seen in other species. 

For the dermal route, great differences were seen between the tested species and the mode of 

occlusion. The rabbit seems to be the most sensitive species with LD50 of about 500 mg/kg 

when administered occlusively. 

For the oral route, available studies considered as reliable show LD50 values upwards from 

1000mg/kg. 

According to the data available, a classification R20/21/22 is needed for all three routes of 

exposure. 

A number of human case studies are available from attempted suicides which suggest that the 

human LOEL is in the region of 400 mg/kg bw. Human data is preferred for risk 

characterisation, especially for EGBE, because its haematotoxicity is more marked in animals 

than in humans. There is sufficient kinetic data available to enable route to route 

extrapolation to be carried out to risk assess the more relevant dermal and inhalation routes. 

These extrapolations are carried out in the risk characterisation section. It should be noted that 

this is a worst case estimation of the LOEL derived from the McKinney paper in which the 

possible range of exposure was between 0.4 and 1.2 g/kg bw. 
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4.1.2.2.3 Specific toxicity: haematotoxicity 

Some studies were performed especially to assess the haematotoxic potential of EGBE in 

various conditions. 

The studies described above are but a few that indicate that EGBE causes acute haemolytic 

anaemia in rats and mice. This response is consistent and probably forms the basis for 

establishing an No Observable Adverse Effects Levels (NOAEL) in species used 

experimentally. It has also been suggested that it is an essential, early step in the development 

of haemangiosarcomas of the liver in male mice (described in 4.1.2.8.3). Clearly, if the 

haemangiosarcomas arising in male mice (see Section 4.1.2.8.3) are ultimately a consequence 

of anaemia induced by EGBE, the species and sex specificity of the neoplastic response must 

be due to some factor(s) subsequent to the anaemia, since this also occurs in rats and female 

mice. Possible elements contributing to the specificity of the response will be examined and 

the strength of the evidence evaluated in order to reach a conclusion on the human relevance 

of the haematological effects and the neoplastic responses observed in rodents. 

In vitro

In vitro and in vivo response of mammalian erythrocytes to EGBE and BAA was assessed 

(Carpenter et al., 1956). The results are summarized in the table 4.54: 

Table 4.54: Summary of the erythrocyte toxicity in Carpenter et al., 1956. 

In vitro response In vivo response 

2.5 % EGBE 0.1 % BAA 

Animal Sex 

Max.time 

without 

haemolysis

, Min 

Max. time to 

haemolysis, 

Min 

Max.time 

without 

haemolysis, 

Min 

Max. time to 

haemolysis, 

Min 

Osmotic 

fragility upon 

inhalation of 

EGBE 

Haemoglobinuria 

upon inhalation of 

EGBE 

2 rats 

3 rats 

2 rats 

F 

M 

F 

46 60-67 

35-40 

35-40 

40-60 

40-46 

Significant ↑

at 62 ppm x 

4h 

Seen at 203 ppm x 

7h but not at 107 

ppm x7h 

2 mice 

2 mice 

1 mouse 

M 

M 

F 

22-30 35-40 

40-45 

45 

50-60 

55 

Significant ↑

at least as low 

as 100 ppm x 

7h 

Seen at 200 ppm 

but not at 100 ppm 

x7h 

2 rabbits 

4 rabbits 

2 rabbits 

M 

M 

F 

110.113 123-125 

60-90 

80-90 

80-100 

85-100 

Significant ↑

at 125 ppm x 

7h 

Not seen as high as 

197 ppm x 7h 

1 monkey F 120 130 No 

Significant ↑

Not seen as high as 
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1 monkey M 103 120 at 200 ppm x 

7h 

200 ppm x 7h 

2 dogs 

2 dogs 

F 

F 

20 40-86 

89-120 

 No 

Significant ↑

at 665 ppm x 

7h x 2 days 

Not seen as high as 

665 ppm x 7h 

1 human 

2 humans 

F 

M 

80 

60-83 

100 

80-94 147-268 

 No 

Significant ↑

at 200 ppm x 

8h 

Not seen at 200 

ppm x 8h 

2 guinea 

pigs 

2 guinea 

pigs 

M 

M 

60-68 80 

360 

 No 

Significant ↑

at 665 ppm x 

8h 

Not seen at 665 

ppm x 8h 

Effects of EGBE and BAA on human, rat, dog and rabbit blood were assessed in vitro (ICI, 

1985). EGBE and BAA solutions were prepared with veronal buffered saline. pH was 

adjusted to 7.2. One ml of erythrocyte suspension was added to 3 ml of the test solution. The 

haemolytic activity was calculated using positive (totally lysed sample) and negative (no test 

substance) controls. Concentration range of 0.01 to 2 % BAA and 0.05 to 0.5 % EGBE were 

tested in the main study. With 2.5 and 1 % EGBE solutions, no analysis could be performed 

due to a discoloration of the test medium. The problem was solved by employing 0.5 % 

EGBE as the maximum permissible concentration for incubation. 

With BAA, in the preliminary study, no effects were seen on human erythrocytes whereas 

marked haemolysis was noted in rat erythrocytes (38.4 %). 

With BAA only rats erythrocytes readily lysed and a very marked haemolysis was seen from 

0.05 %. With EGBE dog erythrocytes seemed to be very sensitive to haemolysis and various 

effects were seen from 0.4 % in other species (see tables 4.55 and 4.56). 

Table 4.55: Haemolytic activity of various concentrations of butoxyacetic acid incubated with erythrocytes from rat, 

human, dog and rabbit 

Haemolysis (%) 

Overall concentration (%) 

Species 

0.01 0.02 0.03 0.04 0.05 0.1 0.2 0.5 2 

Rat 0.8 4.4 8.4 11.1 53 91.9 97.9 98.8 24.9 

Human 0.8 1.4 - - 0.2 0 1.2 0.8 0 

Dog 0 13.6 - - 0 0 0 0 2.6 

Rabbit 4.9 0 - - 3.2 1.2 0 1.1 1.7 
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Table 4.56: Haemolytic activity of various concentrations of EGBE incubated with erythrocytes from rat, human, dog 

and rabbit. 

Haemolysis (%) 

Overall concentration (%) 

Species 

0.05 0.1 0.25 0.4 0.5 

Rat - 0 2.5 51.5 62 

Human - 0 1.5 20.5 70.9 

Dog 46.8 36.2 - 41.2 62.3 

Rabbit - 0 2.8 83.7 72 

A single dose of EGBE was applied dermally (semi-occlusively) to groups of 3 female Wistar 

rat at doses of 500, 375 and 260 mg/kg and to a group of 6 female rats at 200 mg/kg (Bartnik 

et al., 1987). Six hours after dosing, blood was sampled retro-orbitally. Animals were then 

killed and an additional blood sample was taken by heart puncture. Concurrently EGBE was 

injected intravenously to groups of 4 male rats at doses of 25, 37.5, 50, 62.5 and 75 mg/kg. At 

3 and 6 hr following application blood was collected retro-orbitally. After termination, blood 

was also collected by heart puncture. 

In this study, in vitro haemolysis was also studied, male Wistar rats and humans red blood 

cells were treated with various concentrations of EGBE and BAA. Positive and negative 

controls were performed, haemolytic activity was expressed as percentage of totally 

hemolyzed. 

In vitro, BAA showed higher haemolytic potency than EGBE. Hemolytic activity of EGBE 

and BAA was markedly lower on the human erythrocytes compared with rat erythrocytes (see 

tables 4.57 and 4.58). 

Table 4.57: Haemolysis of human and rat erythrocytes by EGBE 

Percentage haemolysis 

 15 min 30 min 45 min 60 min 120 min 180 min 

EGBE (mmol/liter) R H R H R H R H R H R H 

100 0 0 0 0 0 0 0 

125 0 0 0 0 10 64 0 

150 0 0 0 0 0 0 8 0 62 3 92 11 

175 0 0 6 0 41 0 91 2 100 23 100 89 

200 5 100 0 100 2 100 8 100 100 100 100

225 96 0 100 14 100 89 100 100 100 100 100 100

250 100 8 100 100 100 100 100 100 100 100 100 100
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Table 4.58: Haemolysis of human and rat erythrocytes by BAA 

Percentage haemolysis 

 15 min 30 min 45 min 60 min 120 min 180 min 

BAA (mmol/liter) R H R H R H R H R H R H 

1.25 0 0 0 0 0 0 0 0 6 0 25 0 

2.50 0 0 0 0 4 0 20 0 79 0 95 0 

3.75 0 0 0 0 17 0 49 0 86 0 100 0 

5.0 0 0 13 0 59 0 88 0 100 0 100 0 

6.25 0 0 17 0 59 0 89 0 100 0 100 0 

7.5 0 0 25 0 69 0 100 0 100 0 100 0 

10 0 0 0 0 0 0 

15 0 0 0 0 0 0 

In vivo, a dermal dose of 200 mg/kg did not cause any haemolytic effect. Other tested doses 

caused haemolytic effects indicated by increase in mean cell volume, lower erythrocyte count 

and haemoglobin level and haemoglobinuria. No dose response relationship was found but 

according to the authors it might be due to inherent biologic variation in percutaneous 

absorption and haemolytic susceptibility and also to fewer animals per dose group. Following 

a IV injection of 62.5 mg/kg, no haemolysis was detectable whereas a dose of 75 mg/kg 

produced slight effects. 

The effects of EGBE and its metabolites on human and rat erythrocytes were investigated in 

vitro (Ghanayem, 1989). Heparinised blood was collected from a male and a female 

volunteer and cardiac blood was collected from male F344 rats, using EDTA as the 

anticoagulant. Blood-chemical mixtures were incubated in a shaking water bath at 37° for up 

to 4 hr. Incubation of whole blood from rats with 20 mM EGBE was associated with a slight, 

but significant decrease in haematocrit values and significant haemolysis, as determined by 

free plasma haemoglobin concentrations. No significant changes were observed with 5 or 10 

mM EGBE. Similar incubations, but with 2-(1-[14C]butoxy)ethanol, followed by High 

Perfomance Liquid Chromatograpy (HPLC) analysis for metabolites failed to demonstrate the 

presence of either Butoxy Aldehyde (BAL) or BAA. Incubation of rat blood with BAL at 0.5, 

1.0 or 2.0 mM resulted in both time- and concentration-dependent swelling of erythrocytes, as 

shown by an increase in haematocrit values, although significant haemolysis occurred only 

after incubation with 2 mM BAL for 4 hr. Similar incubations of rat blood with BAA at 0.5, 

1.0 or 2.0 mM also induced swelling of erythrocytes and haemolysis, but the highest 

concentration induced its maximum haematocrit response in 2 hr, which was followed by a 

rapid fall as haemolysis was essentially complete by 4 hr. Repetition of the incubations of rat 

blood with BAL, but supplemented with aldehyde dehydrogenase and NAD+

, resulted in a 

significant potentiation of the effect of BAL, whereas addition of cyanamide (an inhibitor of 

aldehyde dehydrogenase) significantly decreased the activity of BAL in both the presence and 

the absence of the exogenous enzyme. Therefore, it appeared that BAA was the haemolytic 

metabolite and that haemolytic activity of BAL was due to its metabolism to BAA by 
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dehydrogenases present in rat blood. It was also found that reduction of blood ATP 

concentrations was more effectively produced by the acid than by the aldehyde, while EGBE 

itself had no effect on ATP concentration, even at a 10-fold higher concentration. It can be 

concluded that erythrocyte swelling precedes haemolysis, strongly suggesting that the 

erythrocyte cell membrane is the target of the haemolytic species. It was not clear, however, 

whether cell swelling was mediated by loss of ATP, or whether this loss was a secondary 

effect. 

In contrast to these data with rat blood, incubation of blood from volunteers with BAA at 

concentrations up to 2 mM had no effect on haematocrit or plasma free haemoglobin. Small, 

but significant increases in haematocrit were observed at 4 and 8 mM after 4 hr incubation 

and there was minimal, but significant haemolysis at 8 mM after 4 hr. A slight decrease in 

blood ATP was observed with 8mM BAA, but there was no time dependent relationship. 

SD rats were administrated EGBE by gavage at 50 and 500 mg/kg (Kurantsin-Mills et al., 

1992). Blood samples were taken at 0.5 – 2 and 4hr after dosage for determination of the 

physical characteristic of RBCs by scanning microscopy (SEM) and frequency distribution of 

their indices by flow cytometry. 

SEM revealed discocyte-spherocyte transformation in treated rats. Flow cytometric analysis 

of the frequency distribution of the RBCs showed that the MCV and Mean Cell Haemoglobin 

Concentration (MCHC) increased and decreased, respectively compared to the controls. 

Whole blood viscosity was evaluated by microviscometry at 37°C over a wide range of shear 

rates. The deformability of RBCs was estimated as the resistance of a cell flowing through 

microchannels that simulated microvessels. Whole blood viscosity increased at shear rates of 

5.75 to 230/s for doses 50 and 100 mg/kg, and then decreased thereafter due to haemolysis. In 

vitro simulation of shear-dependent haemolysis using the microviscometer at shear forces of 

0.62 to 13.20 dynes/cm2

 indicated that the RBCs of EGBE-treated rats hemolyzed 

significantly more than the controls. Flow studies in the microchannels also showed that the 

deformability of RBCs of EGBE-treated rats was significantly less than the control cells due 

to the decreased surface area to volume ratio. These results demonstrate that membrane and 

cellular changes in the RBC due to EGBE and/or its metabolites alter its rheological 

properties, and therefore compromise transit through the microvessels of the microcirculation 

resulting in haemolysis. 

In vitro haematopoietic toxicity of some glycol ethers and derivatives (EGEE, EGME, EGBE, 

EGEEA, EGMEA, EGBEA, diEGBE, EGBEdiA and PGME) was assessed using 

haematopoietic culture assays in murine, rat and human species (Ruchaud et al., 1992). 

Acute and subacute toxicities of the compounds were analysed: on established haemopoietic 

cell lines (either leukemic or growth-factor-dependent cell lines), on haemopoietic progenitor 

growth and differentiation (CFU-c assay) and in bone marrow long-term cultures (Dexter type 

cultures) to determine the effects on both the hemopoietic and the stromal cell tissues. 

Acute and subacute toxicity on haematopoietic cell line. 

EGBE was tested for 48 hr at concentrations between 0.1 M and 0.1 μM. Typical dose 

response curves were obtained after treatment of NB4 cells. For all compound tested doseresponses show the common feature of a very steep slope on a semi-log plot, indicating that 

cell viability went from normal to nil by increasing concentrations by only a factor of 4 to 8. 

This steep slope and the sigmoid shape of the curve suggested that the substance hindered 

some cellular mechanisms essential for cell survival. Combined dose-response/time-course 

analysis were carried out on the human promyelocytic leukaemia celll line NB4 show that the 
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toxicity increased regularly with time over 96 hr treatment. Compared to other glycol ethers, 

EGBE caused a distinct toxicity feature for short incubations (0 to 40 hr) indicating that 

injuries causing cell death had probably multiple origins. The first component of the toxic 

phase worked at doses > 1mM and was over by 48 hr incubation. The second component 

probably worked from the beginning of the incubation, when its effects accumulate with the 

previous ones, but persist for longer incubation times (> 91 hr) and at lower doses. No 

differences were found for the same cell line when growth was supported by one or the other 

of the two growth factors tested, IL3 and G-CSF. 

Acute toxicity on Haemopoietic Progenitor Cells (CFU-c): human umbilical cord blood cells 

were treated with EGBE for 15 hr before CFU-c assay plating, then incubated for 14 days 

with 5637 CM. Growth of haemopoietic colonies was dramatically reduced after treatment. 

Virtually no growth was observed at concentrations > 20 mM. Similar results were found with 

murine bone marrow cells. 

Toxicity on haemopoietic cells and microenvironment in Dexter Cultures: effects of EGBE 

were evaluated on Dexter type long-term cultures two weeks after their establishment. 

Haemopoiesis was followed for 2 months with a constant exposure to various concentrations 

of compound. An acute toxicity of EGBE became obvious from 4 days of exposure at 7.5 

mM. Virtually no haemopoietic cells were found in the culture medium while only a few 

haematopoietic foci were still present within the layer of stromal cells. Increasing the 

concentrations definitely eradicated haemopoiesis, but stromal cells remained unaffected. 

Large macrophages like cells in the adherent layer where more sensitive than fibroblastic or 

preadipocytic stromal cells. After 2 weeks of exposure, aplastic cultures were obtained for 

doses between 7 and 15 mM. In these cultures, the stromal cell layer was apparently 

unaffected but no haemopoiesis persisted. Doses exceeding 30 mM also destroyed the 

haemopoietic microenvironment. Haemopoietic cell production in the liquid phase of cultures 

was evaluated twice a week for 2 months. Results after 4 or 8 days of treatment showed that 

haemopoiesis was very rapidly destroyed within a narrow dose range of the compound: for 

low doses low injury was apparent as judged by cell counts or cell morphology. 

In summary, of these studies, EGBE was the most toxic compound. The IC50 after 96 hr of 

exposure was in the range 50-100 μM. The pattern of toxicity suggested that cell death 

resulted from several mechanisms that had cumulative effects during the earliest period of 

exposure. This result is reminiscent of the haemolytic effect of EGBE reported by the others. 

By targeting the leucocyte membrane, EGBE could be responsible for both irreversible 

damage and cytolysis. 

In vitro haemolytic effects of BAA were assessed on human and rat erythrocytes (Udden, 

2002). Human blood was sampled from healthy volunteers, hospitalised patients and children 

and rat blood from Fisher 344 rats (9-11 weeks old). RBC were isolated, adjusted to a Packed 

Cell Volume (PCV) of 10 % and incubated in the presence of BAA (99 % pure) for 1-4 hours 

at 37° C. Various parameters were assessed: microhaematocrit, haemoglobin, RBC count, % 

haemolysis, erythrocyte deformability, erythrocyte osmotic fragility, density and Na and K+

concentrations in erythrocytes. A microscopic examination of erythrocytes was also 

performed. 

A time dependent increase of deformability was seen after exposure of human erythrocyte to 

10 mM BAA compared to controls. No haemolysis was observed. To compare subhaemolytic effects of BAA, human RBC were challenged with 0-10 mM BAA for 4 hours 

while rat erythrocytes were exposed to BAA at 100 fold lower concentrations. A comparable 

increase in cell rigidity with haemolysis was seen in both species. At the highest concentration 
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of BAA used, there was a 64 % increase in Po of rat RBCs and an increase in Po of 40 % for 

human RBCs. A significant change in human erythrocyte deformability occurred at 7.5 and 10 

mM BAA whereas decrease deformability in the rat erythrocyte was noted first at 0.05 mM 

BAA. In Mean Cellular Volume (MCV), there was a small but significant increase in human 

red blood cell volume after exposure to the highest concentration of BAA. For the size 

distribution, a main peak (very close to the MCV) was seen. A shoulder was also present. 

Both rats and human erythrocytes appeared to shift in size to a larger cell volume, as the 

number of smaller red blood cells diminished. For osmotic fragility, no significant changes in 

human erythrocytes were seen until they had been exposed to either 7.5 or 10 mM. Rat 

erythrocytes demonstrated a remarkable increase in osmotic fragility after exposure to lower 

concentrations of BAA. For erythrocyte density, human red blood cells showed only a very 

small shift toward lower density, however, the rat erythrocytes showed a marked shift to 

lower cellular density. For erythrocyte cations contents, there was a significant increase in 

both human and rat erythrocyte Na concentration with exposure to high and low levels of 

BAA respectively. However, human red blood cell K+

 was slightly decreased and this 

occurred after incubation in the highest concentrations of BAA. Rat red blood cell K levels 

decreased after exposure to 0.025 mM BAA, the lowest concentration used. Although there 

was a trend towards increased total cation contents in the human, only the rat erythrocytes 

showed a significant net change in total cation content, a small net increase after their 

incubation with 0.1 mM BAA for 4 hours. The morphology of human erythrocytes was not 

changed after exposure to 10 mM BAA for 4 hours. However, rat erythrocytes exposed to 0.1 

mM BAA showed increased numbers of spherocytes compared to the controls. 

After corrections, there was no difference in haemolysis of RBCs obtained from hospitalised 

adults and children compared to well adults when exposed to 10 mM BAA. MCV did increase 

as a result of BAA exposure, but no individual had a significant difference in the pattern of 

the frequency distribution of cell size. 

In vivo

A fragility test was performed by adding a drop of whole rat blood delivered with a needle 

into an agglutination tube containing 1 ml of saline (Mellon Institute of Industrial Research, 

1952) . The saline concentrations ranged from 0.36 % to 0.72 % in steps of 0.04 %. After 

addition of the blood the tubes were incubated at room temperature for 2 hr and read. The 

supernatant liquid is examined for any indication of haemolysis. Initial haemolysis was read 

as the highest concentration of saline in which haemolysis occurs and complete haemolysis 

the nearest in which there were no intact red cells visible. Cell detritus will not settle out in 

the 2 hr interval of the test. Initial haemolysis occurred at 0.44 % NaCl and complete 

haemolysis at 0.36 % NaCl in a remarkable consistent manner with rat blood. The exceptional 

normal reading was 0.48 % initial and 0.40 % complete. 

Fifteen males and 15 females were exposed 7 hr/d to 500 ppm of EGBE. 13 out of 15 females 

died during or shortly after the first exposure. Lung congestion and haemorrhage and 

generalised congestion of other organs was found at autopsy. The 2 other rats died after the 

second and the third exposures respectively. Seven out of 15 males died during or after the 

first exposure, one each after 2 and 3 exposures, 3 after 4 exposures and 3 survived 30 

exposures. Two of these survivors had moderate cloudy swelling of convoluted and loop 

tubules of the kidney, one had moderate cloudy swelling of the liver and one had completely 

normal kidneys, liver, lung, spleen and testis. Blood studies after 30 exposures revealed 

anaemia and increased fragility of the red cells. 
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Regarding the high mortality at 500 ppm, another study was carried out with lesser 

concentrations. Animals were exposed to 314 ppm but lethality was found within 3 days of 

exposure in the 15 females exposed. Pathology was comparable to that described for the 500 

ppm dose. 

Another study using 200 ppm doses was performed to the fragility test. Fragility of red cells 

on a group of rats was followed before and after exposure each day for a period of 9 days. 

Each day fragility of the red cells increased from initial haemolysis in 0.48 % saline before 

exposure to initial haemolysis at 0.52 % to 0.68 % after the exposure (following an overnight 

or a 17 hr rest period red cell fragility had returned to the normal value. 

Another group of 6 rats concurrently exposed at 200 ppm was used for enumeration of red 

blood cells and haemoglobin determinations. The protocols showed that the rats became 

anemic after 26 hr of exposure during 4 days. The red cell count was about half of preexposure level and haemoglobin had dropped from about 90 to 65 %. After 5 exposures of 6 

or 7 hours duration followed by a 7-day although they were still below the pre-exposure levels 

in most cases. Thereupon the rats were again subjected to 3 more exposures. Again the red 

counts were depressed and the haemoglobin values lowered even more so than after the first 5 

exposures. A subsequent rest period of 8 days resulted in a measurable increase in both values 

but the red cell counts were still subnormal. Fragility values were normal. 

Further study on rats revealed that at a concentration as low as 25 ppm of EGBE a 7 hr 

exposure produced a rise in red cell fragility whereas after 10 ppm for 7 hr only 1 of 4 rats 

showed any increase. 

Rats fed orally 1.48 g/kg as a 10 % aqueous dilution showed a relatively enormous increase in 

red cell fragility. The results on these four rats are as follows: 

Table 4.59: Effects on rat blood 

Time after dosing Initial and complete 

haemolysis (% Na Cl) 

15 min 0.48 – 0.40 

50 min 0.72 – 0.60 

130 min 0.72 – 0.60 

4 hr 1/2 0.72 – 0.60 

2 Guinea pigs and 2 rabbits were followed for 3 days while being similarly exposed to 200 

ppm EGBE. Guinea pigs were not affected whereas rabbits responded like rats. 

One rabbit was exposed for 10 hours to 200 ppm of EGBE and the fragility or red cells was 

determined periodically until the blood had returned to normal. 
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Table 4.60: Effects on rabbit blood 

Time after dosing Initial and complete 

haemolysis (% Na Cl) 

Pre-exposure 0.48 – 0.40 

3 – 180 min inclusive post-exposure 0.60 – 0.44 

240 – 420 min inclusive post-exposure 0.52 – 0.40 

420 min post exposure 0.48 – 0.40 

25 hours post exposure 0.48 – 0.36 

Two dogs were exposed to concentrations ranging form 200 to 665 ppm to determine the 

sensibility of their red blood cells to EGBE. No effects on RBC fragility were evidenced in 

these studies. 

Two men were exposed to 100 ppm EGBE for a 4 hr period. Rats were concurrently exposed 

to serve as positive controls. RBC fragility tests were performed 4 times in men and twice in 

the rats. No response was seen in humans whereas rats elicited expected positive response 

showing that they are much more sensitive as humans to the haemolytic properties of EGBE. 

Groups of 24 male F344 rats were dosed Per Os (PO) to EGBE (purity 99 %) at doses of 500 

and 1000 mg/kg (Grant et al., 1985). Six rats/group were bled and killed on day 1, 4, 8 and 22 

after the last treatment. Analysis of RBC, White Blood Cell (WBC), Hb, MCV, Haematocrit 

(HCT), reticulocytes and differential white cell count were performed. After termination, 

spleen, thymus, liver, testes and kidneys were weighted and were examined by microscopy. 

Cervical and mesenteric lymph nodes were also examined by microscopy. Femoral bone 

marrow was also examined by electronic microscopy. This study was concurrently performed 

with EGME to compare haematological effects caused by the two glycol ethers. 

Body weight gain was affected for high doses of EGBE. There was also an increase of the 

relative weights of spleen, liver and kidneys and a decrease of thymus weight. These changes 

returned to normal by day 22 apart for liver and kidneys for which the weight was still slightly 

raised. Hyperplasia of marrow without evidence of haemorrhage was seen. Splenic 

extramedullary haemopoiesis was marked in both groups on day 1 but returned to normal by 

day 8. A transient lymphocyte depletion of the thymic cortex was also noted and returned to 

normal by day 4. Animals treated with the high dose had reduced RBC, HCT, HB and 

elevated MCV, reticulocytes counts and Mean Corpuscular Haemoglobin (MCH). Most of 

these changes disappeared over the recovery period, although the MCV and MCH remained 

slightly elevated on day 22. The reticulocytosis was particularly striking on day 1 and 4 but 

had returned to normal by day 8. In this group increased number of normoblastes, pronounced 

anisocytosis, polychromasia and presence of Howell Jolly bodies were found but had resorbed 

by day 8. Leucocytes were also depressed on day 1 only due to a decrease number of 

circulating lymphocytes. These changes were also seen with the lower doses, but less in 

severity. 

The role of Alcohol and Aldehyde dehydrogenases in the haematotoxicity of EGBE was 

assessed (Ghanayem et al., 1987b). Three groups of F344 rats (9-12 weeks old) were 

submitted to different treatments: rats of the first group were treated IP with pyrasole (250 

mg/kg) one hour before gavage administration of water (control group) or EGBE. For the 
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second group, cyanamide (50 mg/kg) was administrated IP to animals 0.5 hr before water 

(control) or EGBE administration by gavage. The third group received only a single dose of 

EGBE by gavage (500 mg/kg). Animals were placed immediately in metabolism cages after 

EGBE treatments to collect urine and faeces. 

Concurrently, BAA or BAL was administrated to rats by gavage at the molar equivalent of 

125 mg/kg EGBE. Rats were also placed in metabolic cages. 

After EGBE administration, blood samples were collected during termination of the study. 

Evaluated parameters were: RBC, Haemoglobin (Hb) concentration, PCV, HCT, MCV, 

MCH, MCHC and platelet. Free Hb was also quantified in the plasma and urine of these rats. 

After collection of blood samples, liver, kidneys and spleen of treated animals were collected 

and weighted. Tissues were collected 24 hr and 48 hr after treatments. 

Gavage administration of EGBE caused severe haemolysis accompanied by an increase in the 

relative spleen weight. The increase was significant at all time points and was maximum 2 hr 

after EGBE administration. Recovery from treatment was not complete even 48 hr after 

dosing. A significant decrease in the number of circulating RBCs and Hb concentration and 

HCT was seen in treated animals. These effects were maximum within 8 to 24 hr after dosing 

and showed a rebound at 48 hr. These finding were accompanied by increase of the free Hb in 

the plasma and haemoglobinuria. In the liver, histopathologic evaluation made 24 hr after 

dosing showed coagulative necrosis and phagocytosis of Hb by hepatocytes and Kupffer cells. 

In contrast, pre-treatment of rats with pyrasole resulted in complete prevention of the increase 

of the relative spleen weight and of the haemolytic effects caused by EGBE alone. Liver 

effects were also completely prevented by pyrasole pre-treatment. With cyanamide treatment, 

significant increases of the relative spleen weight, however the time to maximum effects was 

approximately doubled in rats treated with cyanamide and EGBE as compared to rats treated 

with EGBE alone, a significant decline in number of RBCs, HCT and Hb concentration in 

cyanamide treated rats was also seen but was significantly less than what was found after 

EGBE treatment only. 

After BAA or BAL administration, the same effects were seen on relative spleen weight than 

after EGBE administration. The maximum effects were observed at 4hr after dosing. No 

differences were seen on blood parameters between the three chemicals. Treatment with 

cyanamide before BAL administration resulted in a significant protection against BAL 

induced toxicity. 

Effect of age on the toxicity was assessed (Ghanayem et al., 1987c). EGBE (purity > 99 %) 

was administered orally to F344 rats (16 months old) at doses of 32, 63, 125, 250 and 500 

mg/kg. Immediately after dosing rats were placed in metabolic cages and urine was collected 

at different times. Rats were killed at various time points after administration and blood was 

collected by cardiac puncture. Spleen, liver, kidneys, testes and urinary bladder were collected 

and examined histologically. The following measurements were done on blood samples: 

RBC, WBC, Hb, HCT, MCV, MCH, MCHC and platelet count. Free concentrations of Hb 

were also determined in urine and in plasma. 

Gavage administration of EGBE in young or adults rats resulted in a significant dose related 

increase of the spleen weight evidenced from 125 mg/kg. Young rats were less sensitive than 

older ones. Maximum effects on relative spleen weight were observed at 2 and 4 hr after the 

administration of 500 mg/kg to rats (young or old with a more marked effect in old animals). 

At 125 mg/kg dose, effects were maximum 4 hr after gavage in both age groups. After 

reaching the maximum, controls levels where obtained about 48 hr after treatment. On 
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haematologic parameters, EGBE caused a significant decrease in the number of circulating 

RBCs, Hb concentration and HCT. Other blood parameters showed minimal changes. These 

effects were dose dependent and older rats were significantly more susceptible than younger 

rats. At 125 mg/kg, effects on RBCs, Hb and HCT were only seen in old animals 8 and 24 hr 

after administration. Young animals were not affected by treatment at this dose level. The 

decline in RBCs, Hb and HCT was accompanied by a significant dose dependent increase in 

the concentration of free Hb in the plasma of treated rats. The peak level of free Hb in the 

plasma was lower in young animals compared to the older ones at all doses investigated. 

Maximum free plasma Hb levels were seen after the administration of 500 mg/kg EGBE and 

were approximately 5 times higher in the plasma of the older rats than those of measured in 

the plasma of the younger rats. For the 125 mg/kg dose, no effects were seen on free plasma 

Hb of young animals whereas a significant increase was detected 8 hr after administration of 

the same dose to older rats. Haemoglobinuria was observed and was concomitant with the 

decline of free Hb in the plasma. A difference between old and young rats was also noted (see 

table 4.61). 

Table 4.61: Incidence of haemoglobinuria induced in rats of various ages by gavage administration of EGBE. 

Dose (mg/kg) 

Age 32 63 125 250 500 

4-5 weeks 0/6 0/6 1/11 6/6 12/12 

9-13 weeks 0/6 0/6 12/12 6/6 12/12 

5-6 months 0/6 6/6 6/6 ND ND 

16 months 6/6 6/6 6/6 ND ND 

Histopathologic evaluation of tissues of rats of various ages examined 24 hr after EGBE 

administration revealed that EGBE causes dose and age dependent liver changes. In 9 to 13 

week old rats, EGBE caused focal disseminated coagulative necrosis of hepatocytes. In 

addition there was evidence of phagocytosis of dark pink protein compatible with Hb by 

Kupffer calls and hepatocytes. These effects were observed at an incidence of approximately 

16 and 83 % at 250 and 500 mg/kg doses, respectively. Evidence of regression of these liver 

lesions was seen at 48 hr after EGBE dosing. No liver changes were observed at the same 

doses in 4 to 5 week old rats. In the kidneys, small and dark pink protein droplets, compatible 

with Hb, were seen in the proximal tubular epithelium of treated rats. In addition Hb casts 

were observed in the proximal tubules. The incidence and severity of these effects were dose 

dependent and were observed in 9 to 13 week old rats but not in 4 to 5 week old rats. Similar 

to the liver, kidney lesions were more severe at 24 hr after EGBE administration and 

exhibited signs of regression when examined at 48 hr after dosing. 

The influence of the type of haematology analyser on measured parameters was assessed 

(Ghanayem et al., 1990b). Groups of male F344 rats (9-11 weeks old, 3-5 animals per group) 

were treated by gavage with 125, 250 or 500 mg/kg of EGBE (purity 99.9 %). Animals were 

held for 1, 2, 4, 8 or 24 hr after treatment. Control rats received the vehicle (water). At the end 

of each holding period, blood samples were collected and analysed using both a laser-based 

and an impedance-based haematology analyser. The following parameters were measured : 

RBC and WBC counts, Hb, HCT, MCV, MCH, MCHC and platelet counts. In addition PCV 

were determined by centrifugation. 
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Parameters obtained with the laser-based analyser indicated dose and time-related decreases 

in RBC and Hb. Similar effects were observed with the impedance based analyser. Striking 

differences were seen in HCT MCVs MCH and MCHC of EGBE treated rats. While a dose 

and time dependant increase in HCTs was recorded early after treatment (followed by a 

decline as haemolysis occurs) using the impedance based analyser a dose-dependant decrease 

was consistently recorded using the laser-based analyser. 

By 24 hr HCT significantly decreased in all three dose groups and whichever the analyser 

used. The impedance based analyser also detected marked doses related early increases in 

MCVs followed by a decline to near normal by 24 hr after EGBE treatment. In contrast a 

small decrease in MCV was detected with the laser-based analyser. PCV confirmed the results 

obtained from the impedance-based analyser showing an early increase in PCVs followed by 

a decline as haemolysis of RBCs occurs. 

In the 125 and 250 mg/kg dose groups, changes in MCHs recorded by both types of 

instruments were the same. However 4 hr after the high dose, a much greater increase was 

seen in MCHs from the impedance-based than from the laser-based analyser. Finally, while 

the laser-based analyser recorded a dose-dependent increase in MCHCs the impedance-based 

analyser recorded a dose-dependent decrease in MCHCs in the same blood samples. 

This study demonstrated the importance of the analyser used to determine the early effects 

described for EGBE haematotoxicity: increase of HCT and MCV. The impedance-based 

analyser is therefore more appropriate. 

EGBE (99 % pure) was administrated via oral route to groups of male SD rats at doses of 50 – 

100 - 250 and 500 mg/kg (CMA, 1990). Blood samples were collected at 0.5 – 2 and 4 hr 

after treatment for various analysis: 

- Cell morphology was examined by microscopy and red cell indices were recorded: RBCV 

and Hb concentration distribution. Plasma and urine Hb concentrations were also 

measured. 

- The measure of the erythrocyte density distribution was performed. This parameter reflect 

changes in the volume of the red cells and the profile of the changes (entire population or 

a fraction of them). 

- The ATP content of the red cells was determined to assess the ability of EGBE to perturb 

the energy state of the RBC and consequently the function of ionic pumps. 

- Effects on red cell lipid auto-oxydation was measured to see if haemolysis could be 

produced by a mechanism of peroxidation of unsaturated fatty acids of the cell membrane, 

MDA (malonyldialdehyde) served as a marker of lipid auto-oxidation. 

- Viscosity and deformability of RBC was also measured after treatment. 

The morphology of RBC showed a dose related trend to the formation of spherocyte. For 

RBC indices, the MCV frequency distribution curve broadened significantly. The mean cell 

volume increased from 53.8 fL to 86.9 fL. The frequency distribution of MCHC in contrast 

narrowed, and the mean value decreased from 35.2 gm/dl to 25.1 gm/dl. Similar data were 

obtained for all animals studied, and the MCV, MCHC, RDW and HDW values, as well as 

the other indices showed similar trends depending on the dose of EGBE administered and 

time of exposure prior to the sampling of blood. The red cell MCV increased steadily whereas 

the MCHC decreased as a function of the dose of ingested EGBE, suggesting an increase in 

cell water content. The mean value of MCV increased with increasing dose of ingested 
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EGBE. The EGBE-treated animals had significantly higher MCV than the control group. The 

dose-response relationship noted for MCV was most apparent for 500 mg group which 

showed a significantly higher mean MCV values among the EGBE-treated rats as compared 

with the control group. Erythrocytes from the control animals had higher MCHC values than 

all EGBE-treated animals, with higher doses of the glycol ether resulting in lower MCHC 

values. No clear dose response relationship was observed for other parameters. Using a 2-way 

ANOVA with time and dose as factors, a significant difference between 50 and 500 mg/kg 

dose group was observed in the following parameters: MCV, MCHC, HCT, RDW, HDW, Hb 

and RBC at 0.5 and 2 hr observation times. Haemoglobin in the plasma and urine was 

demonstrated 2 hours after EGBE administration. Dose-dependency was demonstrated 2 

hours after the EGBE administration. 

The median density of rat RBCs treated with EGBE decreased as a function of the dose of 

ingested compound. These changes in the profile indicated an increase in cell volume and a 

decrease in cell Hb concentration (perhaps as a result of an increased water cell content). 

There was no specific effect on any subpopulation of cells according to the density profiles. 

No differences in the RBC ATP levels were seen in EGBE treated animals. 

The MDA concentration decreased to about 50 % regardless of the administered dose. The 

decline in MDA concentration is suggestive of the arrest of autooxidative processes in the 

cells by EGBE or its metabolites. There was no evidence for an increase in membrane lipid 

peroxidation. 

An increase of 72 % of the plasma viscosity was seen in treated animals (at dose of 500 

mg/kg EGBE). At equal haematocrit of 40 %, the mean blood viscosity of the rats that 

received 50 and 100 mg/kg was 2 to 3-fold higher than that of he control rats. The mean 

viscosity of the rat that received 250 and 500 mg/kg was lower than that at the other two 

doses, but higher than that of control rats. 

The effects of a daily dose of EGBE on haematological parameters were assessed in rats 

(Ghanayem et al., 1992). EGBE (purity 99 %) was administrated by gavage to groups of male 

Fischer 344 rats (10-14 weeks old – 6 rats/group). Five groups of animals received 125 mg/kg 

EGBE during 1, 2, 3, 6 or 12 consecutive days. Twenty four hours after the last dose animals 

were killed and 2 blood samples were collected, one with EDTA/dipotassiom salt as 

anticoagulant and the other with an acetate citrate dextrose (ACD) solution (to be used for the 

determinaion of blood ATP concentration). Livers and spleens were collected and weighted. 

Blood samples were analysed for the determination of RBC count, HB, HCT, MCV, MCH, 

MCHC and platelet counts. In addition PCV were determined on all blood samples. Other 

mechanistic studies were performed both in vitro and in vivo. 

Some studies were performed in parallel to try to draw a mechanism of action of RBC 

toxicity: 

- Experiment 1: Haematotoxicity of EGBE in pretreated/recovered rats in vivo was 

assessed: one group of rats received 125 mg/kg/d for 3 consecutive days and another 

group of untreated rats served as controls. Animals were then allowed to recover for a 

period of 7 days. Treated and untreated rats were divided into 3 groups, the first receiving 

only water, the second and the third receiving EGBE at doses of 125 and 250 mg/kg 

respectively. Blood samples were collected 2, 8 and 24 hr after dosing (the same analysis 

than the previous described study were performed). Animals were killed after blood 

collection and the spleen was collected and weighed. 
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- Experiment 2: The effects of BAA on blood from EGBE pretreated/recovered rats were 

also studied in vitro. Rats were treated with 125 mg/kg for 3 concecutive days and 

allowed a 7-day recovery period. Blood was incubated with BAA at concentration of 0 – 

0.5 – 1 or 2 mM for 0.5 – 1 – 2 or 4 hr. At the end of the incubation period, PCV and 

blood ATP concentrations were measured. 

- Experiment 3: The effects of EGBE in bled/recovered rats in vivo were also studied. Rats 

were bled and allowed a 7-day recovery period. After this period, treatments with 250 and 

125 mg/kg via oral route were administered. Blood samples were collected 2, 8 and 24 hr 

after dosage and analysed as described below. After termination, the spleen was removed 

and weighted. 

- Experiment 4: Effects of BAA in bled/recovered rats in vitro were also assessed. Rats 

were bled and allowed a 7-day recovery period. Animals were then killed and blood 

collected and pooled. The effects of BAA on PCV and ATP were investigated as 

described in the previous experiments. 

Following repeated exposure (experiment 1), haemolysis evidenced by a decrease RBC, HB 

and HCT was seen and become more pronounced following the third day of dosing but 

gradually recovery was observed thereafter. With continued repeated exposures to EGBE 

beyond day 3, rats exhibited a gradually increase in RBC and HB and approached pretreatment levels by day 12. HCT followed a similar pattern as RBCs and HB as dosing 

continued. On the other hand, MCV increased as the dosing regimen was extended to 6 days 

and declined to remain above control levels throughout the 12 day study. ATP concentrations, 

MCV and the number of reticulocytes increased after 6 days and then declined by day 12. 

Reticulocytosis and increased ATP concentration were evident after the second day of dosing 

and persisted in EGBE treated rats. The relative weight of the spleen increased to a maximum 

after 6 days from which it declined between days 6 and 12. Relative liver weight was 

minimally affected by treatment and following a moderate decline on day 3 and 6, it was 

increased on day 12 compared to controls. 

In the second experiment, EGBE pretreated/recovered rats were found to be less sensitive to 

the haemolytic effects of EGBE than control rats. The RBC and HB declined in a dose and 

time dependent manner, moreover there was a minimal decline of these parameters in rats 

pretreated with the low dose of EGBE. HCT increased at 2 hr and remained near or slightly 

below controls in EGBE pretreated/recovered rats. In contrast, rast which were not previously 

exposed to EGBE exhibited an increase in HCT early after treatment which later declined in a 

dose and time dependent manner. Treatment of pretreated animals with the high EGBE dose 

(250 mg/kg) caused more pronounced haematotoxicity than the low dose (125 mg/kg), this 

effect remained less than that observed in untreated rats of the same dose level. Pretreated 

animals were also less sensitive to the increase in MCV and ATP depletion caused by EGBE 

than non exposed rats. In addition the number of reticulocytes was significantly higher in rats 

pretreated with EGBE than in controls. Comparison of the relative spleen weight showed that 

the increase caused by EGBE is less in pretreated animals than in untreated. 

In vitro (experiment 3), blood from pretreated rats was significantly less sensitive to BAA 

than untreated rats: increase in PCV was greater in untreated rats than in treated rats. 

Similarly ATP concentrations in blood showed that pre-treatment decrease the susceptibility 

of erythrocytes to the effects of BAA in vitro. Both effects of BAA became more dramatic as 

the concentration of BAA and the incubation time increased. 
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Bled/recovered rats (experiment 4) were significantly less sensitive to the haemolytic effects 

of EGBE than controls. The RBCs and HB declined in a dose and time dependent manner, 

however the decline in bled/recovered rats treated with the 125 mg/kg dose was minimal. 

HCT increased at 2 hr and remained near pre-treatment levels in bled/recovered rats. In 

contrast, EGBE administration to controls resulted in an early increase in HCT and then 

declined in a dose and time dependent manner. Although the number of reticulocytes in 

bled/recovered rats prior to the exposure to EGBE was significantly higher than in the 

corresponding controls. Treatment of bled/recovered animals with the high dose of EGBE 

caused more pronounced haematotoxicity than the low dose, however these effects continued 

to be less pronounced than those observed in control rats receiving the same EGBE dose. 

Comparison of the relative spleen weights demonstrated that there was a significantly greater 

increase caused by EGBE in control rats compared to bled/recovered rats. 

In vitro, blood obtained from bled/recovered rats was less sensitive to BAA–induced increases 

in PCV and ATP depletion. These effects were more pronounced with high BAA 

concentrations and with longer incubation times. 

Some studies tried to demonstrate that a low dose of EGBE administrated a few days before 

the administration of a lethal dose of EGBE can protect from haematological effects and then 

from death (Sivarao and Mehendale, 1995). In these studies, 3 groups of 12 female SD rats 

were treated orally with EGBE 500 mg/kg for groups 1 and 2 whereas group 3 served as 

controls and was administrated with distilled water. On day 7 groups 1 and 3 were 

administrated LD50 dose of EGBE (1500 mg/kg) while group 2 received distilled water. 

Haematocrit of all rats was monitored on a daily basis as an index of toxicity. Rats were 

observed 14 days. 

The effect of increasing the time gap between pre-treatment and lethal challenge on animal 

survival was studied by pretreating rats with 500 mg/kg EGBE on day 0. Rats were randomly 

divided into 3 groups (n=10, 8 and 6). On day 7, 14 and 21 days, one group of these rats 

received a single administration of 1500 mg/kg. Rats were observed twice daily for 14 days. 

In vivo effects of pyrasole (ADH inhibitor) were tested with about the same experimental 

protocol (7 rats in each group): the treatment group received pyrasole 250 mg/kg one hour 

before treatment with 500 mg EGBE. HCT were recorded before pyrasole administration, 24 

hr following EGBE pre-treatment and on day 7 of the experiment. 

In these studies, the in vitro haematotoxicity of EGBE was also assessed. Dose response 

curves were constructed by incubating 1 – 10 mM of BAA in saline to rats blood. PCV 

(Packed Cell Volume – a marker of haematotoxicity found to be specific to BAA) were 

determined at various time points (1 - 4 hr). PCV are expressed as percent of controls. 

Another experiment was carried out to investigate if the loss of autoprotection in vivo could 

be related to a corresponding loss in resilience of RBC in vitro. Rats were administrated a 

single dose of 500 mg/kg EGBE following which they were randomly divided in 2 groups of 

4 rats each. On 7 and 21 days one of the groups was killed and blood was incubated with 

BAA and PCV was determined in different concentrations of BAA. 

To test the hypothesis that newly formed RBCs are primarily responsible for the EGBE 

induced autoprotection, bled/recovered rats were used. Rats were bled 2.5 ml for 3 days. 

Blood letting was followed by a 7-day period of recovery. Recovery was ensured by the 

return of depleted haematocrit values to pre-bled levels. Control rats underwent a sham 

treatment without blood letting. Both the bled/recovered rats as well as their sham treated 

controls were challenged with a lethal dose of EGBE (1500 mg/kg), and their survival was 
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recorded, twice daily for 14 days. Incubation studies with BAA, were also conducted using 

RBC from these rats, to corroborate the in vivo studies. 

Moderately toxic symptoms were seen with the “protective” dose of EGBE: ataxia, 

haematuria, paleness of skin and morbidity. Haematocrit values fell to 18.6 twenty four hr 

after treatment. Recovery was rapid. HCT reached control values level by 7 days. Rats 

received pre-treatment were fully protected against the subsequent lethal challenge with 1500 

mg/kg EGBE while “non protected” rats experimented high (> 90 %) mortality. Survivors 

showed an average HCT of 13.5 whereas the “protected” rats experienced a much lesser fall 

in their HCT with no mortality. 

Regarding the effects of pyrasole pre-treatment on autoprotection, the initial haemolytic 

episode due to EGBE pre-treatment was somehow attenuated (HCT recorded 24 hr after 

EGBE pre-treatment did not fall in the pyrasole treated rats) but following the administration, 

rats experienced profound ataxia that lasted several hours more than observed in control rats. 

Following the lethal challenge, pyrasole pretreated rats suffered high mortality (87 %). 

With increasing time between the pre-treatment and the lethal dose, the percentage of survival 

decreased indicating that the autoprotection is gradually lost with time. 

RBC isolated after EGBE pre-treatment showed a significantly higher susceptibility to BAA 

induced swelling than the cells isolated 7 days after EGBE treatment indicating that the 

resilience decrease with time. 

When bled/recovered rats were challenged with lethal dose of EGBE, 87 % survived in sharp 

contrast to the 6 % survival of the corresponding sham treated controls. These findings 

suggested that newly formed RBCs were innately resistant to the haemolytic action of BAA. 

Morphological changes of rat erythrocytes after administration of EGBE in vivo or BAA in 

vitro were assessed (Udden, 2000). Fisher 344 rats were dosed orally with 125 and 250 mg/kg 

EGBE. Blood samples were collected before and 30 minutes, 1 hr, 2 hr and 4 hr after 

administration. Approximately 75 μl of blood anticoagulated with EDTA was collected for 

microhaematocrit determination, fixation and for preparation of blood smears on glass slides. 

For in vitro studies blood samples EDTA anti-coagulated were exposed to 1 or 2 mM BAA. 

Samples were taken at intervals for determination of a spun haematocrit, preparation of blood 

smear and fixation. Washed red blood cells were prepared by centrifugation followed by 

removal of the plasma and buffy coat and re-suspension in buffer. Cells were washed 4 times 

and then re-suspended to 50 % haematocrit. BAA was added to achieve a final concentration 

of 1 or 2 mM. 
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Table 4.62: In vivo and in vitro results 

In vivo results In vitro results 

30 min / 125 mg/kg 

Erythrocytes with mouth shaped central pallor 

or stomatocytes. 

2 hr / 125 mg/kg 

Increase of the stomatocytes nb and increase 

of sphero-echinocytes. 

4 hr / 125 mg/kg 

Prevalence of stomatocytes and spherocytes 

1 hr / 250 mg/kg 

Impressive spherocytosis 

HCT increased in a dose dependant manner. 

30 min / both doses 

Cupping and spherocytic changes more 

pronounced after 1 hr similar than EGBE 

administrated PO. 

2 hr / 1 mM 

Most of RBC were spherocytes. Wright 

stained: spherocytosis but only minimal 

stomatocytose. 

Sphero-echinocytosis was frequently 

observed after 1-2 hr of incubation. 

Effects were more marked with 2 mM BAA. 

Washed erythrocytes incubated with 1 or 2 

mM BAA demonstrated haemolysis with 

ghost formation and agglutination. 

Increase of HCT comparable to EGBE treated 

animals (1 hr / 125 mg/kg equivalent to 1hr / 

1mM BAA). 

 No changes in human erythrocytes. 

The difference between males and females Fischer 344 rats after exposure to an oral dose of 

EGBE was assessed (Ghanayem et al., 2000). Fischer rats (12-16 weeks old) received an oral 

dose of 250 mg/kg EGBE (purity > 99 %). Control rats received 5 ml water/kg. 

Haematological parameters were analysed after termination (RBC – WBC – HB – HCT – 

MCV – MCH – MCHC – PCV). Blood smears were prepared from all samples. 

Morphological changes were evaluated by light microscopy. 

The effects of BAA were also evaluated in vitro. Blood samples were exposed to 

concentrations of 0.25 – 0.5 and 1 mM. Samples of blood were also incubated with 

physiological saline to serve as controls. PCV was determined at 0.25 – 0.5 – 1 – 2 and 4 hr 

after incubation. 

EGBE resulted in early swelling of erythrocytes as evidenced by an increase in HCT and 

MCV. In comparison to males, female rats exhibited a greater increase in MCV at all time 

points. However HCT declined in both genders at 8 and 24 hr after dosing. At 4 and 8 hr after 

dosing, RBC, HCT and HB were significantly lower in female rats than in males, however the 
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difference in these parameters were not statistically significant at 24 hr. This reflects a faster 

onset of erythrocytes haemolysis in female after dosing compared to males. This is also 

confirmed by the greater increase of the relative spleen weight in females compared to males. 

Other haematological parameters were also consistent with a faster onset of haemolysis in 

female rats. In parallel reticulocyte counts were greater in females than in males at all time 

points, indicating a faster regenerative response. Morphological alterations were seen in blood 

smears prepared from rats at 4, 8 and 24 hr after EGBE administration. At 4 hr after 

administration blood smears exhibited evidence of erythrocyte destruction, schistocytes, 

stomatocytes, ghost cells and other deformed erythrocytes. A decrease in the nb of RBC and 

absence of schistocytes with hypochromic RBC remaining at 8 and 24 hr was also observed. 

Aggregation of RBCs was evident by the formation of rouleaux at 24 hr after administration. 

In males alteration in RBCs were quantitatively similar than in females but these changes 

were observed more frequently and developed at a faster onset in female rats. 

EGBE (purity > 99 %) was administered by gavage to groups of Fisher 344 rats at doses of 

250 mg/kg for 1, 2 or 3 consecutive days (Ghanayem et al., 2001). Animals were sacrificed 

24 or 48 hr after the last dose. Blood samples collected at termination were analysed for RBC, 

WBC, HB, HCT, MCV, MCH, MCHC and platelet count. In addition PCV were determined 

on all blood samples. Erythrocytes morphological changes were evaluated by light 

microscopy. A complete necropsy examination was performed on all rats. The following 

tissues and organs were collected and examined: sternum, femur, vertebral column, tail, heart, 

lung, liver, spleen, kidneys, left eye and brain. 

Severe acute regenerative anaemia in both sex in all treated animals were seen. However the 

progression of haemolytic anaemia as a function of the number of days of exposure varied and 

suggested a faster onset of haemolysis in females. After 24 hr following a single dose, the 

haematological parameters were relatively similar in both males and females except for the 

reticulocyte count. Female rats exhibited approximately a 2 fold increase in reticulocytes with 

a minimal increase in males. In addition, haemolysis was more severe in females at 24 and 48 

hr after the second daily dose, as evidenced by the continued decline of the haematological 

parameters at these time points. Reticulocyte counts in females doubled and quadrupled at 24 

and 48 hr after treatment respectively. The progression of haemolytic anaemia was also more 

severe in females after the second daily exposure. At 24 hr after the third dose, the 

haematological parameters calculated as percentages of controls, were relatively similar in 

both sexes of rats and were indicated severe regenerative anaemia. 

Significant morphological changes in erythrocyte were also observed in treated animals. 

Slight macrocytosis with moderate rouleaux formation and stomatocytosis were observed in 

female rats at 24 hr after a single dose. These morphological changes became progressively 

more severe as dosing continued and included the occurrence of occasional schistocytes and 

ghost cells and an increased number of reticulocytes (micronuclei). Erythrocyte 

morphological changes were qualitatively similar in male rats after 1 or 2 doses and were 

most severe after 3 daily doses of EGBE. 

Macroscopic abnormalities were noted in both males and females: watery blood, yellowish 

discoloured livers, dark brownish kidneys and multiple petechial haemorrhages on the sclera. 

In females, only bluish discoloration was noted on the tip of the tail. In the 2 females that died 

within 24 hr after the first dose, thrombosis was noted only in submucosa of the most anterior 

section of the nasal cavity. Other lesions noted in these rats included focal hepatocellular 

necrosis and renal tubular necrosis associated with haemoglobin casts. Thrombosis was 

evident in females sacrificed 24 hr after a single dose. The vascular thrombi were occlusive 

and consisted of amorphous granular material or partly organized fibrin with varying numbers 
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of red blood cells. Widespread thrombi formation noted in the ocular ciliary processes and in 

the limbus associated with retinal haemorrhage and necrosis were suggestive of an ischemicinfarctive process. Thrombosis was also observed in the submucosa of the most anterior 

section of the nasal cavity, mostly located in the nasal and maxillary turbinate as well as 

the septum. Evidence of EGBE-induced thrombi was also observed in the pulmonary 

small caliber vessels and the right ventricle of the heart in association with foci of 

myocardial necrosis and inflammation. In agreement with earlier findings, thrombosis 

also affected the dental pulp of the incisors in conjonction with focal stromal and 

odontoblastic coagulative necrosis. Lesions consistent with acute infarction were noted 

in the coccygeal vertebrae and femur, where histopathological changes included 

intramedullary thrombosis, and necrosis of bone marrow cells, bone-lining cells, and the 

cortical and trabecular osteocytes. Dying cells exhibited nuclear pyknosis and/or 

fragmentation to complete loss of nuclear staining, often appearing as ghost cells. Soine 

of the lacunae were empty. The changes in the femur were usually confined to the 

diaphysis (medullary cavity bone marrow and trabecular bone) and inner one-third of the 

cortex, while in the coccygeal vertebrae, the metaphyseal bony trabeculae and epiphysis 

were also necrotic. In rats sacrificed 48 hr after two or three daily EGBE doses, bone 

marrow regenerative erythroid hyperplasia was also observed in addition to the 

consistent presence of the fore mentioned lesions. Other noted changes included 

multifocal hepatocellular necrosis in the liver, extramedullary haematopoiesis in the 

spleen, and haemoglobinuric nephrosis. 

In male rats treated with EGBE for one or two days, pathological changes were limited 

to those associated with haemolysis, consisting of renal haemoglobinuric nephrosis and 

splenic extramedullary haematopoiesis. However, the 3-day EGBE dosing induced 

thrombosis and tissue infarctions similar to those seen in female rats. In particular, 

disseminated thrombosis was noted in the longs, nasal submucosa, liver, heart, bones, 

tail and teeth. Degeneration, necrosis and/or infarction were noted in the heart, eyes, 

teeth and bones. 

Three groups of 3 female GP were treated with a unique subcutaneous dose of EGBE at 0.1 – 

0.5 or 1 ml/kg (Unilever, 2001). Blood samples were taken before dosing and on day 1, 2 and 

5 after dosing prior termination. On blood sampled, the following parameters were measured: 

* Haematology: PCV – Hb – RBBC – WBCC – MCHC – MCV – MCH and reticulocytes 

* Biochemistry: K+

 - Mg2+ - Ca2+ - free haemoglobin 

At necropsy the following organs were examined macroscopically: liver, spleen and kidneys. 

No effects were seen on any measured parameters. 

In a recent publication (Koshkaryev et al., 2003), the mechanism of EGBE-induced 

haemotoxicity was studied. Male and female Fischer 344 rats were exposed to two, three or 

four daily doses of EGBE (purity > 99 %) at 250 mg/kg. Blood samples were taken from rats 

before sacrifice on days 2, 3 and 4 (groups 2, 3 and 4 respectively), each time 2h after 

administration and from group 5 before sacrifice 24 days after the last dosing. After isolation, 

RBC were subjected to determination of RBC flow properties on the day of collection 

(aggregability, adherence and deformability). 

RBC aggregability was not affected by the treatment. The effects on deformability were 

inconclusive. The main effect was an increase of the RBC adherence to Extra Cellular Matrix. 
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There were no differences between male and female. The adherence was highest at day 2 (the 

first to be examined) after which it decreased sharply with time. 

Further investigations were performed to elucidated the mechanism of thrombosis formation 

(Nyska et al., 2003). Eyes from female Fischer 344 rats exposed to EGBE were studied 

histologically and immunohistochemically using specific monoclonal antibodies to determine 

the in situ protein expression of vascular cell adhesion molecule-1 (VCAM-1), endothelial 

intercellular adhesion molecule-1 (ICAM-1), and P-selectin. Groups of female rats (4 per 

treated groups, 8 for control group) were treated with 250 mg/kg EGBE (100 % purity) in 

drinking water for 2, 3 or 4 consecutive daily treatment. Sacrificeof the animals was done 2 

hours after the last scheduled treatment. 

Histopathologically, lesions in the retina consisted of retinal thrombosis, haemorrhage, 

degeneration and necrosis. Changes were mainly located in proximity to capillaries and 

consited of multifocal haemorrhage within the retinal pigment epithelium (RPE) and 

choriocapillaries and between the RPE and photoreceptors (leading to retinal detachement) 

and the photoreceptors and inner and outer nuclear layers. Haemorrhage in the RPE was 

associated with degeneration, exfoliation, loss of epithelial cells and presence of neutrophils. 

Haemorrhage whitnin the receptor layer caused organizational disruption and loss of 

photoreceptor cells. In the inner and outer nuclear layers, the haemorrhages and thrombosis 

were associated with degeneration and loss of cells resulting in thinning, disorganisation, and 

fusion of these layers. Thrombi were also noted within blood vessels of the ciliary processes, 

in the limbus and in the retina. Positive VCAM-1 expression was observed only in eyes of rats 

exposed to 3 and 4 doses of EGBE (mainly in the iris, ciliary processes and retina). Only 

weak effects were seen after one or two doses. No effects were seen for ICAM-1 and Pselectin. 

Studies with metabolites

The effects of BAA on human and rat blood cells were investigated (CMA, 1993). Human 

erythrocytes were obtained from healthy volunteers and rats erythrocytes from Fischer 344 

rats. Red cells were isolated and incubated in the presence of 0, 0.2 or 2mM BAA for 1-4 hr at 

37 °C. Erythrocyte suspensions were sampled at intervals for determination of 

microhaematocrit, Hb, RBC count and percentage of haemolysis. Morphology of RBC was 

also observed. The deformability of erythrocytes was assessed. 

A 30 % haemolysis was demonstrated in rat erythrocytes when incubated with 2 mM. For 0.2 

mM only little haemolysis was seen (when incubated for 6 hr or longer, mild haemolysis was 

detected). Less than 1 % haemolysis was seen in human erythrocyte when incubated for 4 hr 

with 2mM. For deformability, exposure of rats erythrocytes to 2 mM for 1 hr resulted in a 

dramatic decrease in red blood cell deformability (increase in cell rigidity). A longer 

incubation (4 hr) in the presence of 0.2 mM BAA also produced significant changes in the 

deformability curve in the absence of significant haemolysis. No deformation was seen in 

human erythrocytes exposed to 2 mM for 4 hr. 

Distinct changes in the morphology of rat RBCs exposed to BAA were observed. Rat RBCs 

incubated in buffer alone for 4 hr were found to be mostly normal appearing discocytes with 

occasional crenated cells identified. Incubation in the presence of 0.2 mM BAA resulted in 

striking spherocytic and spheroechinocytic RBCs. Also apparent was a tendency for BAA 

incubated RBCs to adhere to each other. Incubation with 2 mM BAA resulted in dramatic 

haemolysis with the appearance of RBC ghosts (which appear to be mutually adherent). 
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Another finding was the presence of precipitates of rat Hb. Human RBC morphology was 

unaffected by a 4 hr incubation in 2mM BAA. 

Effects of BAA on RBCs from 10 mammalians were studied in vitro (Ward et al., 1992 ; 

Ghanayem and Sullivan, 1993). To validate in vitro finding, the haematotoxic effects of 

EGBE was compared in vivo using one representative from each of the sensitive and less 

sensitive animals. Various animals were used to collect blood samples (see table 4.63) 

Table 4.63: Effects of BAA on blood from different species 

Species Strain Average age 

Mouse B6C3F1 9 wks 

Rat F344 10 wks 

Hamster Syrian (LVG:LAK) 27 wks 

Rabbit New Zealand 37 wks 

Guinea pig Hartley 28 wks 

Canine Hound 18 wks 

Feline Dom. Short Hair 7 yrs 

Pig Poland China 2 yrs 

Baboon P. cynocephalus 5 yrs 

Human Adult 

Erythrocytes were incubated with BAA at concentrations of 0, 1 or 2 mM. Complete Blood 

Counts (CBCs) and packed cell volume (spun haematocrit) were measured at 0, 1, 2 and 4hr 

after incubation. CBCs included white blood cell count, platelet count, RBC, HB, HCT, 

MCV, MCH and MCHC. 

Male rats and male Guinea Pigs were administrated EGBE at dose of 250 mg/kg. Blood 

samples were taken at 2, 8 or 24 hr after treatment for rats and 1, 2, 4, 8 hr for Guinea Pigs. 

CBC and spun HCT were performed at each time point. 

All three rodent species were relatively susceptible to the effects of BAA: It caused a time and 

concentration dependant increase in MCV and HCT. At high concentrations, swelling of 

RBCs was occasionally followed by a decline in the number of RBCs. Haemolysis was most 

significant in mouse blood incubated with the high concentration of BAA. The 2 mM dose of 

BAA produced greater than 45 % increase above the corresponding control in MCV at 2 hr, 

with a 6 % decrease below control in the RBC count. At 4 hr after incubation, MCV declined 

from the 2 hr levels but remained significantly higher than controls. The number of RBCs 

decreased but 30 % below the corresponding control levels. HB concentration declined in a 

manner parallel, but to a lesser extend, to the number of RBCs. MCV increased by greater 

than 15 % and 35 % above controls after incubation with 1 or 2 mM BAA for 4 hr. In 

addition, HCT increased in parallel to MCV. Although significant swelling of RBC occurred, 

no significant haemolysis was observed in hamsters. 
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For guinea pigs, there was no significant changes in MCV, HCT, RBC count or HB 

concentration in the blood of animals with vehicle, 1mM or 2mM BAA over the 4 hr study. 

Blood from rabbits exhibited an increase of MCV and HCT in a concentration and time 

dependent manner. At the 2 mM BAA, a greater than 20 % increase in the MCV was 

observed above the corresponding control at 2 hr which further increased to 39 % above 

control levels at 4 hr. HCT increased in a manner parallel to MCV and was time and 

concentration dependent. Despite this extensive swelling, the RBC count and HB 

concentration remained essentially unaffected throughout the incubation. 

Two species of carnivores were investigated for the haematological effects of BAA. Both 

canine and feline RBCs were found to be essentially insensitive to BAA. 

Pig cells exhibited no significant change in size or number. 

Two species of primates were investigated: baboon and human. In baboon blood incubated 

with BAA, MCV increased in a time and concentration dependent manner approaching a 35 

% above control increase after 4 hr incubation with 2 mM BAA. HCT increased in a fashion 

parallel to MCV and this increase was time and concentration dependent. The RBC counts 

declined slightly and this haemolysis was significant in blood incubated with either BAA 

concentration for 4 hr. In contrast, incubation of human blood with the high concentration of 

BAA demonstrated minimal or no effect. The changes in red cells size in the 2mM dose 

varied slightly from one human to another, with MCV increased of about 3.5 % above 

corresponding controls at 1, 2 and 4 hr. HCT also increased 2-4 % above controls in the 4 hr 

incubation period. The effects of BAA on MCV and HCT were not time dependent and were 

not statistically significant at any time point during the incubation. These minimal effects 

were not statistically significant and were not associated with any significant changes in RBC 

count or HB concentration. 

In vivo, gavage administration of EGBE caused an early increase in MCV and HCT in rat 

blood followed by a decline as a function of time. These effects wee associated with 

haemolysis and a decline in HB concentration and the number of RBCs. MCH and MCHC 

were affected in a manner reflective of the changes in RBC and HB. In contrast to the effects 

in rats, EGBE caused no significant changes in any parameters of guinea-pigs. These findings 

were in full agreement with the effects seen in the in vitro experiments. 

The effects of BAA on rat and human erythrocytes were also studied by Udden and Patton, 

(1994) and Udden (1994). Heparinised human blood was obtained from healthy volunteers 

and rat blood was obtained by cardiac puncture of male F344 rats. Erythrocytes were washed 

free of plasma, re-suspended in buffer to a packed cell volume of 10 % and incubated in 

buffer alone or buffer containing 0.2 or 2mM BAA (Shell Chemicals Development Co., purity 

99 %) for up to 4 hr. BAA at 0.2 mM did not cause haemolysis of rat erythrocytes, but the 

2mM concentration had a rapid effect resulting in ca. 30 % haemolysis after 4 hr. Addition of 

glucose to the medium gave no protection. In contrast, incubation of human erythrocytes for 4 

hr in the presence of 2mM BAA resulted in no haemolysis over that slight amount (< 1 %) 

owing to incubation of the cells in buffer alone. Rat erythrocytes also demonstrated decreased 

deformability (or increased cell rigidity), using a nucleopore filtration technique, and an 

increased mean cellular volume in the presence of 0.2 or 2mM BAA, whereas these effects 

were not observed with human erythrocytes. 

Study of the effects in vitro of BAA on human blood samples was extended to include 

heparinised blood obtained from 9 healthy adults (mean age 41.6 years, range 31 – 56; 5 men, 

4 women), 9 older people (mean age 71.9 years, range 64 – 79; 5 men, 4 women), 7 patients 
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with sickle cell disease and 3 people with hereditary spherocytosis (Udden and Patton, 1994). 

Erythrocytes were washed free of plasma, re-suspended in buffer to a packed cell volume of 

10 % and incubated in buffer alone or buffer containing 2mM BAA (Shell Chemicals 

Development Co., purity 99 %) for up to 4 hr. Erythrocytes from the younger healthy subjects 

showed no significant increase in haemolysis in presence of BAA compared to that observed 

in the incubations in buffer alone. There was a greater tendency for erythrocytes from the 

other volunteer groups to haemolyse more readily in buffer alone than erythrocytes from the 

younger healthy group. However, none of the groups showed an increase in haemolysis due 

to the presence of 2 mM BAA. Erythrocyte deformability was measured using a nuclepore 

filtration technique, but no changes due to BAA exposure was detected (Udden and Patton, 

1994). 

Mechanistic studies were performed by Udden and Patton, 2005 to elucidate the mechanism 

of BAA induced haemolysis. Red Blood Cells of Fischer 344 rats (9-11 week old) were added 

to the following culture medium: 10 mM TRIS, pH 7.4; 140 mM KCl and 10 mM glucose 

with or without 2 mM CaCl2. The RBCs were incubated in the presence of BAA at 37° C. In 

some experiments the inhibitor charybdotoxin (inhibitor of the Ca activated potassium 

channel) was added 30 minutes before the addition of BAA. 

To determine the effect of osmolarity, RBC were incubated with 1 or 2 mM BAA for 2 hours 

in the presence of 0 – 0.1 sucrose. Results are summarised in the table 4.64. 

Table 4.64: Effect of external sucrose on BAA-induced cell swelling 

RBC was determined before (MCVt0) and after (MCV2hrs) a two hour incubation with 0 (•), 

1 (c) or 2 mM () BAA in the presence of 0-0.1 M sucrose. The ratio MCV2hrs/MCVt0 is 

shown to demonstrate the relative decrease in cell swelling as a function of the increased 

external concentration of sucrose. The inset figure shows impedance size distributions 

determined by a particle counter for rat red blood cells incubated in buffer, buffer with 0.1 M 
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sucrose, 2.0 mM BAA, or 2.0 mM BAA with 0.1 M sucrose after 2 hour incubation and 

resuspension in isotonic saline without sucrose or additional BAA. 

Haemolysis as a result of incubation with 2.0 mM BAA and 0-0.1 M sucrose. 

Influence of K and Na content in the suspending medium was also assessed. Rat red blood 

cells were incubated for 4 hours in calcium free media with external sodium concentration 

varying from 0-140 mM. Potassium was added to achieve a total external cation concentration 

of 140 mM.Results are summarised in the figure 4.65. 

Rat red blood cells were incubated for 2 hours in the presence of 0-2.0 mM BAA in low 

sodium (-o-) buffer containing 110 mM potassium and 30 mM sodium or normal sodium (-•-) 

buffer containing 140 mM sodium Potassium was added to achieve a total external cation 

concentration of 140 mM.Results are summarised in the table 4.65. 

Table 4.65: Effect of replacement of external sodium with potassium on haemolysis and MCV changes induced by 

2.0 mM BAA. 

RBC were incubated in the presence of 2 mM BAA for two hours in the presence of 0-4 mM 

CaCl2 (-o-) or MgCl2 (-•-). Results are summarised in the table 3a. In another experiment 
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EGTA (Ca chelating agent ) (-o-) was added to the medium containing 2 mM CaCl2 (-•-). 

Results are summarised in the table 4.66. 

Table 4.66: Effects of divalent cathions and Ca chelation on BAA-induced haemolysis 

When rat red blood cells were incubated for 2 hours with BAA at concentrations between 0 

and 2 mM there was a dose related increase in haemolysis and MCV when calcium was 

present (-•-) (Figure 4a). The dose response for haemolysis showed a greater effect of BAA 

when calcium was absent (-o-). However, a dose response effect for BAA for increased MCV 

was not apparent in the cells treated in the absence of calcium (Table 4.67). 

Table 4.67: Dose response relationship for BAA induced haemolysis and cell swelling 
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The morphology of rat red blood cells suspended in calcium free or calcium containing buffer 

was similar. Incubation with BAA in the presence of calcium produced spherocytes. In the 

absence of calcium, spherocytosis was more prominent as was ghost formation and 

fragmentation of the cells. 

To confirm that the effect of Ca was to delay the onset of haemolysis, RBC were incubated 

with 1 (figures 68a and 68c) or 2 mM (figures 68b and 68d) BAA in the presence (-•-) or 

absence (-o-) of 2 mM Ca for four hours. 
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Table 4.68: Effect of Ca on haemolysis 

These figures show that Ca delayed the onset of haemolysis caused by BAA. During the first 

hour of exposure, the initial increase in cell size did not depend on the presence of absence of 

Ca (fig c and d). At the onset of significant haemolysis more cells were destroyed when Ca 

was absent and the MCVof the remaining cells appeared to be much smaller. 

An increased in RBC intracellular Ca was confirmed by measurements after exposure to 0-2 

mM BAA in the presence of Ca for 4 hours. 

To verify that effects were due to an activation of a specific Ca activated K channel (causing 

cells to lose K and prevent cell swelling – Gardos pathway), RBC were incubated with a 

specific inhibitor of this channel (charybdotoxin). Charybdotoxin in the absence of BAA did 

not cause haemolysis, but haemolysis was greatly increased in presence of BAA. When Ca 

was present in the middle containing charybdotoxin, the increase of haemolysis was 
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equivalent to the haemolysis seen in the previous experiment when there were no Ca in the 

middle (figures 4.68a and 4.68b). 

From these experiments, the authors suggested that the mode of action of BAA begins with an 

influx of sodium that is not fully compensated by potassium loss and ends with osmotic lysis 

of the red cell because of an influx of water accompanying sodium. Our data also points to a 

role for calcium entry in the red blood cells. 

Summary haematotoxicity 

In studies performed in vivo, the same signs of toxicity seen in acute toxicity studies (LD50 

studies) were recorded. Thrombosis were observed in various localisations sometimes leading 

to necrosis due to an infarction mechanism (tail, dental pulp, pulmonary, ocular). 

Mechanistic studies have shown that EGBE causes haematotoxicity in vivo in rats and that 

BAA causes the same effects in vitro at very low concentration. When metabolic pathways 

leading to the formation of BAA were blocked, no effects were seen on RBC. It can be 

concluded that BAA is responsible of haematotoxicity in vivo. 

Some species were very sensitive to EGBE- or BAA-induced haemolysis: rat, mouse, hamster 

baboon whereas other species were resistant to these effects: dog, guinea pig, pig, cat, rabbit 

and humans (30 x less sensitive than rats). In one study, dogs were very sensitive to EGBE 

but not to BAA. 

In these studies, an increased sensitivity to haemolysis was seen in old animals and in females 

in vitro and in vivo with BAA, showing that the differences of metabolism between male and 

female could not explain totally sex difference. 

In one study, it was demonstrated that the type of analyser was important to detect early 

changes in blood parameters (increase of HCT and MCV). An impedance based analyser was 

more sensitive than a laser based analyser. 

In vivo or in vitro, haemolysis was due to a decrease of erythrocyte deformability due to 

erythrocyte swelling (this also explains the formation of thrombosis). Newly formed 

erythrocytes were more resistant than old ones. It was also showed that EGBE pre-treatment 

gave a relative “protection” against higher doses administered later. Moreover, a study 

(Lomonova and Klimova, 1977) showed that repeated exposure to EGBE, 3 hours a day, 6 

days a week for 4 months was more haematotoxic than the exposure to the same dose of 

EGBE, 6 hours a day, 3 consecutive days a week for 4 month. This study demonstrates an 

adaptive mechanism of “protection” when animals have a period of recovery time before a reexposure to EGBE.

The mechanism leading to erythrocyte swelling and loss of deformability is for the moment 

unknown. Apparently, there is no evidence of oxidative mechanism on erythrocyte 

membrane. Udden, 2002 showed that, at low doses of BAA in rats, the increase of Na+

 intraerytrocytaire was not balanced by decrease in K+

. It has been demonstrated that Ca could also 

play a role in the mechanism. This mechanism lead to osmotic regulation causing an increase 

in the size and the cell volume of erythrocytes, a decrease of the density and of the 

deformability and an increase of the osmotic fragility. 

In humans, slight effects were seen with doses of 8 mM and 4 mM of BAA in vitro 

(Ghanayem, 1989). 
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For the effects of EGBE in dog (and the absence of effects when treated in vitro with BAA), 

another mechanism could be involved in EGBE induced haemolysis. 

4.1.2.3 Irritation 

4.1.2.3.1 Skin 

Studies in animals

Guinea pigs

Primary skin irritation was determined by application of the compound to the depilated 

abdomen of Guinea pigs at doses of 1, 5, 10 or 20 ml/kg (Eastman Kodak, 1981b). 

Skin responses were compared with those seen in rabbits in the same study. A dose of 0.3 

g/kg in rabbit lead to a moderate irritation whereas a dose of 4.5 g/kg in guinea pigs lead to a 

strong irritation. 

Rabbits

A skin irritation study was performed according to the method described in the US code of 

Federal Regulations (Huntingdon Life science, 1979b). EGBE was administered on the 

abraded and intact skin of 6 rabbits during 24 hours under semi-occlusive conditions. After 

exposure, scoring was performed (according to the Draize method) immediately and 48 hr 

after the first reading. 

At the first reading time, 5 out of 6 animals exhibited a slight to moderate erythema and 4 out 

of 6 exhibited a slight oedema. At the second reading time, 4 out of 6 animals exhibited a very 

slight to moderate erythema and 3 out of 6 exhibited a slight oedema. 
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Rabbit 24 hours 72 hours 

Intact skin Abraded skin Intact skin Abraded skin 

1 Erythema 0 0 0 0 

Oedema 0 0 0 0 

2 Erythema 1 1 2 2 

Oedema 0 0 0 0 

3 Erythema 1 1 0 0 

Oedema 1 1 0 0 

4 Erythema 2 2 1 1 

Oedema 1 1 1 1 

5 Erythema 1 1 1 1 

Oedema 1 1 1 1 

6 Erythema 1 1 1 1 

Oedema 1 1 1 1 

According to Draize scoring. 

In this study, EGBE can be considered to be slightly irritant to rabbit skin (primary irritation 

index: 1.5). 

EGBE was only mildly irritating to rabbit skin when applied and left uncovered for a 4 hr 

period on rabbit skin (Bushy Run Research Center, 1980b). More prolonged exposure to the 

undiluted chemical (24 hr) can lead to moderate to severe irritation characterised by erythema, 

oedema and necrosis (as seen in acute dermal toxicity test). 

EGBE and others glycol ethers were tested to assess their cutaneous irritation properties. 

(Zissu, 1995). Test substance (purity > 99 %) was administrated dermally to rabbits according 

2 test methods for assessing cutaneous irritation: the EEC testing method and the Draize 

protocol. Three New Zealand white rabbits were used in the EEC protocol and 6 in the Draize 

protocol. 0.5 ml of the test substance was applied occlusively on the flank of each animal, 4 hr 

for the EEC protocol and 24 hr for the Draize protocol (for the later, EGBE was also applied 

on the second scarified flank). Animals were observed for erythema and oedema. At 72 hr, 

histopathological control of the skin at the site of application completed the macroscopic 

observation. 

According to the EEC method, EGBE is classified irritant and according to the Draize 

method, EGBE is classified as severely irritant. These results were confirmed by histological 

observations. No detailed observations were available in this publication. 

Within the framework of the elaboration of a new EEC directive covering all preparations, 

different classes of substances were tested in different dilutions according to the experimental 

protocols and interpretation criteria laid down in Annex V and VI, part ID of directive 

79/831/EEC (Annex VI, 1983, Annex V, 1984) on dangerous substances. In Jacobs and 
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Martens, 1985 and 1987, EGBE and other ethylene glycols (purity > 99 %) were 

administrated dermally to rabbits according the EEC testing method. Five New Zealand white 

rabbits were used. 0.5 ml of EGBE was applied under an exposure chamber on the flank of 

each animal, during 4 hrs. Maximal mean erythema was 1.8 but the results per animal were 

very divergent. (From not irritating to very irritating). When diluted in polyethylene glycol 

400 (50%), no sign of any irritation was observed.

Summary on skin irritation: 

All the studies performed on rabbits and Guinea-pigs have shown that EGBE have caused 

moderate irritation (erythema and oedema) when applied occlusively on the skin for a period 

of 4 hours (in studies corresponding to the EU criteria for classification). If the substance was 

applied on scarified skin or for a longer period of time, signs of severe irritation sometimes 

leading to necrosis were reported. 

Overall, in animals, EGBE can be considered to be a skin irritant. Classification R38 is 

needed. 

4.1.2.3.2 Eye 

Animal studies

Marked irritation (Erythema and oedema) was seen when rabbits were instillated with one 

drop of pure EGBE (Von Oettingen and Jirouch, 1931). The corneal reflex was absent after 

treatment. 

In an old study poorly reported, EGBE produced only moderate irritation when 0.02 mL was 

instilled into a rabbit eye (Carpenter and Smyth, 1946). 

Eye irritation was assessed after administration of EGBE into a rabbit eye, pure or diluted 

(1/10 and 1/100) (Hoechst, 1966). Animals were examined 1, 3, 7 and 24 hours after 

application. 

For pure substance, corneal and conjuctival injuries were noted (severe redness and chemosis 

and corneal opacification). For the 1/10 dilution, only a slight conjuctival redness was seen. 

And for the 1/100 dilution, no effects were seen. 

An eye irritation study was performed according to the method described in the US code of 

Federal Regulations (Huntingdon Life science, 1979a). Six animals were instillated with 0.1 

ml of pure EGBE and observed during 72 hours. A scoring of the effects at 24, 48 and 72 

hours according to the method of Draize was performed. 

Effects on cornea, conjunctivae and iritis were seen in 5, 2 and 6 out of 6 animals at 

observation time of 24, 48 and 72 hours respectively and were not reversed after the 72 hr 

observation period. According to the EU classification system, EGBE could be considered as 

a severe eye irritant due to the irreversible effects seen at the end of the observation period 

although this period was only 72 hr. This classification rule is only relevant when no other 

data are available from other eye irritation tests for the period after 72 hours. This study 

cannot be relevant to support R41 classification. 

Instillation of 0.005 mL of undiluted EGBE into rabbit eyes produced severe corneal injuries 

with iritis (Bushy Run Research Center, 1980b). Moderate corneal injury was observed when 
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0.5 mL of a 15 % aqueous dilution of EGBE was instilled into the eyes of rabbits and no 

injuries was noted for a 5 % aqueous dilution. No more details about this study are available, 

the value of 0.005mL reported in the study is quite surprising in relation with the effects 

observed and the other doses tested. 

EGBE (99 % purity) was instilled in the right eye of 6 New Zealand white rabbits (Jacobs and 

Martens, 1987 “Evaluation of the enucleated eye test against the in vivo eye irritation test in 

the rabbit, Jacobs G. and Martens M.” IHE-report 01/12/1987). - Parent, 1992). The test was 

performed in two phases. The test was performed according to OECD guideline 405 (version 

1981). In the second phase, 3 other chemicals were tested and besides the clinical 

examination, corneal swelling was also measured. 

Erythema, chemosis, iritis and corneal opacity were scored according to the Draize scores at 

4, 24, 48, 72, 96 and 168 hr after treatment. The mean erythema, chemosis, iritis and corneal 

opacity values (average of the 24, 48 and 72 hour observation) are: 2. 59, 0. 78, 1.00 and 1. 

33, respectively. The mean corneal upperlayer damage (loss of epithelium measured by 

fluorescein retention on the cornea using a hand-slit lamp) was 95% after 4 hrs. Pain response 

was measured and described as “a few blinks only, normal within one or two minutes; 

animals didn’t squeal or rubbing the eyes”. The substance caused marked pannus in all 6 

rabbits. 

EGBE, according to this study, should be classified as an irritant. (mean erythema score >2.5, 

iritis =1). Pain sensation after contact with the product seems to be low, what can lead to 

underestimation of the irritating properties. Studies with fluorescein retention revealed a 90% 

loss of the upperlayer of the cornea after instillation. Generally speaking, the pannus reported 

does not disappear in rabbits and remain through the lifetime. In contrast, pannus response 

when seen in humans is usually associated with chronic disease and is not an acute irritative 

effect. Pannus generally disappears in humans. For those reasons pannus was not considered 

in the past as an irreversible effect for EC-classification however it must be noted that under 

the GHS it will be considered as leading to a classification of “severe irritating”. 

The study was redone some years later under the same protocol (Jacobs, 1992). The authors 

came to nearly the same mean scores (average of the 24, 48 and 72 hour observation of: 2.47, 

0.83, 0.83 and 1.73 respectively.), but since the mean erythema score and iritis score is lower 

than 2.5 and 1 respectively, no classification requirement is triggered. 

Table 4.69: Irritation scores in the Parent, 1992 Jacobs, 1992 study 

Time after end of application 4 hr 24 hr 48 hr 72 hr 96 hr 

Mean score of conjunctivitis (3 max) 1.4 2.4 2.6 2.4 1.6 

Mean score of chemosis (4 max) 1.7 1.0 0.8 0.7 0.2 

Mean score of iritis (2 max) 1.0 1.0 1.0 0.5 0.2 

Mean score of corneal opacity (4 max) 0.7 1.5 1.5 2.2 1.2 

Mean surface of corneal damage (100 % max) 95 94 81 60 23 

EGBE (99 % purity) was instilled in the eye of 3 rabbits (ECETOC, 1998). The test was 

performed according to OECD guideline 405 (version 1981) and GLP. Erythema, chemosis, 

iritis and corneal opacity were scored according to the Draize scores at 1, 2,3,7,14, 21 days 
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after treatment. The EC scores for erythema, chemosis and corneal opacity values (average of 

the 24, 48 and 72 hour observation) are 2.33, 2.78 and 2.33 respectively. Opacity resolved in 

all 3 animals althought very slight redness (score 1), resist in 2 of 3 animals and chemosis 

(score 1) in one animal. According to the scores a classification is needed as irritant. 

Six New Zealand white rabbit were tested in a Draize irritancy test with about 100 chemicals 

including EGBE (purity > 99 %) (Kennah et al., 1989). The cornea, iris and conjunctivae 

were scored at 24, 48 and 72 hr and at 7, 10, 14 and 21 day. Ketamine was injected 

intramuscularly just prior to measurement at a dose of 40 mg/kg to sedate the rabbits. Corneal 

thickness was measured before instillation and thereafter at intervals coincident with the 

Draize scoring. Three measurements were made on each cornea and averaged. Results were 

expressed as swelling: corneal thickness on observed day / corneal thickness prior to 

instillation x 100. (a 100% swelling value represents no change). 

Draize score obtained for EGBE are summarized in table 4.70 

Table 4.70: Draize scores in the Kennah study 

concentration Draize score % swelling (%- value untreated cornea)

100 66, severely irritating, resolution of 

ocular irritation within 14 days. 

181 (81) 

70 49, moderately irritating 146 (46) 

30 39, moderately irritating; relative 

high scores are due to conjunctival 

damages 

114 (14) 

20 2, minimal irritating 113 (13) 

10 1, minimal irritating 91 (-9) 

Based on all the substances studied, a significant linear correlation was established between 

Draize score and % swelling (r = 0.86). A great majority of chemicals showed agreement 

between the Draize score and the corneal swelling when compared with class irritancy rating 

(mild, slight, moderate, severe, extreme). 

For EGBE, Draize scoring was more severe than % of swelling. These differences are due to 

conjonctival damage, more heavily weighted by Draize scoring. But, swelling seems to detect 

more persistent irritation than Draize score. Resolution of ocular irritation was within 14 days. 

Draize score is a “weighted scoring system involving multiplication factors” on a mix of all 

effects reported (e.g. discharge+chemosis+redness+opacity,+area of opacity and iritis). It is 

not possible to convert this scoring as such to the EC system. 

In a similar study over 32 substances (Jacobs and Martens, 1989) using the EC method of 

calculation, a better linear correlation was obtained when comparing specific effects like 

corneal swelling and corneal opacity (r=0.94, n=32) as well as corneal swelling and erythema 

(r=0.93, n=32). When all eye lesions are taken into account, a threshold value of corneal 
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swelling of 60 (untreated cornea is set to 0%) can be accepted for classification purpose. For 

EGBE, the mean value of corneal swelling over 3 animals and 24,48,72 hrs was 70%. (In this 

study the observed pannus was not taken into consideration for the threshold value). 

Application of this threshold value for classification (60% of corneal swelling) to the Kennah 

study results in: 

EGBE (>99% pure) ; Irritating (Xi, R36) 

EGBE (70%), EGBE (30%), EGBE(20%), EGBE (10%) not classified 

In a paper reporting only the results of some studies, the instillation of a 5 % solution of 

EGBE according to Draize procedure did not exhibited any irritant properties (Bagley, 1994) 

A GLP study was performed to assess the eye irritation potential of EGBE according to 

OECD guideline 405 (Safepharm laboratories, 1994b). 6 New Zealand white rabbits were 

instilled 0.1 ml EGBE into the conjunctival sac of the right eye. Amounts of ocular 

damage/irritation were noted 1, 24, 48 and 72 hr following treatment. Additional observations 

were made on days 7, 14 and 21 to assess the reversibility of the ocular effects. No 

information on test substance purity was included in the study report. The eyes were not 

rinsed 24 hours following treatment. 

Areas of corneal opacity were noted in all treated eyes on 24 and 48 hr after treatment. 

Reversibility was not obtained in 21 days for 1 animal. Iridial inflammation was noted in all 

treated animals 1, 24 and 48 hr after instillation. Reversibility was obtained in 14 days. 

Moderate conjunctival irritation was observed at 1, 24 and 48 hr. Effects were present till 

D14. Petechial haemorrhage was seen in some animals at each observation time from 1 to 72 

hr. Ectropion was noted from 72 hr observation time in some animals and not reversible for 1 

animal at the end of the study. One animal showing signs of discomfort was therefore killed 

for humane reasons immediately after the 14th day. The scores are shown in the table below. 

Table 4.70 bis: Individual score for Safepharm laboratories study, 1994b) 

Animal Time (hrs) Opacity Iritis Redness Chemosis 

24 1 1 2 2 

48 1 1 2 2 

72 1 1 2 2 

1 

7 day 0 0 0 0 

24 1 1 2 2 

48 1 1 2 2 

72 1 1 2 2 

2 

14 days* 2* 1 2 2 

24 1 1 2 2 

48 1 1 2 2 

72 1 1 2 2 

3 

7 days 0 0 0 0 

24 1 1 2 2 

48 1 1 2 2 

72 2 1 2 2 

4 

21 days 2* 0 0 0 

24 1 1 2 2 

48 1 1 2 2 

72 2 1 2 2 

5 

14 days 0 0 0 0 

24 1 1 2 2 

48 1 1 2 2 

72 1 0 2 1 

6 

7 days 0 0 0 0 

* vascularisation. Animal 2 sacrificed after observation at 14 days
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EGBE is considered to be a severe irritant to the eye in this study. 

EGBE 5 % in water solution was tested in various in vitro assays: SIRC cytotoxicity assay, 

Neutral red uptake assay (48hr), cell protein assay, fibroblast cytotoxicity assay, corneal 

plasminogen activator assay (CEPA) (Harbell et al., 1997). 

Given that these assays are not yet (or will never be) validated, the results obtained cannot be 

used in this risk assessment. (However, the average Draize score obtained in vivo can be used: 

2.7). 

In a GLP study performed according to OECD guideline 405, 3 New Zealand white rabbits 

were instilled with EGBE (purity 99.6 %). Animals were observed 21 days (BASF, 2000). 

Eye washing was performed about 24 hours after treatment, before the 24 hour reading. 

One animal exhibited a slight corneal opacity reversible in 21 days. Signs of a slight iris 

injury was described, reversible in 7 days. A medium to severe irritation of the conjunctivae 

was also observed and was reversible in 21 days. According to criteria for assessment of 

ocular lesions, EGBE can be considered as an eye irritant. The individual animal scores are 

shown below: 

Table 4.70 ter: Individual score for BASF study, 2000 

Animal Time (hrs) Opacity Iritis Redness Chemosis 

24 1 0 2 2 

48 1 0 2 2 

72 1 1 3 2 

1 

14 days 0 0 0 0 

24 0 1 3 2 

48 1 1 3 2 

72 1 1 3 2 

2 

21 days* 0 0 0 0 

24 1 1 3 2 

48 1 0 2 1 

72 1 0 2 1 

3 

21 days 0 0 0 0 

Human data: 

After exposure to 200 ppm of EGBE, immediate irritation of the nose and throat, followed by 

ocular irritation and disturbed taste was experienced by all three subjects (Mellon Institute of 

Industrial Research, 1955). 

Summary of eye irritancy data 

A lot of studies have been performed to assess the eye irritation properties of EGBE. Most of 

them were not performed according to guidelines, but overall, all studies have shown that 

EGBE is irritant or severely irritant to the eyes of rabbits with effects both on conjunctivae, 

iris and cornea. In one well performed study EGBE produced irreversible effects on the 

conjunctivae and on the cornea in at least one treated rabbit. According to the EU 

classification system, these effects would trigger a classification R41, “severely irritant to 

eyes” because of the irreversibility of the effects at the end of the observation period. But 

these studies were performed according “old” guidelines. In the new guidelines, it is possible 

to rince the treated eye for animal welfare purposes and to more closely simulate the human 

situation where lacrimation would clear the substance from the eye. In a test, performed 
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according the recent guidelines, EGBE was only irritant (R36). This result was given 

precedence by the C&L group who confirmed a classification of R36. 

It was also demonstrated that dilution of EGBE in water decrease its irritant properties as well 

as rinsing of the eyes in case of exposure. 

4.1.2.3.3 Respiratory tract 

Animal data: 

Sensory irritation was evaluated using an in vivo method with mice (Kane et al., 1980). Male 

Swiss-Webster mice (4/group) were exposed whole body to a series of chemicals (including 

EGBE). The measured response was the maximum percent decrease in respiratory rate, 

averaged over 4 mice, simultaneously exposed for 10 minutes. The responses obtained for 

various concentrations of solvents were utilized to develop a concentration-response 

relationship, for this, the concentration associated with a 50 % decrease in respiratory rate 

(RD50) was calculated. 

Time response relationship: after an initial decrease observed within a few seconds and 

characterized by a pause during the respiratory rate, a phase of recovery occurred but 

respiration rate was lesser than normal until the end of the exposure period. 

The response obtained within the concentration range tested was less than 50 % decrease in 

respiratory rate. The RD50 calculated by extrapolation was 2825 ppm (confidence limit 1695 

– 7278). These values are well in excess of the saturated vapour pressure. When compared 

with the criteria of evaluation, 0.01 RD50 (about 28 ppm) would cause minimal or no sensory 

irritation whereas 0.1 RD50 (about 280 ppm) would cause definite but tolerable sensory 

irritation. 

An estimate of the RD50 of EGBE was reported using modelling based on physicochemical 

properties (Alarie et al., 1995). Calculated log RD50 was 3.45 (2818ppm). This is in good 

agreement with the experimental data. 

Human data: 

Three volunteers were exposed to 100 and 200 ppm of EGBE for periods of 2 or 4 hours, 

separated by a 2 hour period of non-exposure (Mellon Institute of Industrial Research, 1955). 

Immediate irritation of the nose and throat, followed by ocular irritation and disturbed taste 

was reported by all three subjects Whether such ‘irritation’ was physiological or merely 

discomfort is not clear.. 

Some human studies by inhalation for toxokinetic studies (Johanson et al., 1986a, Kumagai et 

al., 1999) have shown that EGBE was not irritant to the eyes or respiratory tract at doses of 20 

ppm during 2 hours or 25 ppm for exposure periods of 10 minutes. A study performed by 

Johanson and Bomanin 1991 in which human volunteers were exposed to 50 ppm EGBE 

whole body (but breathing pure air) and mouth only did not describe irritant effects, but 

according to the description of the study, this kind of effects were not recorded. 

In a recent study (Jones et al., 2003) no signs of irritation were reported after exposure to 50 

ppm EGBE (see § 4.1.2.1.2: Four volunteers (2 males and 2 females, aged 28-33) were 

exposed on 9 separate occasion, each occasion separated by at least 3 weeks. The exposure 

was 50 ppm EGBE for 2h. To record any physiological changes in the volunteers under the 

different conditions, physiological monitoring equipment was used.). The published study 
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paper does not mention if these signs were checked except the recording of the physiological 

changes. However, the author did indicate in a written communication that the volunteers 

were asked to report any adverse effects and none were reported (Jones, personnal 

communication). 

Summary respiratory irritation: 

Animal studies available (including repeated dose toxicity studies performed by inhalation on 

rats and mice) did not show any signs of significant respiratory irritation. No classification is 

required for this end point. 

From the human data it is apparent that the NOEC is >50ppm whilst the NOEC (based on 

effects of discomfort) is <100-200ppm. A NOEC of 50ppm is taken forward for risk 

characterisation. 

4.1.2.4 Corrosivity 

According to EU classification criteria EGBE cannot be considered as a corrosive substance. 

Some signs of necrosis were seen in some studies but only when animals were exposed intradermally or for a long period of time by occlusive wrapping. 

4.1.2.5 Sensitisation 

4.1.2.5.1 Studies in animals 

Skin

In vivo studies 

In a GLP study performed according to OECD guideline 406, EGBE was tested in Guinea 

Pigs for sensitisation potential (Unilever, 1994). Induction was made with a dermal injection 

of 0.5 % EGBE in 0.9 % saline and a topical application of a 25 % EGBE in 0.9 % saline. 

Challenge was made with a topical application of 10 % EGBE in saline. All topical 

applications were made occlusively. Concentrations tested were based on results found in 

preliminary studies (showing a Maximal Concentration with No Irritation (CMNI) of 10 %). 

Two challenge applications were made at one week interval. 

No evidence of sensitisation was seen in treated animals both at challenge and re-challenge. 

EGBE was tested in a Maximised Magnusson & Kligman test (Zissu, 1995). After induction 

(test substance concentrations not reported) a 1 % concentration of EGBE was used for 

challenge. 

No animals exhibited signs of sensitisation. 
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4.1.2.5.2 Studies in humans 

Skin

In a GCP study, the sensitising potential of 10 % (v/v) EGBE in aqueous solution was 

assessed in human on 201 volunteers (TKL Research, 1992). Induction phase consisted in 9 

consecutive occlusive application of EGBE. Patches were removed 24 hr after application. 

Assessments of the sites of application were made at 48 hr intervals and after new patches 

were reapplied. Following the ninth evaluation, the subjects were dismissed for a 14-day rest 

period. The challenge phase was initiated during the sixth week of the study, identical patches 

were applied to sites previously unexposed to EGBE. They were removed after 24 hr of 

exposure and the sites graded after additional 24 hr and 48 hr periods. Re-challenge if 

required was to be performed whenever there was evidence of possible sensitisation. The rechallenge was conducted on naïve sites on the back under both occlusive and semi-occlusive 

conditions approximately one or two weeks after challenge had been completed. Patches were 

applied for 24 hr, the patches removed and the sites evaluated 48, 72 and 96 hr after patches 

applications. 

Only 7 of the 48 hr evaluations and 12 of the 72 hr evaluations showed slight erythema. One 

subject at the 72 hr reading had definite erythema. More severe responses which would have 

been expected if delayed contact sensitisation had occurred were not observed. In conclusion, 

according to the authors, none of the 201 subjects completing the study showed evidence of 

sensitisation. Re-challenge was not required for any subject. 

Two cases of dermatitis herpetiformis (DH) temporally related to the use of a cleaning 

solution containing EGBE (Snider and Maize, 1993). Analysis of the mixture showed the 

presence of EGBE, trisodium phosphate, nonylphenol polyethylene glycol ether, 

anthraquinone, lemon fragrance and an antifoam agent. Patch testing with the individual 

chemicals at the same concentration than in the mixture (except for EGBE which was diluted 

to avoid irritation) and at the same time precipitated a flare of DH in both patients. Causal 

agent remained unknown because each individual ingredient was not separately patch tested. 

These results are hardly reliable for EGBE risk assessment because of co-exposure. 

Respiratory tract

Considering SAR in the glycol ether family, the wide dispersive use of them and that no 

glycol ether has even been associated with cases of respiratory sensitisation, it can be 

considered that this toxicological property cannot be expected and is not relevant for risk 

assessment. 

4.1.2.5.3 Summary of sensitisation 

No signs of skin sensitisation were seen in two animal studies or in a human patch test. 

Moreover, considering Structure Activity Relationship (SAR) in the glycol ether family, the 

wide dispersive use of EGBE and that EGBE has never been associated with cases of skin 

sensitisation, it can be considered that skin sensitisation cannot be expected and is not relevant 

for risk assessment. No classification is needed for this end point. 
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4.1.2.6 Repeated dose toxicity 

4.1.2.6.1 Studies in animals 

Inhalation 

Rat

Three groups of 23 Wistar rats were exposed to EGBE in an inhalation chamber at 

concentrations of 0, 135 and 320 ppm (0, 652 and 1546 mg/m3

), 7 h/day, 5 day/week for 5 

weeks. Eight animals/group were used for pathological examination and were sacrificed by 

pair after 1, 3 and 5 weeks of exposure (Werner et al., 1943b). The remaining animals were 

used for growth and haematological studies. Haematological examinations were made before 

– during and after the exposure (week 0-2-4-6-9) and consisted of red and white cell counts – 

differential counts, reticulocytes count and Hb estimations. Three weeks after the end of 

exposure and after a last blood analysis animals were sacrificed and organs examined (liver, 

kidney, heart, spleen and lung). 

No effects on body weight gain were seen in either group of EGBE treated animals. No 

mortality or signs of systemic action were seen in the group of treated animals. The 

erythrocyte counts and haemoglobin concentration were significantly decreased at the end of 

1 week of exposure to the high concentration of EGBE. Circulating reticulocytes were also 

increased over the same period. 

For this study, transient effects on haematological parameters were seen at the high dose. The 

No Observable Adverse Effect Concentration (NOAEC) was 135 ppm based on transient 

haematological effects at 320 ppm. 

Mortality of rats after two 8-hr exposures to 500 ppm (2415 mg/m3

) of EGBE was 3/6 

animals (Mellon Institute of Industrial Research, 1952). At 250 ppm (1208 mg/m3), 0/6 

animals died after or during six 8 hr exposure whereas 1/5 animals died after or during four 8-

hour exposure. No deaths occured when 6 animals were exposed to six 8-hour exposure of 

125 ppm (604 mg/m3

) EGBE. No effects were seen in the control group. Due to the 

experimental conditions, this study cannot be taken into account for the Risk Assessment. 

Groups of Alderly Park rats (4 males and 4 females/group) were exposed (whole body) to 

various concentrations of EGBE and for different periods of time (Gage, 1970). Rats were 

maintained in the exposure chamber for periods of up to 6 hours, and between repeated daily 

exposures they were returned to their cages where food and water were freely available. In the 

initial experiments the concentrations were selected to produce, if possible, acute effects after 

short exposures. Thereafter the exposure period was extended and the concentration lowered 

until the animals could survive 6-hour exposures, five days a week, for up to four weeks. With 

liquids, the concentrations which could be tested in these acute and subacute experiments. The 

rats were weighted and examined each morning, and their conditions and behaviour were 

recorded throughout the exposure period. Urine was collected overnight after the last 

exposure day for biochemical tests. Blood was collected during euthanasia for haematological 

tests. After a gross examination of the organs, the lung were collected and examined. The 

following organs were also taken for microscopic examination: lungs, liver, kidneys, spleen, 

adrenals and occasionally heart, jejunum, ileum and thymus. 
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Table 4.71: Experimental pattern of exposure for Gage study, 1970 

Dose Number of exposures Symptoms 

250 ppm 

(1208 mg/m3) 

4 x 6-hr exposures initial haemaglobuniria and lethargy, low HB 

and MCHC, weight loss 

100 ppm 

(483 mg/m3) 

15 x 6-hr exposures no toxic signs, urine and blood tests normal 

apart from increased red cell osmotic 

fragility: autopsy, organs normal 

50 ppm 

(242 mg/m3) 

15 x 6-hr exposures as 100 ppm experiment 

20 ppm 

(97 mg/m3) 

15 x 6-hr exposures no toxic signs, blood normal: autopsy, organs 

normal 

In this study, the Lowest Observable Adverse Effect Concentration: LOAEC based on 

haematological signs was 50 ppm (15 exposures) leading to a NOAEC of 20 ppm. Other 

effects than those seen on haematological parameters were seen only at 250 ppm (4 

exposures) 

Eighty five rats divided in three groups were exposed to EGBE (purity unknown) at doses of 

0 or 17 ppm (84 mg/m3

). The first group was exposed 3 hr day on 6 days per weeks and the 

second group was exposed to 6 hr/day on 3 consecutive days per week (Lomonova and 

Klimova, 1977). The third group served as control. It is noted that this study was not 

conducted according to GLP ans was poorly reported. Body weight of animals was measured 

regularly. A standardised battery of general parameters and the level of blood 

cathecholamines were checked. For specific toxicity, blood parameters and erythrocyte 

fragility tests were performed. Catalase activity was also measured. Some functional tests 

were also performed: hexenal test, “cold test” and water maze. After the 4 months of 

exposure, an unique dose of 800 mg/m3 of EGBE was administered to assess the adaptative 

effects (if any) of regular administration of small doses of EGBE. 

Overall, the results of all parameters assessed has shown that the toxicity of EGBE was more 

severe in the first group (3 hr/d 6 days/week) than in the group 2. Moreover, the charge dose 

of 800 mg/kg administered at the end of the 4 month exposure period enhanced less effects in 

the second group than in the first one. Significant toxic signs were limited to haemolysis and 

narcosis. Due to the experimental conditions and the specificity of the test, data obtained can 

only be qualitative and therefore is not suitable for risk characterisation. No NOAEC can be 

derived. 

Groups of Fisher 344 rats (8 animals per sex in each group with an addition of 7 females to 

the high exposure and control groups, 7 males to the control group and 8 males to the high 

exposure group - the additional animals were used to determine, if necessary, the reversibility 

of any effect - these animals were observed during 14 days after the last exposure) were 

exposed to 0, 20, 86 and 245 ppm (0, 97, 415 and 1183 mg/m3

) of EGBE 6 hr/day for 5 

consecutive days –followed by 2 days without exposure and then 4 days of additional 

exposure (Bushy Run Research Center, 1981a). All animals were observed before, during and 

following exposure for any signs indicative of toxic effects. The appearance and demeanor of 
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all animals were recorded at these times. The modified Irwin Screen test was performed on 

five rats per sex from the 245 ppm exposure group and five rats per sex from the control 

group for signs indicative of behavioural and/or neuromuscular abnormalities. Animals were 

observed on exposure days 1, 2, 5, 6 and 7 prior to following exposure and preceding 

sacrifice. During the post-exposure period, reversibility groups were screened on days 4, 7 

and 14. Evaluations were made on the following: corneal response, pupil response, tail pinch, 

toe pinch, righting reflex, locomotor activity, impaired gait, respiration, tremors, convulsions, 

salivation, piloerection, diarrhea, tail elevation, lacrimation, stereotypy. All rats had a 

ophthalmologic examination prior to group assignments. Rats were culled if abnormalities 

were observed. Except for reversibility groups, an eye examination was also done on all rats 

at sacrifice. The body weight for each rat was determined on the morning preceding the 1st

, 

2nd, 5th, 6th and 7th exposure days and again prior to sacrifice. During the post-exposure period 

the body weights of the reversibility group were recorded on post-exposure days 4 and 7 and 

prior to sacrifice, day 14. Haematological tests were performed on blood samples collected 

from all rats on the day prior to sacrifice. The analysis included: haemoglobin, haematocrit, 

erythrocyte count, white blood cell count, mean corpuscular volume, mean corpuscular 

haemoglobin and mean corpuscular haemoglobin concentration. Reticulocyte and white blood 

cell smears were made for each animal, but were only evaluated for high exposure and control 

animals. All rats were killed for necropsy on the day following the final exposure (except the 

rats of the reversibility groups which were sacrificed on the 14th day following the final 

exposure. Liver, lungs, kidneys and testes were weighed. 

No deaths occurred during the study. After the first and second exposure, only red staining of 

urine was seen in animals of the high dose group. A slight increase over the control values for 

audible respiration and nasal discharge were observed in male rats of the 245 ppm group. 

Urogenital area wetness was observed in a few females of the 245 ppm exposure group. Male 

rats in the 245 ppm or control groups appeared normal during the modified Irwin Screen 

testing sessions. One female rat of the 245 ppm exposure group exhibited an abnormal 

righting reflex following exposure. A decrease in body weight gain was observed for animals 

treated with 245 ppm and for females treated with 86 ppm. During post exposure observation, 

animals recovered and no differences were seen with controls at post exposure day 14. 

Erythrocyte count, haemoglobin and MCHC values were significantly lower in males and 

females of the 245 ppm group compared to controls. These animals also exhibited a 

significant increase in MCV, nucleated red blood cells, reticulocytes and in males only, 

lymphocytes. Haematological values in both males and female rats exposed to 86 ppm were 

significant for haemoglobin (decrease) and MCV (increase). The females of the 86 ppm group 

also had significant decreases in MCH and MCHC and an increase in the haematocrit values. 

All effects seems to be dose-related. No haematological effects were seen in the 20 ppm 

group. In the reversibility group, animals exhibited a substantial recovery following 14 day 

post exposure period. However a significant decrease in erythrocyte count (male only) and a 

significant increase in MCV (all animals) and in MCH (male only) were still present in 

treated animals. The leukocyte count returned to control value during this recovery period. 

The mean liver weight values (relative and absolute) for the female of the 245 ppm group 

were higher than the controls. In addition, the relative liver weight for the females of the 86 

ppm group and for the males of the 245 ppm group were higher than the controls. The mean 

relative testes weight for the males of the 86 ppm group was lower than controls, this can be 

considered to be an isolated finding not related to the treatment. After 14 day recovery period, 

no significant differences from controls in male or in female organ weight were observed. At 

necropsy, no finding could be specifically related to EGBE exposure. In this study, a NOAEC 

of 20 ppm can be indentified, according to the effects seen on haematological parameters at 

86 ppm. 
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EGBE (purity 99.4 %) vapour was administered by whole body exposure to Fischer 344 rats 

at concentrations of 0, 5 – 25 or 77 ppm 0, (24 – 121 – 372 mg/m3), 6 hr/day, 5 day/week for 

approximately 13 weeks (16 males and 16 females/group) (Bushy Run Research Center, 

1981b). Animals were observed for signs of toxicity before, during and after exposures. A 

modified Irwin screen test was performed on 5 rats/sex/group from the high concentration and 

controls (see 9 day study for details, ref: Bushy Run Research Center, 1981a) and a sub-group 

was killed after 2 weeks (9 exposures) for interim examination. Body weight determination 

were performed regularly. Food consumption was determined weekly. All rats had an 

ophthalmologic examination prior to group assignments, male rats of the 77, 25 ppm and 

controls were re-examined prior to terminal sacrifice. Only the females of the 77 ppm group 

were examined. Haematologic tests were performed on blood samples collected from all rats 

on the day prior to sacrifice. Clinical examinations included urinalysis, haematology and 

serum clinical chemistry. Urine from rats of the high exposure group was analysed for 

haemoglobin and red blood cells. Urine from the same 5 rats per sex was analysed prior to 

exposure for the first 5 exposure days and prior to exposure each Wednesday thereafter. Urine 

was also collected and a more comprehensive analysis was performed one week prior to the 

terminal sacrifice from all rats. An interim sacrifice was performed on 6 rats per sex per 

exposure level. All remaining rats were necropsied at the final sacrifice on the day following 

the last exposure. After sacrifice, a gross dissection and an evaluation were performed on each 

animal. The following organs were weighed at terminal sacrifice: liver, kidneys, lungs, and 

testes. No organ weights were obtained for the rats at the interim sacrifice. At the time of the 

necropsy, histopathological examinations were performed on the usual tissues selected for 

such studies. 

No death occurred related to treatment. No effects were seen concerning ophthalmologic 

examination and Irwin Screen test. For rats that were killed at the interim sacrifice time, after 

9 exposures, the females of the 77 ppm group had lower body weight gain values comparing 

to the controls. No differences were found among the females of the lower exposure groups or 

males of any exposure group. For the main group, similar results were obtained for the same 

periods of time. However, following this time period the depression in body weight gain 

noted in the females was reversible and returned to values similar to the values of the control 

group. The decreases of food consumption are consistent with the changes noted in body 

weight gain. Slight signs of haemolytic anaemia were seen in females exposed to high doses 

of EGBE. Subtle haematological effects noted in females were characterised by minimal 

depression of red blood cell counts, haemoglobin and haematocrit with an attendant slight 

increase in MCH. These effects were noted at the interim sacrifice time and persisted 

throughout the study without increasing in severity. There was no indication of red blood cells 

or haemoglobin in the urine collected daily during the first exposure week or weekly 

thereafter from the male and female rats of the high exposure level. Moreover, there was no 

treatment related alteration in erythrocyte fragility at either the interim or terminal sacrifice 

times. No other haematological, urinary or clinical chemistry findings of toxicological 

significance were observed among the rats. Several incidental lesions were present in various 

organs, however no treatment-related, gross or microscopic lesions were found in either the 

male or female rats sacrificed at the end of the study. On organ weights, the only significant 

finding was lower relative kidney weight among the males of all treated groups. This finding 

is not believe to be related to the treatment. 

The NOAEC was 25 ppm, based on the non-progressive haematological effects in females 

and a transient reduced body weight gain at two weeks seen in females of the 77 ppm dose 

group. 
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EGBE was administered to groups of 10 male and 10 female F344/N rats by whole-body 

exposure to 0, 31, 62.5, 125, 250 or 500 ppm (0 – 150 – 302 – 604 – 1208 – 2416 mg/m3) 

EGBE vapour (lot no. QP-921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 

%) for 6 h per day, 5 days per week for 14 weeks (NTP, 2000 ; Nyska et al., 1999a ; Long et 

al., 2000). 

Rats were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All rats were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study and then weekly until the 

end of the experiment. Clinical observations also were recorded weekly throughout the 

experiment. Haematological examinations were performed at the end of the experiment, using 

an Ortho ELT-8/ds 9000 analyser. Differential leukocyte and nucleated erythrocyte counts 

were made by microscopic examination of blood films 

Autopsies were made on all study rats. Organ weights measured were for the heart, right 

kidney, liver, lungs, right testis and thymus. A complete histological examination was 

performed on the chamber control and 500 ppm males and females and on the 250 ppm 

female rats. 

Six female rats were found moribund and killed during the study: five in the 500 ppm group 

(four in week 1, one in week 5) and one in the 250 ppm group (in week 8). By the end of the 

study, body weight gains were significantly reduced in females of the 500 ppm group, but 

were unaffected in all other groups. Clinical findings were most prevalent in rats of both sexes 

exposed to 125, 250 or 500 ppm and included abnormal breathing, pallor, red urine stains, 

nasal and eye discharge, lethargy and either increased salivation or lacrimation. All females of 

the 500 ppm group, particularly during the first two weeks, developed alternating blue and 

white bands on their tails that caused them to self-mutilate and loose the distal portion of their 

tails. 

Haematological examination showed that inhalation of EGBE resulted in the development of 

a persistent and exposure-related macrocytic, normochromic, responsive anaemia, as 

indicated by decreased haematocrit values, haemoglobin concentrations and erythrocyte 

counts in the 125 ppm or greater group males and in all groups of exposed females. The 

effects were dose related. This evidence of a sex difference in the severity of the anaemia was 

also seen in the 500 ppm group, in which the indicators were slightly more severe in the 

females than in the males. Evidence of an erythropoietic response was shown by increases in 

reticulocyte and nucleated erythrocyte counts in males of the 125 ppm or greater groups and 

females of the 62.5 ppm or greater groups. Other haematological changes were decreases in 

lymphocyte and monocyte counts in males of the 125 ppm or greater groups and increased 

platelet counts in females of the 125 or 500 ppm groups. Some organ weight changes were 

observed. These were: increases of the kidney of males in the 500 ppm group and females in 

the 125 ppm or greater groups; increases of the liver of males in the 250 or 500 ppm and 

females in the 125 ppm or greater groups; and decreases of the thymus of females in the 500 

ppm group. Thrombosis was observed in the tail vertebrae, femur, incisors, nasal cavity, lung, 

heart and liver of most females in the 500 ppm group, but not at all in the 250 ppm group. 

Additionally, in the 500 ppm group females, there was degeneration of the hepatic 

centrilobular areas and renal tubules (4/5 in each case) and atrophy of the thymus (4/5) and 

spleen (1/5). Haematopoietic cell proliferation was observed in the spleen of 2/5 group same 

group. Bone marrow hyperplasia was recorded in males of the 250 and 500 ppm groups and 

females in all groups from 62.5 ppm. The severity of this response was dose related In the 

forestomach of females in the 500 ppm group, but not in lower dose groups, observations 
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made were of inflammation (3/5), necrosis (2/5), ulcers (2/5) and hyperplasia (1/5). Effects 

also observed in the 250 ppm group females were hepatic necrosis, pigmentation of Kupffer 

cells and renal tubule cells and bone marrow hyperplasia (NTP, 2000). No NOAEC was 

found for female rats. LOAEL was 31 ppm based on haematological effects seen at all doses 

tested.A NOAEL of 62.5 ppm was found for male rats, based onhaematotoxic effects seen at 

125 ppm.. 

EGBE was administered to groups of 50 male and 50 female F344/N rats by whole-body 

exposure to 0, 31.2, 62.5 or 125 ppm (0 – 150 – 302 – 604 mg/m3

) EGBE vapour (lot no. QP921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %)for 6 hr per day, 5 days 

per week for 104 weeks. For haematological and bone marrow analyses, additional groups of 

27 male and 27 female F344/N rats were exposed to 0, 62.5 or 125 ppm for evaluation at 3, 6 

and 12 months and 9 male and 9 female rats were exposed to 31.2 ppm for 3 months (for 

haematological examination only) and 6 months (NTP, 2000). 

Rats were held in the testing laboratory for 18 days and were 7 – 8 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All rats were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study, after which they were 

recorded monthly from week 5 until week 89 and then every two weeks from week 92 until 

the end of the experiment. Clinical observations also were recorded monthly from week 5 

until week 89 and then every two weeks until the end of the experiment. Haematological 

examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 6-months and on 

a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated 

erythrocyte counts were made by microscopic examination of blood films. 

Autopsies and microscopic examinations were made on all of the main study animals that had 

not been used for blood and bone marrow sampling. Organ weights were not measured. At 

autopsy, all organs and tissues were examined for grossly visible lesions and all major tissues 

were fixed and processed for histological examination. A quality assessment pathologist 

evaluated the slides from all tumours and potential target organs, which included bone 

marrow, forestomach, kidney, liver, lung, nose and spleen of males and females and the 

adrenal and clitoral glands of females. The slides and the preliminary quality assessment 

report were then submitted to a National Toxicology Program Working Group of pathologists. 

Survival of exposed male and female rats was similar to the chamber control groups, the mean 

numbers of survivors at the end of the experiment in each of the 0, 31.2, 62.5 and 125 ppm 

groups were 19, 11, 21 and 24 males and 29, 27, 23 and 21 females, respectively. No clinical 

signs were attributed to EGBE exposure. Body weights of male and female rats in the 31.2 

and 62.5 ppm groups and male rats of the 125 ppm group were generally similar to the 

controls throughout the experiment. Body weights of female rats of the 125 ppm group were 

generally lower than those of the chamber control from week 17 until the end of the 

experiment. 

Neoplastic effects observed in these experiments are described in section 4.1.2.8.1.3. 

Haematological examination showed that inhalation of EGBE resulted in the development of 

a persistent and exposure-related macrocytic, normochromic, responsive anaemia, as 

indicated by decreased haematocrit values, haemoglobin concentrations and erythrocyte 

counts. These changes occurred at 3, 6 and 12 months in 62.5 ppm group females and 125 

ppm group males. Some anaemia also occurred at 3 and 6 months in the 31.2 ppm group 

females and at 12 months in the 62.5 ppm group males. Increases in circulating reticulocyte 
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and nucleated erythrocyte counts are consistent with an erythropoietic response to the 

anaemia. In bone marrow there were approximately 15 % - 35 % decreases in the 

myeloid/erythroid ratio in the 125 ppm rats of both sexes, but particularly in females. Bone 

marrow hyperplasia was recorded in males of the 250 and 500 ppm groups and females in all 

groups from 62.5 ppm. The severity of this response was dose related. 

Non-neoplastic effects of exposure were observed in the nose, liver and spleen. 

In the nose there were significant increases in the incidences of hyaline degeneration of the 

olfactory epithelium in all groups of males (chamber control, 13/48; 31.2 ppm, 21/49; 62.5 

ppm, 23/49; 125 ppm, 40/50) and in females exposed to the two higher concentrations of 

EGBE (chamber control, 13/50; 31.2 ppm, 18/48; 62.5 ppm, 28/50; 125 ppm, 40/49). The 

severity of the lesion was minimal and did not change with increasing exposure concentration. 

It is the most common age-related change of the nasal passages of rats (St. Clair and Morgan, 

1992) and it has been proposed to have an adaptive or protective role (Buckley et al., 1985). 

Incidences of Kupffer cell pigmentation of the liver increased significantly in all exposed 

groups of male rats (chamber control, 23/50; 31.2 ppm, 30/50; 62.5 ppm, 34/50; 125 ppm, 

42/50) and in the two higher exposure groups of female rats (chamber control, 15/50; 31.2 

ppm, 19/50; 62.5 ppm, 36/50; 125 ppm, 47/50). The severity of the lesion increased in the 125 

ppm group of both sexes. 

In the spleen, incidences of fibrosis were significantly increased in the two higher exposure 

groups of male rats (chamber control, 11/50; 31.2 ppm, 14/50; 62.5 ppm, 19/50; 125 ppm, 

20/50), but not in females (NTP, 2000). No NOAEC could be derived for non-neoplastic 

effects in this study. The LOAEC for both male and female rats was 31 ppm, based on an 

increased incidence of minimal Kupffer cell pigmentation and on significant haematological 

effects in females. 

Mouse

EGBE (purity unknown) was administered by whole body exposure to dogs, monkeys, guinea 

pigs, mice and humans (Mellon Institute of Industrial Research, 1955). Groups of 70 male 

C3H mice were exposed to 0, 100, 200 or 400 ppm (0 – 483 – 966 – 1932 mg/m3

) EGBE 7 

hr/day for up to 90 days. Body weights were recorded weekly. Serial sacrifices were 

performed on 15 mice from each group after 30 and 60 exposures, on 10 mice after 90 

exposures, and on 11 to 18 mice after 42 days rest following 90 days of exposure. Ten mice 

from each group were used for serial determination of erythrocyte fragility after various 

exposure periods. The blood samples were obtained by section of the cervical cord. Liver and 

kidney weight were measured. Only the kidneys were cut for histological examination 

(because repeated exposures to even fatal concentrations of EGBE produced undramatic 

micropathology in rats. No mortality or gross pathology or renal tissue changes were 

observed. Statistically significant alterations of liver weights occurred in the mice exposed to 

400 ppm for 30, 60 and 90 days. A similar occurrence was noted in the mice exposed for 60 

days to 200 ppm. The number of mice which had bloody urine immediately after exposure 

was proportional to the exposure concentration. No case of bloody urine was noted after the 

third exposure. Increased erythrocyte fragility was found in the mice exposed to all three 

concentrations. The increase in erythrocyte fragility appeared to be as great after the first 

exposure as it was after the 89th exposure. In all cases the erythrocyte fragility value fell to 

essentially normal levels when blood samples were taken from mice after 17 hr rest. 
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Sixty mice divided in three groups were exposed to EGBE (purity unknown) at doses of 0 and 

17 ppm (84 mg/m3). The first group was exposed 3 hr/day on 6 days per weeks and the second 

group was exposed to 6 hr/day on 3 consecutive days per week (Lomonova and Klimova, 

1977). The third group served as control. It is noted that this study was not conducted 

according to GLP and was poorly reported. Body weights of animals were recorded at regular 

intervals. A standardised battery of general parameters and the concentrations of blood 

cathecholamines were checked. For specific toxicity, blood parameters and erythrocyte 

fragility tests were performed. Catalase activity was also measured. Some functional tests 

were also performed: hexenal test, “cold test” and water maze. After the 4 months of 

exposure, an unique dose of 800 mg/m3 of EGBE was administered to assess the adaptative 

effects (if any) of regular administration of small doses of EGBE. 

Overall, the results of all parameters assessed have shown that the toxicity of EGBE was more 

severe in the first group (3 hr/d 6 days/week) than in the group 2. Moreover, the charge dose 

of 800 mg/kg bw administered at the end of the 4 month exposure period enhanced less 

effects in the second group than in the first one. Significant toxic signs were limited to 

haemolysis and narcosis. 

EGBE was administered to groups of 10 male and 10 female B6C3F1 mice by whole-body 

exposure to 0, 31, 62.5, 125, 250 or 500 ppm (0 – 150 – 302 – 604 – 1208 – 2416 mg/m3) 

EGBE vapour (lot no. QP-921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 

%)for 6 hr per day, 5 days per week for 14 weeks (NTP, 2000). 

Mice were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All mice were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study and then weekly until the 

end of the experiment. Clinical observations also were recorded weekly throughout the 

experiment. Haematological examinations were performed at the end of the experiment, using 

an Ortho ELT-8/ds 9000 analyser. Differential leukocyte and nucleated erythrocyte counts 

were made by microscopic examination of blood films. 

Autopsies were made on all study mice. Organ weights measured were for the heart, right 

kidney, liver, lungs, right testis and thymus. A complete histological examination was 

performed on the chamber control and 500 ppm male and female mice. 

In the 500 ppm group, in week 2 of the experiment, two male and two female mice died and 

two male and two female mice were found moribund and killed. All other mice survived. By 

the end of the study, body weight gains were significantly reduced in males of the 125, 250 

and 500 ppm groups, but were unaffected in all other groups. Clinical findings were found 

only in males and females of the 500 ppm group that died or were killed and included 

abnormal breathing, red urine stains and lethargy. 

Haematological examination showed that inhalation of EGBE resulted in the development of 

a persistent and exposure-related normocytic (unlike rats), normochromic, responsive 

anaemia, as indicated by decreased haematocrit values, haemoglobin concentrations and 

erythrocyte counts in the 125 ppm or greater group males and in all groups of exposed 

females. Normocytic and normochromic erythrocytes were demonstrated by the lack of 

change in the mean cell volumes and mean cell haemoglobin concentrations, respectively. 

Other haematological changes were increased numbers of polychromatophilic erythrocytes in 

the 500 ppm group and increased platelet counts in males of the 500 ppm group and females 

of the 250 and 500 ppm groups. Increases were found in absolute and relative liver weights 
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of males of the 500 ppm group and in relative liver weights of males of the 250 ppm group 

and females of the 500 ppm group. Haemosiderin deposition in Kupffer cells was the only 

change observed in livers of the 500 ppm group males and the 250 and 500 ppm group 

females. Haemosiderin pigmentation was also increased in renal tubule cells of both males 

and females of the 500 ppm group. Extramedullary haematopoietic cell proliferation 

(primarily erythroid) and haemosiderin pigmentation were present in males exposed to 125 

ppm or greater and females exposed to 250 or 500 ppm. In the forestomach, there were 

significant increases in females only in the incidence of inflammation in the 250 and 500 ppm 

groups and epithelial hyperplasia in the 125 ppm and greater groups. Effects observed in the 

males and females of the 500 ppm group that were either killed or died included forestomach 

inflammation, necrosis and ulceration, suppurative inflammation of the peritoneum and 

mediastinum, atrophy of the spleen, thymus and lymph nodes, renal tubule degeneration and, 

in males, testicular degeneration and epididymidal necrosis (NTP, 2000). No NOAEC for 

female mice could be derived for non-neoplastic effects in this study, the LOAEC for female 

mice being 31 ppm, the lowest dose tested, based on significant haematological effects. The 

NOAEC for male mice was 62.5 ppm, based on haematological effects at 125 ppm. 

EGBE was administered to groups of 50 male and 50 female B6C3F1 mice by whole-body 

exposure to 0, 62.5, 125 or 250 ppm (0 – 150 – 302 – 604 mg/m3) EGBE vapour (lot no. QP921215-26D2, Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days 

per week for 104 weeks. For haematological and bone marrow analyses, additional groups of 

30 male and 30 female B6C3F1 mice were exposed to 0, 62.5, 125 or 250 ppm for evaluation 

at 3, 6 and 12 months (NTP, 2000). 

Mice were held in the testing laboratory for 18 days and were 7 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All mice were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study, after which they were 

recorded monthly from week 5 until week 93 and then every two weeks from week 94 until 

the end of the experiment. Clinical observations also were recorded monthly from week 5 

until week 93 and then every two weeks from week 94 until the end of the experiment. 

Haematological examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 

6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and 

nucleated erythrocyte counts were made by microscopic examination of blood films. Bone 

marrow cellularity was measured using a Coulter Model ZH counter, while cytological 

evaluations of bone marrow cell morphology and myeloid/erythroid ratios were made 

microscopically. 

Autopsies and microscopic examinations were made on all of the main study mice that had 

not been used for blood and bone marrow sampling. Organ weights were not measured. At 

autopsy, all organs and tissues were examined for grossly visible lesions and all major tissues 

were fixed and processed for histological examination. A quality assessment pathologist 

evaluated the slides from all tumours and potential target organs, which included bone 

marrow, forestomach, kidney, liver, lung, nose and spleen of males and females and the 

preputial gland, prostate, skin (prepuce), testis and urinary bladder of males. The slides and 

the preliminary quality assessment report were then submitted to a National Toxicology 

Program Working Group of pathologists. 

Survival of male mice exposed to 125 or 250 ppm was significantly less than that of the 

chamber control group, whereas survival in the lower dose group was similar to the chamber 

control group. The mean numbers of survivors at the end of the experiment in each of the 0, 

62.5, 125 and 250 ppm groups were 39, 39, 27 and 26 males and 29, 31, 33 and 36 females, 
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respectively. No clinical signs were attributed to exposure to EGBE. Body weights of exposed 

male mice were generally less than those of the chamber control during the last 25 weeks of 

the experiment. Body weights of female rats of the 250 ppm group were generally lower than 

those of the chamber control from week 30 until the end of the experiment, the difference 

being about 20 % for much of that time. Body weights of the 62.5 and 125 ppm group females 

were generally lower than the chamber controls from about week 60 until the end of the 

experiment. 

Neoplastic effects observed in this experiments are described in section 4.1.2.8.1.3. 

Haematological examination showed that inhalation of EGBE resulted in the development of 

a persistent and exposure-related normocytic and normochromic, responsive anaemia, as 

indicated by decreased haematocrit values, haemoglobin concentrations and erythrocyte 

counts; it lacked, in general, changes in mean cell volumes and mean cell haemoglobin 

concentrations. These changes occurred at 3, 6 and 12 months in 125 and 250 ppm group 

male and female mice. Some anaemia also occurred at 6 months in the 62.5 ppm group 

females and there was a minimal increase in cell volume, suggesting a macrocytosis, in 

females of the 250 ppm group at 12 months. Increases in circulating reticulocyte counts that 

were observed in 125 and 250 ppm male and female mice at 3 and 6 months and 250 ppm 

female mice at 12 months, are consistent with an erythropoietic response to the anaemia. In 

bone marrow there was no change in cell counts and no decrease in the myeloid/erythroid 

ratio, unlike the observations reported for rats (above). Additionally, thrombocytosis 

developed, as shown by increased platelet counts in the 250, 125 and 62.5 ppm exposed 

groups of males and females at 12 months, in the 250 ppm group males at 6 months and 

females at 6 and 3 months and in the 125 ppm group females at 6 months. 

Incidences of haemosiderin pigmentation in Kupffer cells of the liver was significantly 

increased in males of the 125 and 250 ppm groups and females of all groups exposed to 

EGBE in a dose-dependent manner (see Table 33 in 4.1.2.8.1.3). 

Haematopoietic cell proliferation in the spleen was increased in males exposed to 125 and 250 

ppm and females exposed to 250 ppm, but it was not accompanied by any change in 

myeloid/erythroid cell ratio. Incidences of haemosiderin pigmentation in the spleen were 

significantly increased in all exposed groups of males and in the 125 and 250 ppm groups of 

females. These observations were attributed to the primary haemolytic effect of EGBE that 

was followed by regenerative hyperplasia of the haematopoietic tissue. Increases in the 

incidence of hyperplasia were also observed in the bone marrow of males exposed to 125 and 

250 ppm. 

In the nose there were increases in the incidences of hyaline degeneration in all groups of 

female mice of the olfactory epithelium (chamber control, 6/50; 62.5 ppm, 14/50; 125 ppm, 

11/49; 250 ppm, 12/50) and in the respiratory epithelium (chamber control, 17/50; 62.5 ppm, 

35/50; 125 ppm, 26/49; 250 ppm, 23/50). The severity of the lesion was minimal and did not 

change with increasing exposure concentration. There was no clear dose response 

relationship. In males, the incidence was similar to the chamber controls. 

Non-neoplastic lesions of the forestomach consisted of ulcer (full thickness defect of the 

forestomach epithelium), particularly in females, epithelial hyperplasia (an increased 

thickness of the stratified squamous epithelium sometimes accompanied by an increased 

thickness of the keratinised layer) that was usually focal and, particularly in females, 

frequently associated with ulceration ( see Table 5 in 4.1.2.8.1.3). 
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There were a number of inflammatory changes in the urogenital system (NTP, 2000). These 

are shown in detail in the table below. Effects were only seen in male mice. In the kidney, 

incidences of glomerulosclerosis and hydronephrosis were significantly increased at 125ppm 

(but not at 250ppm) whilst the incidence of chronic inflammation was increased significantly 

at 250ppm. The incidences of inflammation of the preputial and prostate glands in males 

exposed to 250ppm were significantly increased as were incidences of inflammation of the 

bladder. The incidence of ulcer of the transitional epithelium of the urinary bladder was 

significantly increased at 125ppm but not at 250ppm. The NTP authors concluded that these 

effects were indicative of an ascending urinary tract infection described as mouse urologic 

syndrome. The occurrence of this syndrome is particularly common in inhalation studies with 

mice, in which they are housed individually in wire mesh cages (Everitt et al., 1988). It is 

likely that the condition was exacerbated by the irritative effects of EGBE or its metabolites 

but is not primarily mediated by the substance. 

Table 4.71 bis: Incidence of non-neoplastic lesions in the urogenital system of male mice following 2-years 

exposure to EGBE 

 Control 62.5ppm 125ppm 250ppm 

Kidneya 50 50 47 50 

 Glomerulosclerosis 4 (1.3) 4 (1.3) 11* (1.3) 9 (1.4) 

 Hydronephrosis 1 (2.0) 0 6* (1.8) 5 (2.2) 

 Inflammation 0 1 (2.0) 2 (2.5) 4* (3.) 

Preputial glanda 49 49 49 49 

 Inflammation 2 (1.0) 7 (2.3) 6 (2.3) 8* (2.1) 

Prostate glanda 49 49 48 45 

 Inflammation 0 2 (2.0) 3 (2.3) 4* (1.8) 

Skin, prepucea 50 50 49 50 

 Inflammation 2 (3.5) 3 (3.7) 13** (3.7) 8* (3.9) 

 Ulcer 0 3 (2.7) 11** (2.8) 8* (2.5) 

Urinary bladdera 50 49 5046 45 

 Inflammation 0 2 (3.5) 5* (3.4) 4* (3.8) 

 Transitional epithelium, ulcer 0 1 (3.0) 4* (2.8) 2 (3.0) 

Notes: Figures in brackets are severity of lesion. 1=minimal, 2=mild, 3=moderate, 4=marked a

=number of animals examined. 

*=significant p<0.05. **=significant p<0.01 

No NOAEC could be derived for non-neoplastic effects in female mice in this study, the 

LOAEC being 62.5 ppm, the lowest dose tested, based on marginal, but significant 

haematological effects. The NOAEC for male mice was 62.5 ppm, based on body weight 

gain decrement, marginal, significant haematotoxic effects and inflammation of the kidney, 

urinary bladder and prepuce at 125 ppm. 

Guinea-pig

EGBE (purity unknown) was administered by whole body exposure to guinea pigs 7 hr/day 

(Mellon Institute of Industrial Research, 1955). Groups of 10 males were exposed to doses of 

0,375 and 500 ppm for 30 days. In addition 6 males and 6 females were added to the above 

groups for the study of sex differences in susceptibility. Bw was recorded weekly. Liver and 

kidney weight were measured after euthanasia. Liver, lung, kidney were submitted to 

histological examination. 

In the 500 ppm group, 2 male guinea pigs died within 12 exposure and one animal died after 7 

exposures to 375 ppm. The mean body weight of both groups of animals fell slightly by the 

third exposure but only the weights of the guinea pigs exposed to the lower level were 
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significantly below the controls after 30 exposures. A significant elevation in the kidney 

weights of both groups was also observed. No haematuria was observed and only the 3 guinea 

pigs that which died during exposure showed any major tissue damage (lung haemorrhage and 

congestion). 

Dog

Groups of 2 dogs were exposed to 415 ppm EGBE (42 % saturation) (purity unknown) 7 

hours daily, five days a week for 12 weeks (Werner et al., 1943c). Erythrocyte, reticulocyte, 

leucocyte and differential counts and haemoglobin estimations were made routinely in the 

morning and in the afternoon. These determinations were made twice during the pre-exposure 

period, 3 or 4 times during the post-exposure period and 8 times during the 12 weeks of 

exposure. Sedimentation rates, haematocrit determinations and fragility tests were made only 

during the latter part of the experiment on afternoon samples of blood. Urine was analysed for 

albumin, sugar and acetone. Urinary sediments were examined microscopically and average 

daily urine volumes were determined. Estimation of urea were made on blood obtained in late 

afternoon immediately before feeding. After sacrifice, histological examinations of the 

following organs were performed: lungs, liver, kidney, spleen, heart, urinary bladder, 

pancreas and large and small intestines. Additional examinations of the spleen were 

performed using a different treatment (acidulated ferrocyanide) in order to demonstrate the 

presence or absence of iron-bearing pigment. 

Dogs exposed to EGBE lost 6 and 9 % of their body weights during the study. Signs of central 

depression and slight increased of eyes and nose secretion were the only clinical effects 

detected. Signs of haemolytic anaemia (significant decreases in haematocrit and haemoglobin) 

were seen in treated animals. 

EGBE (purity unknown) was administered by whole body exposure to dogs 7 hr/day (Mellon 

Institute of Industrial Research, 1955). Dogs were exposed to 0, 100, 200, 385 or 617 ppm 

EGBE (0, 483, 966, 1860 or 2980 mg/m3

). For the 617 ppm dose, one black mongrel female 

was treated for 2 days. For the 385 ppm dose, one male and one female Basenji were exposed 

7 hours a day for 28 and 8 days respectively (haematological tests were performed daily but 

biochemistry only in male and after 26 and 27 exposure). For the 200 ppm group, one male 

and one female Basenji were exposed 7 hours a day for 31 days. For the 100 ppm group, one 

male and one female wire-haired terrier were exposed 7 hr/day for 91 days, usual blood 

chemistry were performed before the first exposure and after the last exposure. Haematocrit 

values and icterus indices were observed in add to the regular haematological tests were 

performed weekly. Bw was recorded weekly. Erythrocyte count, leucocyte count, Hb level, 

erythrocyte fragility and haematocrit value were measured immediately after exposure. For 

biochemistry, bromosulfalein retention time, sedimentation rate, icterus index, blood urea 

nitrogen and plasma fibrinogen levels were recorded. Erythrocyte permeability test was made 

with the method of Bootman and Moses only for the 200 ppm group. Coloured photographs 

of the visceral organs as well as any striking abnormality were performed. Histological 

examinations of adrenals appendix, gall bladder, gonads, heart, intestine, kidneys, liver, lung, 

pancreas, spleen, stomach, thyroid, urinary bladder, uterus and tissue found abnormal at 

autopsy were realised. 

For the 617 ppm group, the only dog exposed died after 2 days of exposure. Vomiting and 

weakness were seen during exposure. Autopsy revealed congestion of the kidneys and lungs. 

For the 385 ppm group, male and female died after 28 d and 8 d respectively. Similar 

symptoms were seen in both male and female: loss of weight, increase in erythrocyte fragility, 
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weakness, apathy, anorexia and increase leucocyte count. The erythrocyte fragility of male 

reached a maximum in 7 days and decrease progressively through 27 days. Analysis of urine 

after the 25th exposure indicated the presence of 55 mg of BAA. The only abnormality found 

in biochemistry was an elevated plasma fibrinogen concentration. At autopsy congestion of 

the lungs and of liver were observed in both animals. Congestion of kidneys were seen female 

only. For the 200 ppm group, slight evidence of toxicity were seen after 31 days of exposure. 

Erythrocyte fragility increased slightly and the erythrocyte fragility was higher than controls 

in both animals. Erythrocyte count and HB decrease slightly through exposure. BAA was 

present in urine after the 14th exposure: 100 and 42.5 mg in male and female respectively. For 

the 100 ppm group, only slight effects on haematocrit were seen. BAA in urine reached 100 

and 94 mg in male and female respectively after 81 exposures. 

Monkey

EGBE (purity unknown) was administered by whole body exposure to monkeys 7 hr/day 

(Mellon Institute of Industrial Research, 1955). Two Rhesus monkeys (1 male and 1 female) 

were treated with 100 ppm (483 mg/m3

) EGBE for 90 days (group 1). Another group of 2 

Rhesus monkeys, were treated with 100 ppm, after 10 days of treatment no effects were seen 

and the dose was increased to 200 ppm (group 2). No control group was used. Bw was 

recorded weekly. Erythrocyte count, leucocyte count, HB level, erythrocyte fragility and 

haematocrit value were measured immediately after exposure, bromosulfalein retention time, 

sedimentation rate, icterus index, blood urea nitrogen and plasma fibrinogen levels were also 

recorded. Coloured photographs of the visceral organs as well as any striking abnormality 

were noted. Histological examinations of adrenals appendix, gall bladder, gonads, heart, 

intestine, kidneys, liver , lung, pancreas, spleen, stomach, thyroid, urinary bladder, uterus and 

tissue found abnormal at autopsy were performed. 

In group 2, of 2 monkeys, 1 died of causes unrelated to treatment. The erythrocyte fragility 

values which were unchanged after 10 days exposure to 100 ppm rose after 4 days of 

treatment with 200 ppm. Erythrocyte fragility values were increased at the beginning of 

exposures. Values returned to normal until the end of the study. A difference was seen 

between male and female: females were more sensitive than males. For group 1, erythrocyte 

fragility values rose to 0.56 – 0.48 (0.46 – 0.40 for pre-exposure values) several times after 

the 18th exposure. A greater rise (0.62 – 0.54) was seen for the female after 7 exposures. 

These high values of the female returned to normal by the end of the 90-exposure period. 

Summary inhalation route: 

Many studies are available on rats, and mice. A few short studies using dogs, guinea-pigs and 

non human primates have also been conducted. 

In rats and mice, common toxicity signs together with effects similar to those observed in 

acute administration. The main effect was haemolysis, which was consistently observed and 

sometimes associated with secondary hepatic effects (Kupffer cells pigmentation and absolute 

and relative liver weight increases). Other effects were decreases of body weight gain, hyaline 

degeneration of the olfactive epithelium, effects on the forestomach and effects on the WBC 

sub-populations (T lymphocyte). In these studies, a NOAEC of 25 ppm in rats and a LOAEC 

of 31 ppm in mice and rats can be established (based on haemolysis, as the only significant 
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primary effect). The LOAEL of 31 ppm (coming from a six month satellite group in the NTP, 

2000 104-week study) is taken into account for the risk characterisation. 

Table 4.72 : Summary of the studies on animals performed by inhalation route 

Study NOAEC (ppm) Effects Reference 

Rats 

5 weeks. Doses 0, 

135, 320 ppm. 

135 (for haematological 

effects) 

Poorly reported Werner et al., 

1943b 

Variable Number 

of exposures . 

Doses 0, 250, 500 

ppm. 

- Poorly reported Mellon Institute of 

Industrial 

Research, 1952 

15 exposures to 0, 

20, 50 and 100 

ppm. 

4 exposures to 250 

ppm. 

20 ppm (haematological 

effects). 

Haematological 

effects 

Gage, 1970 

4 months, 0, 17 

ppm , 6 hours a 

day three days a 

week or 3 hours a 

day six days a 

week. 

- Poorly reported Lomonova and 

Klimova, 1977 

9 days, 6 hours a 

day. Doses: 0, 20, 

86, 245 ppm 

20 ppm (haemotoxicity) Haematological 

effects and effects 

on liver and body 

weight gain. 

Bushy Run 

Research Center, 

1981a 

13 weeks. Doses: 

0, 5, 25, 75 ppm 

25 ppm (haematological 

effects only) 

Haematological 

effects only 

Bushy Run 

Research Center, 

1981b 

13 weeks. Doses: 

0, 31, 62.5, 125, 

250, 500 ppm 

LOAEC of 31 ppm Haematological 

effects. 

Study performed to 

describe the 

vascular and bone 

lesion observed in 

moribund female. 

Nyska et al., 1999a 

Long et al., 2000 

NTP, 2000 

104 weeks. Doses: 

0, 31, 62.5, 

None identified for 

haematological effects; 

31.2 ppm may be 

Haematological 

effects. Effects on 

liver (Kupffer cell 

NTP, 2000 
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125ppm considered as an LOAEC. pigmentation). 

Mice 

Doses: 0, 100, 200, 

400 ppm. 

Duration: 30, 60 or 

90 exposures 

< 100 ppm Haematological 

effects at all doses 

Mellon Institute of 

Industrial 

Research, 1955 

4 months, 0, 17 

ppm, 6 hours a day 

three days a week 

or 3 hours a day 

six days a week. 

 Poorly reported Lomonova and 

Klimova, 1977 

14 weeks. Doses: 

0, 31, 62.5, 125, 

250, 500 ppm 

None identified for 

haematological effects; 

LOAEC of 31 ppm 

Haematological 

effects. Effect on 

body weight gain. 

Irritant effects on 

the forestomach. 

NTP, 2000 

104 weeks. Doses 

0, 62.5, 125, 

250ppm 

none identified 

LOAEC of 62.5 ppm 

Haematological 

effects. Effects on 

liver (Kupffer cell 

pigmentation). 

NTP, 2000 

Guinea pigs 

30 days 

Dose: 0, 375 and 

500 ppm 

none identified Mortality, effects 

on body weight and 

on kidneys. No 

effects on blood 

parameters. 

Mellon Institute of 

Industrial 

Research, 1955 

Dogs 

12 weeks 

Dose: 415 ppm 

< 415 CNS depression, 

haemolytic 

anaemia. 

Werner et al., 

1943c 

Duration between 

2 and 90 days. 

Dose: 0, 100, 200, 

385 and 617 

- CNS depression, 

haemolytic 

anaemia. Effects on 

lungs, kidneys and 

liver. 

Mellon Institute of 

Industrial 

Research, 1955 

Monkeys 

90 days, 

Dose: 0, 100, 200 

ppm. 

- Haematological 

effects (haemolysis)

Mellon Institute of 

Industrial 

Research, 1955 
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Dermal 

Mice

EGBE was administered to BALB/c mice (5-6 weeks old) to assess the effects on the immune 

system (Singh et al., 2001). Doses of 0, 100, 500, 1000 and 1500 mg/kg/day (about 25μl in a 

4:1 mixture of acetone and olive oil vehicle) were applied during 4 days to the backs of the 

mice. The numbers of animals per dose level is not indicated in the study report. Except for 

one group of mice tested for IgM plaque-forming cell (PFC) response, animals were killed 24 

hours after the last dose. Body weights were recorded. Thymus and spleen were removed and 

weighed prior to preparation of cell suspensions. For evaluation of the PFC response, mice 

were immunised by Intraperitoneal injection with sheep erythrocytes on the third day of 

dosing and killed 72 hours after the final dose. Cell suspensions of lymphoid organ tissue 

were prepared for spleen and thymus, cellularity and viability were assessed. 

Lymphoproliferative assays (Non-specific mitogenic stimulation of either B or T 

lymphocytes), mixed lymphocyte response (MLR) assay, activity of natural killers (NK) cells, 

cytotoxic T lymphocyte (CTL) activity and plaque-forming cell response to sheep red blood 

cells were assessed using standard procedures. 

No effects on body weight gain or thymus weight were seen at any dose tested. At the 1500 

mg/kg dose, an increase in the relative spleen weight and an increase of splenic cellularity of 

29 % were seen. There was no changes in the lymphoproliferation assay in the B cells 

population whereas the T lymphocyte sub-population exhibited a decrease of the proliferative 

responses significant only at doses of 500 and 1000 mg/kg (a decrease was also seen for the 

1500 mg/kg dose but not statistically significant, this weaken the relevance of this finding). 

The same results were seen in the MLR assay (decreased responses at the high doses, 

significant only at 500 and 1000 mg/kg). NK, CTL activity and T-dependent PFC response 

was unchanged after treatment. The lack of a dose-related response in these observations 

throws doubt on their significance for EGBE toxicology. The NOAEL for immunological 

effects was 1000 mg/kg bw, based on spleen weight reduction at 1500 mg/kg bw per day. 

Rabbits

EGBE was administered occlusively via dermal route to 4 groups of New Zealand (NZ) white 

rabbits (5 animals/sex) at concentrations of 100 - 50- 25 and 5 % (1 ml/kg of mixture 

corresponding to 900 – 450 – 225 and 45 mg/kg bw) during 6 hr/day for 9 days (dosed 5 days, 

no dosed 2 days and dosed 4 days) (Bushy Run Research Center, 1980c). Control group was 

treated with vehicle only. Dermal irritation score were determined after each test exposure. 

Body weight of animals was measured regularly: all males were submitted every day (and 

also after exposure period) to an ophthalmologic examination (discoloration, discharge, gross 

abnormality, reddening). Urine samples were collected prior to exposure on the 2nd, 5th and 9th

day of exposure and on the day of sacrifice. The following parameters were measured: colour, 

pH, protein, glucose, ketones, urobilinogen, bilirubin and blood. Blood samples were 

collected before dosing the last day of dosing and the day of sacrifice and assessed of RBC 

count, haematocrit, MCV, MCH, MCHC, WBC count. Reticulocytes and WBC differential 

smears were prepared for each animal but not evaluated. Fourteen days after the last dosing, 

animals were euthanised. Tissues were collected and fixed, only kidneys were examined 
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histopathologically. Liver and kidneys of all animals were weighted, in males testes were also 

weighted. 

In the preliminary studies: LD50 was calculated for males: 0.707 ml/kg and for females: 

0.630 ml/kg. In the main study, no male rabbits died during the study. One female rabbit was 

found moribund on day 5, the condition was apparently unrelated to treatment. No body 

changes were seen in male whereas female rabbits treated with undiluted EGBE exhibited a 

decrease in bw gain throughout the direction of the study. All ophthalmologic examinations 

were negative for the male rabbits. The females were not examined. Severe necrosis and 

moderate oedema were observed in all animals dosed with undiluted EGBE by the 6th day of 

treatment. The 50 % dilution produced necrosis of the skin of 1 out of 5 males as compared to 

4 out of 5 females. Erythema preceded necrosis in every instance. Slight oedema was also 

observed in those rabbits exhibiting necrosis. Dosage related erythema was observed at the 

lower dosage levels. Very slight reddening was seen in the male rabbits in the water control 

group. No statistical comparisons were performed on the skin irritation readings. On day 2 of 

treatment there was haemoglobin in the urine of two of four male rabbits dosed with undiluted 

EGBE. There also was a slight increase in urinary protein in four males dosed with undiluted 

material on day 2 of treatment. Both of these conditions returned to normal by treatment day 

5. On day 2 of treatment there was blood in the urine of three of five female rabbits dosed 

with undiluted EGBE. There was a corresponding increase of urinary protein in two of these 

animals. On day 5 of treatment, four of the five females dosed with undiluted material had 

blood in their urine, as well as one dosed with the 50 % dilution. By day 9 of treatment only 

one dosed with undiluted sample still had blood in the urine; however, at this time all five 

females dosed with the 50 % dilution had blood in their urine. There was no evidence of 

haemoglubinuria at any dosage level at the time of sacrifice (14 days post exposure). No 

abnormalities were seen in any of the other measures of urinary effect assessed and there was 

no occult found in the urine of rabbits dosed with the 25 % or the 5 % dilutions. Statistically 

significant depressions of mean erythrocyte count, haemoglobin and mean corpuscular 

haemoglobin concentration were present on treatment day 9 in females dosed with undiluted 

substance and the mean corpuscular haemoglobin was significantly increased. Although not 

statistically significant, the contemporaneous decreased haematocrit and increased mean 

corpuscular volume are consistent changes. The presence of larger red blood cells with more 

haemoglobin is indicative of response to red cell loss by early release of less mature cells into 

the circulation. These effects were no longer seen after the 14-day observation period. 

Females in the three lower dosage groups had no indication of biologically significant 

haematologic effects. The increased mean red blood cell counts on day 9 of treatment in the 5 

% dilution group and on day 14 after exposure in the 25 % dilution group are considered to be 

statistical artifacts. No changes were noted in any organ weight measured in all animals. 

Gross findings included some thickening of the skin in male rabbits given undiluted substance 

and possibly dose related patchy colour change of the kidneys of the three females. 

For this study, the NOAEL of 450 mg/kg can be taken into account mainly based on gross 

findings observed in animals dosed with pure substance. Haematological effects seen during 

the study were transient and recovery was complete after the 14-day observation period. . 

In a GLP study, 4 groups of 10 males and 10 females NZ white rabbits were treated dermally 

(occlusively) with EGBE (high purity) at concentration of 0 % - 2.8 % - 14.3 % and 42.8 % in 

distilled water (corresponding to dose levels of 0 – 10 - 50 - 150 mg/kg) for a 6 hr period/day 

5 d/w for 13 weeks (Wil Research laboratories inc., 1983). Animals were observed for clinical 

symptoms twice daily. Prior to treatment, the site of application of each animal were graded 

using the Draize skin reaction scoring system. Scoring was performed daily for the first 3 
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weeks of the study, the once weekly thereafter. Bw and food consumption were measured 

weekly. Blood was collected before the study, during week 4 and just before termination. The 

following parameters were determined: 

Haematology: white blood cell count, mean corpuscular haemoglobin concentration, 

erythrocyte Count, mean corpuscular haemoglobin, haemoglobin, differential white blood cell 

count, haematocrit, erythrocyte fragility (termination only), mean corpuscular volume. 

Clinical chemistry: glucose, direct bilirubin, total bilirubin, albumin, blood urea nitrogen, total 

bilirubin, alkaline phosphatase. 

All rabbits were subjected to a gross necropsy examination. The following organs were 

weighted at sacrifice for at least 6 animals/sex/level: heart, thymus, testes, spleen, liver, 

ovaries and kidneys. The following tissues were also examined briefly and conserved: 

adrenals, aorta, bone (sternebrae), brain (3 transverse sections), epididymis (both, tail), 

oesophagus, eyes, gall bladder, heart, intestines (caecum, duodenum, jejunum, ileum and 

colon), kidneys, liver (left & median lateral lobes), lung (left), lymph node (mesenteric & 

thoracic), mammary gland, ovaries, pancreas, parathyroids, pituitary, prostate, sciatic nerve, 

seminal vesicles, skeletal muscle, skin (test site & adjacent normal skin), spleen, stomach 

(pylorus and fundus), submandibular salivary gland, testes, thyroids, thymus, tongue, trachea, 

urinary bladder, uterus (both horns), vagina, All gross lesions. 

Red coloured faeces and red liquid material on cage paper (probably blood) were seen in 

some treated animals (each group). Some irritant effects were sometimes seen in animals of 

both control and treated groups. There was no increase in the severity of these effects in 

treated animals versus controls. There were no treatment related deaths. No significant effects 

were seen on body weight gain or food consumption in any group treated compared to control 

animals. Sporadic changes in haematology parameters and RBC fragility values were noted 

but values were within normal ranges for this laboratory and probably not related to the test 

material. Sporadic changes were also noted in WBC counts but were not related to test 

material administration. No changes in biochemistry were related to treatment. No test 

material related effects were noted on absolute or relative organ weights or final body 

weights. 

The NOAEL for this study was equal to 150 mg/kg bw, the highest tested dose. The actual 

NOEL may be of a higher value. 
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Summary dermal dose: 

Table 4.73: Summary of the studies performed by dermal route 

Study (rabbits) NOAEL (mg/kg bw/d) Effects Reference 

Mice 

4 days. Doses: 0, 100 – 

500 – 1000 – 1500 

mg/kg bw/day 

NOAEL=1000 Effects on splenic 

cellularity at 1500. 

Singh et al., 2001. 

Rabbits 

9 days. Doses: 45 – 

225 – 450 - 900 mg/kg 

bw/d 

NOAEL = 450 Transient 

haematological 

effects 

Bushy Run Research 

Center, 1980c 

13 weeks. Doses: 10 – 

50 – 150 mg/kg bw/d 

NOAEL > 150 No effects Wil Research 

laboratories inc., 1983 

Two studies are available on rabbits to assess the toxicity of repeated doses of EGBE 

administered dermally. In only one study, signs of toxicity were recorded and were limited to 

transient signs of haemolysis. This study led to a NOAEL of 450 mg/kg bw/d due to 

haematological effects seen at 900 mg/kg bw/d. 

Given that this study was performed only during 9 days, the NOAEL of the second study, 

which was performed during 13 weeks, could be more reliable for the risk characterisation. 

This NOAEL was 150 mg/kg bw/d. 

The mouse study design for the assessment of EGBE effects on the immune system, give a 

NOAEL of 1000 mg/kg. 

Oral 

Rat

Five groups of Sherman rats (5/sex/group) were fed with diet containing 0, 0.03, 0.125, 0.5 

and 2 % Butyl cellosolve (EGBE purity unknown) corresponding to doses of 0, 18, 76, 310 

and 1540 mg/kg/d (Mellon Institute of Industrial Research, 1952) for 90 days. 

No deaths were seen attributable to the direct action of EGBE. Appetite was not affected and 

no pertinent micropathology was discovered. Tests for blood in pooled urine sample from the 

2 %, 0.5 % and controls groups after 3 and 6 days were negative. The mean weight gain was 

lower than controls for the 2 % group. Relative kidney and liver weight were increased at 2 % 

dose and relative liver weight only was increased at 0.5 % dose. 

The NOAEL can be considered to be 0.125 % (76 mg/kg/d) but considering that the test 

conditions were poorly reported (no control of the administrated substance, evaporation ?) this 

NOAEL will not be taken into account for the risk characterisation. 

Groups of young DW albino rats (10 males and 10 females/group) were given food with 

EGBE (purity unknown) at doses of 1.25 – 0.25 – 0.05 or 0.01 % (corresponding to 919, 188, 

38 and 7 mg/kg in males and 976, 222, 41 and 9 mg/kg in females respectively) during 3 
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months (Mellon Institute of Industrial Research, 1963). A control group was given food 

without EGBE Animals were weighted 4 times during the first week and once a week 

thereafter. After euthanasia liver and kidneys were weighted. Organs were examined for signs 

of pathology, the urinary bladder was examined for concretions. Histology was performed on 

representative tissues. 

One animal died at day 73 after the beginning of the study in the high dose group. Food 

consumption was decreased in both males and females at 1.25 %. In males a significant 

decrease of food consumption was also observed at 0.25 % dose. A significant decrease of 

body weight gain was seen in animals dosed with 1.25 % EGBE. It was also seen in males at 

doses of 0.25 %. A small decrease was seen on the 2 first days of dosing only with the 0.05 % 

dose. The mean liver and kidney weight of males and females were definitely increased at the 

1.25 % dose. At both 1.25 % and 0.25 % testes were atrophied. The NOAEL for this study 

can be considered to be 0.05 %. The purity of the EGBE used in this study is not given, nor 

does there appear to be analytical verification of EGBE doses, homogeneity and stability. 

This makes this study difficult to interpret given for example that testicular atrophy is not 

reported in other oral, repeated-dose studies even at higher doses of EGBE greater than 0.25% 

(e.g., 14-week drinking water study in rats and mice from NTP, 1993; continuous breeding 

study reported in Morrissey et al., 1989). Given the availability of numerous other more 

recent and robust studies, this one cannot be considered reliable to derive the critical NOAEL. 

Groups of 10 Albino rats (CR, COBS, CD-BR) were treated with various glycol ethers 

(including EGBE - purity > 99.5 %) via oral gavage, 5 days per week for 6 weeks (Eastman 

Kodak, 1982). Doses administrated were: 0, 222, 443 and 885 mg/kg. Body weight were 

recorded twice a week during the first week of exposure and every week after until 

termination of the study. Animals were observed daily for clinical signs. Blood samples were 

taken before euthanasia for the determination of haematology indices and biochemistry: 

SGOT, SGPT, alcaline phosphatase (ALP), LDH, urea nitrogen, creatinine and glucose. After 

termination, histological examinations were performed on the following organs: lung, heart, 

thymus, kidneys, liver, spleen, brain, salivary glands, stomach, intestinal tract, pancreas, 

oesophagus, adrenal glands, pituitary, thyroid, parathyroid, trachea, mesenteric lymph node 

(LN), testes and annexes, bone marrow, tongue, nasal cavities and eyes. Before fixation, liver, 

kidneys, heart, testes, brain and spleen were weighted. 

2 out of 10 animals were found dead in the high dose. No mortality was seen in any other 

groups. A significant decrease in body weight gain was seen from D13 and after in animals 

treated with the high dose of EGBE. A significant decrease of food consumption was also 

seen in this group but only from D3 to D20. In other groups there was a trend to a decrease in 

bw gain compabable to controls and a lower food consumption in the middle dose group (not 

significant). Clinical symptoms were limited to bloody urine in the mid and high dose groups. 

Only one of the ten rats given the low dose of EGBE had bloody urine. This persisted through 

the third week of treatment. Other clinical signs at the mid and high dose were lethargy, 

unkempt hair coats, piloerection, rales, slight weakness and inactivity. All doses produced 

significant effects on RBC parameters, showing a characteristic pattern of haemolytic 

anaemia. No effects were seen on WBC. For biochemistry: a significant increase of alkaline 

phosphatase (PAL) was seen from the middle dose. Moreover, in the high dose, a significant 

increase of SGPT and a decrease of glucose (GLU) were noted. These changes were slightly 

different compared to controls so their toxicological significance is uncertain. Absolute and 

relative spleen weights were increased for the middle and high dose of EGBE. A small 

increase of the liver weight (only significant for relative weight) was seen for the high dose 

group. No effects were seen on testes weight (relative or absolute) at any dose group. For 
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gross pathology, only enlarged, dark spleen were seen for high and intermediate dose. Histopathological lesions were hyperkeratosis and acanthosis in the stomach epithelium in all 

treated animals. Hepatomegaly was also seen in 4 out of 10 animals of the high dose group. 

Haemosiderin deposition was seen in some animals (7/10 and 6/10 in the high and mid dose 

group respectively). Thymus atrophy was seen in one animal of the high dose group. 

Congestion of the spleen was seen in all treated animals. Extramedulary haematopoiesis was 

reported for one animal in the high dose group and haemosiderin deposit in the majority of 

animals of the high and mid dose group. For this study, the NOAEL can be considered to be 

lower than 222 mg/kg (which is the Low Observable Adverse Effect Level: LOAEL) based 

on effects seen on spleens and RBC parameters. 

Toxic effects on the immune system of SD rats after 21 days of exposure to EGBE and 

EGME were assessed. Groups of 6 rats of each sex were exposed to EGBE (purity 97 %) in 

drinking water at concentration of0, 2 000 and 6 000 ppm corresponding to about 0, 180 and 

500 mg/kg respectively for males and 1600 – 4800 ppm for females corresponding to 200 and 

444 mg/kg respectively (Exon et al., 1991). All rats were injected SC at the base of the tail 

with 1 mg aqueous KLH in a 0.2-ml volume 7 days after initiation of glycol ether treatment. 

A second injection of aqueous KLH was given at the base of the tail on Day 13 to initiate an 

IgG antibody response. All animals were injected into the right footpad with 100 μl of heataggregated KLH on Day 20. The left footpad was injected with 100 μl of sterile saline. At the 

time of termination serum samples were collected for analysis of antibody levels to KLM. 

Spleens were used as the cell source to assess natural killer (NK) cell cytotoxicity and 

interleukin 2 (IL2) and interferon (IFN) production. All assays were performed on each rat on 

experiment. 

The 24 hr Delayed-Type Hypersensitivity reaction (DTH) response was assessed on Day 21 

by measuring the thickness of the heat-aggregated KLH-injected footpad and substracting the 

thickness of the saline-injected footpad. The reaction was expressed as millimetre difference 

in swelling between the two footpads. 

Serum collected by cardiac puncture at time of termination (Day 21) was analysed for antiKLH IgG antibodies by an indirect enzyme-linked immunosorbent assay (ELISA). 

Natural killer cell cytotoxicity: suspension of spleen cells were prepared. Splenic natural killer 

cell cytotoxicity was assessed by the 4-hr 51Cr release from YAC-1 tumour target cells as 

previously described (Exon et al., 1991). Specific 51Cr release was calculated by: cpm 

experimental release - cpm spontaneous release x 100 / cpm maximum release by 2 % SDS - 

cpm spontaneous release. Spontaneous release of 51Cr by target cells was less than 10 % in all 

assays. 

Interleukin 2 production by rat spleen cells was measured by the capacity to support the 

growth of the IL2-dependent CTLL-2 cell line in vitro. This assay does not distinguish 

between IL2 and IL4 production. A sample of spleen cells was centrifuged and the cells were 

resuspended in RPMI (supplemented with penicillin, streptomycin, 5 % heat-inactivated 

foetal bovine serum). Samples were incubated 24 hr with concanavalin A and the cell-free 

supernatants were harvested and tested for IL2 activity. Briefly, 4 x 103

 IL2-dependent murine 

CTLL-2 cells (in RPMI, 5 % foetal bovine serum, 5 x 10-5 M 2-mercapto-ethanol) were 

cultured in replicate 200-μl volumes in flatbottomed 96-cell culture plates in the presence of a 

log-2 dilution series of putative IL2- containing supernatants. After 18 hr, 1 μCi[3

H]TdR was 

introduced to each well for 4 hr, after which the cells harvested onto glass fiber filters, and the 

radioactivity measured by scintillation countering. 
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Interferon concentrations in supernatants of spleen cells cultured for 48 hr with 1.0 μg/ml 

concavalin A were assessed by the capacity to block the cytopathic effects of vesicular 

stomatitis virus on rat RT fibroblasts. 

For pathological examination, the thymus, spleen, liver and right kidney and testis were 

collected and weighted at necropsy. The tissues were examined by light microscopy. 

Body weights were decreased in male rats treated with 6000 ppm EGBE and in females 

treated with 1600 or 4800 ppm. No effects were seen in thymus or in testes (macroscopically 

or histologically). Relative liver weights were increased in male rats treated with the low dose 

of EGBE. Relative kidney weight was decreased in female rats treated with the low dose of 

BE. Microscopic changes were not observed in sections of liver and kidney taken from the 

BE-exposed rats. Histopathologic examination of the spleen was not done since all of this 

tissue was used as a source of cells for the immune function assays. Immune Function 

assessment: Natural killer cytotoxic responses were enhanced in male and female rats treated 

with the low dose of BE. No effect on antibody production was observed in the BE-treated 

rats. No significant effects of glycol ether treatment were observed on DTH reactions of these 

rats. The cellular infiltrate in the KLH-injected area of the footpad consisted mainly of 

mononuclear phagocytes, typical of a Type IV hypersensitivity reaction. The authors 

concluded that the immune system is not a sensitive target for EGBE. 

EGBE (lot no. BT00504LP, Aldrich Chemical Co., USA, purity ≈ 99 %) was administered in 

drinking water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 

male and 10 female F344/N rats for 13 weeks. These concentrations provided target dose 

levels of 0, 100, 150, 250, 400 or 650 mg/kg bw per day. Estimates of compound 

consumption based on water consumption by rats were 69, 129, 281, 367 and 452 mg/kg/day 

for males and 82, 151, 304, 363 and 470 mg/kg/day for females. Supplemental groups of 10 

rats/sex/group/time point were included for haematology and clinical chemistry observations 

at weeks 1 and 3 (NTP, 1993). 

Rats were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study 

began. They were housed 5/cage, water and feed were available ad libitum. All rats were 

observed twice daily throughout the study. Body weights were recorded at the beginning of 

the study and then weekly until the end of the experiment. Clinical observations also were 

recorded weekly throughout the experiment. Haematological examinations were performed 

using a Series 7000 cell counter and a Series 810 whole blood platelet analyser (Baker 

Instruments). Clinical chemistry variables were measured with a Cobra Fara analyser (Roche 

Diagnostics). Evaluations of vaginal cytology and sperm morphology were done for the three 

highest doses tested. Results concerning these examination are reported in the reprotoxicity 

section. 

Complete autopsies were made on all study rats. Microscopic examinations were made on all 

control group and treated rats. 

No rats treated with EGBE died or were killed before the scheduled end of the study and there 

were no clinical signs of toxicity. Body weight gains were reduced in both male and female 

rats of the 4500 and 6000 ppm treatment groups. In males and females there were reductions 

in drinking water consumption in the higher dose groups, this being clearly concentration 

related in females, from a mean of 18.8 ml/day in the control group to 10.7 ml/day in the 6000 

ppm exposure group. A markedly macrocytic and mildly hypochromic anaemia was observed 

at each time point and reticulocyte counts were moderately increased in weeks 1 and 13. For 

males there was a decrease in erythrocyte counts at all time points in the 3000 ppm and 
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greater groups, while in females the decrease occurred in the 1500 ppm and greater groups. A 

consistent thrombocytoapenia was observed at all time points in males and females of the 

4500 and 6000 ppm groups; it also occurred in females of the 3000 ppm group at week 13. 

The most consistent blood chemistry observation was an increase in alkaline phosphatase, 

particularly in the high dose groups. This observation is consistent with mild cholestasis. 

Thymus weights were significantly reduced in males of the 4500 ppm group and males and 

females of the 6000 ppm group. Other organ weight changes were found, but these appeared 

to be secondary to body weight gain reduction (spleen was not weighed). Histopathological 

lesions occurred in the liver, spleen and bone marrow and male and female rats. Hepatic 

changes included primarily centrilobular hepatocellular degeneration and primarily 

centrilobular Kupffer cell pigmentation. These changes were present in the majority of dosed 

rats, but they were more prevalent in the 3000 ppm and greater dose groups and were slightly 

more severe in females. In addition, the cytoplasm of hepatocytes of rats at all dose levels was 

more eosinophilic and lacked the ampholytic-to-basophilic granularity typical of the controls. 

In the spleen there was an increase in haematopoiesis and deposition of haemosiderin. In bone 

marrow there was an hyperplasia characterised by an increase of haematopoietic cells and 

decrease in marrow fat cells. All of these lesions were present in the majority of dosed rats, 

but they were more prominent in the 3000 ppm and greater groups (NTP, 1993). There was no 

treatment effect on testis weights but there was a reduction in the size of the uterus in females 

at 4500 and 6000 ppm. Changes in uterine weight were considered by the authors to be 

secondary to the reduction in body weight gain rather than a direct effect of EGBE. The only 

spermatozoal measurement that showed significant change relative to the control group was 

sperm concentration which was slightly decreased in all groups of treated males; however, 

this effect was not dose-related. There were no significant differences from the control group 

in oestrous cycle length for treated females, although females treated at 4500 and 6000 ppm 

spent more time in dioestrous than the other groups. This correlates with the smaller uterine 

size, which was attributed to a secondary consequence of reduced body weight gain. These 

effects and their no relevance for risk assessment are more developed in the section 4.1.2.9.1 

“other studies”. No NOAEL was identified in this study, based on cytoplasmic alterations in 

hepatocytes of both male and female rats at 750 ppm, equal to 69 mg/kg bw per day and 82 

mg/kg bw per day in males and females respectively. 

EGBE (purity > 99 %) was administered to female Fischer 344 rat (10 – 12 w old) via oral 

route (Nyska et al., 1999b). Two groups of 8 animals were treated with EGBE at dose of 250 

mg/kg or vehicle during three days. Animals were sacrificed 24 hours after the last treatment. 

Complete necropsy examination were performed on all rats, the following tissues and organs 

were examined microscopically: sternum, femur, head, vertebral column, tail, heart, lungs, 

liver, spleen, kidneys, eyes and brain. Both eyes were bisected mid-sagitally and 12 sections 

were prepared (6 stained with HE and six stained with PAS). 

Gross abnormalities were noted only in EGBE treated animals and included bluish tail 

discoloration, watery blood, discoloured liver, dark kidneys and multiple Petechial 

haemorrhages on the sclera. Some ocular changes were observed in 5/8 treated rats. Retinal 

changes were bilateral and occasionally located in proximity to capillaries. Lesions included 

multifocal haemorrhage within the retinal pigment epithelium (RPE), the choriocapillaris 

between the RPE and the photoreceptors leading to retinal detachment, and the photoreceptors 

and inner and outer nuclear layer. The haemorrhage in the RPE were associated with 

degeneration, exfoliation and loss of these cells. The haemorrhages within the photoreceptors 

layers, sometimes associated with minimal polymorphonuclear cell infiltration, caused 

organizational disruption of the rods and cones and, at time, resulted in their loss. In the inner 

and outer nuclear layers, the haemorrhages were associated with degeneration and loss of cell 
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resulting in thinning, disorganization and fusion of these layers In a single rat, a patent retinal 

vessel was still surrounded by necrotic cells of the outer and inner nuclear layers, suggesting 

an ischemic-infarctive process. In another case, an area of necrosis was present in the outer 

retinal layers, probably related to occlusion of the regional blood vessel of the ciliary 

processes and in the limbus. 

In the organs other than the eyes, a range of histopathological changes were observed. 

Occlusive trombi were noted in the submucosa of the most anterior section of the nasal cavity 

and the small calibre pulmonary vessels. Thrombosis was also noted in the dental pulp of the 

incisors in association with focal stromal and odontoblast coagulative necrosis. The femur and 

coccygeal vertebrae at intermedullary locations also exhibited thrombosis in association with 

necrosis of marrow cells, bone-lining cells, and both cortical and trabecular osteocytes. In the 

femur, the changes were usually confined to the diaphysis (medullary cavity and inner third of 

the cortex), while in the coccygeal vertebrae, the metephysis and epiphysis were also 

involved. Usual changes seen with EGBE were also noted in other organs: multifocal nonzonal hepatocellular necrosis, splenic extramedullary haematopoiesis, bone marrow erythroid 

hyperplasia and haemoglobinuric nephrosis. 

These effects, seen in female rats only, seem to be closely related to haemolytic toxicity. As 

the rats are one of the most sensitive species to haemolytic effects of EGBE and humans are 

resistant to these effects such symptoms could occur but at very high doses (hardly obtainable 

in normal use conditions). The design and objectives of this study make it unsuitable to derive 

a NOAEL for Repeated Dose Toxicity. 

Mouse

EGBE (lot no. BT00504LP, Aldrich Chemical Co., USA, purity ≈ 99 %) was administered in 

drinking water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 

male and 10 female B6C3F1 mice for 14 weeks. These concentrations provided target dose 

levels of 0, 100, 150, 250, 400 or 650 mg/kg bw per day (NTP, 1993). Estimates of compound 

consumption based on water consumption by mice were 118, 223, 553, 676 and 694 

mg/kg/day for males and 185, 370, 676, 861 and 1306 mg/kg/day for females. 

Mice were held in the testing laboratory for 11 - 12 days and were 6 weeks old when the study 

began. They were housed individually, and water and feed were available ad libitum. All mice 

were observed twice daily throughout the study. Body weights were recorded at the 

beginning of the study and then weekly until the end of the experiment. Clinical observations 

also were recorded weekly throughout the experiment. Complete autopsies were made on all 

study mice. Microscopic examinations were made on all control group and treated mice. 

No mice treated with EGBE died or were killed before the scheduled end of the study and 

there were no significant clinical signs of toxicity. Body weight gains were slightly reduced in 

both male and female mice of the 3000 and 6000 ppm treatment groups. No particular pattern 

was evident in drinking water consumption. Organ weight changes that were found were 

secondary to body weight gain reduction. No treatment related gross or microscopic lesions 

in male or female mice were found (NTP, 1993). The NOAEL of 223/370 mg/kg is not very 

reliable because no haematological analysis were performed on animals during or after the 

study. 

In a series of studies, the mechanism of EGBE (purity 99 %) accumulation in forestomach 

was assessed (Poet et al., 2003). Five experiment were conducted to examine various endpoints: 
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- target tissue histology / forestomach irritation 

- tissue dosimetry and pharmacokinetics 

- fur deposition 

- salivary excretion 

- retention in stomach content 

For target tissue histology assessment, B6C3F1 adult mice (16/sex/dose) were treated with 

EGBE orally at doses of 400, 800 or 1200 mg/kg for 4 days (in fact due to an excessive 

unexpected mortality at these dose levels, surviving animals after 2 days of dosing were 

administered half of their corresponding original target dose). Half the animals were targeted 

for forestomach toxicity evaluation, the remainder were examined for liver effects. To 

investigate if parenteral administration could also produce forestomach lesions, extra groups 

of mice were administered saline solutions of EGBE by either SC or IP injection of 400 

mg/kg for 4 days (6 animals/route of administration). 

Following oral administration, neat EGBE caused dose-related forestomach lesions in all 

animals. This lesions were mainly epithelial hyperplasia and inflammation. Minimal lesions 

similar to those produced in the lower dose oral gavage groups were observed in 1/6 of the IP 

dosed mice and 2/6 of the SC dosed mice. 

Summary oral route: 

Table 4.74: Summary of studies performed by oral route 

Study NOAEL 

(mg/kg bw/d) 

Effects Reference 

Rats 

90 days in food 0, 18, 76, 310 and 

1540 mg/kg/d 

NOAEL = 

76 mg/kg/d, 

LOAEL = 

310 mg/kg/d 

Poorly reported Mellon Institute of 

Industrial Research, 

1952 

90 days in food 1.25 – 0.25 – 0.05 or 

0.01 % 

919, 188, 38 and 7 

mg/kg in males and 

976, 222, 41 and 9 

mg/kg in females 

NOAEL = 0.05 % 

(38 and 41 mg/kg in 

male and females 

respectively) 

Decrease in food 

consumption and 

body weight gain in 

males. Testes 

atrophy. 

Mellon Institute of 

Industrial Research, 

1963 
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6 weeks gavage 0, 222, 443 and 885 

mg/kg 

LOAEL = 222 mg/kg

Haematological 

effects at all doses 

and irritant effects on 

the stomach 

Eastman Kodak, 

1982 

21 days in drinking 

water 

0, 180 and 500 

mg/kg for males and 

200 and 444 mg/kg 

for females. 

Not suitable for RA 

No remarkable 

effects on the 

immune system 

Exon et al., 1991 

13 weeks in drinking 

water 

69, 129, 281, 367 and 

452 mg/kg/day for 

males and 82, 151, 

304, 363 and 470 

mg/kg/day for 

females 

LOAEL of 69 and 82 

mg/kg bw/day for 

males and females 

respectively. 

Slight decrease in 

body weight gain and 

haematological 

effects. Liver effects. 

NTP, 1993 

3 days 250 mg/kg 

Not suitable for RA 

Haematological 

effects, trombi 

formation. 

Nyska et al., 1999b 

Mice 

4 days Not suitable for RA Irritation of the 

forestomach 

Poet et al., 2003 

14 weeks in drinking 

water 

118, 223, 553, 676 

and 694 mg/kg/day 

for males and 185, 

370, 676, 861 and 

1306 mg/kg/day for 

females. 

NOAEL = 223 and 

370 mg/kg bw/d for 

males and females 

respectively 

LOAEL = 553 and 

676 mg/kg bw/d for 

males and females 

respectively 

Slight decrease in 

body weight gain 

NTP, 1993 

Six studies on rats and two on mice are available. Effects seen by oral route were body 

weight reduction, haemolysis, hepatic effects and local irritation effects. Irritation to the 
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forestomach was seen after gavage dosing and, to a far lesser extent, after subcutaneous and 

intraperitoneal injection. This difference is most likely due to the higher local concentration 

after gavage dosing. Overall, a LOAEL of 69 and 82 mg/kg bw/d (in males and females 

respectively) can be fixed in a three-month study. 

Other routes 

EGBE was administered IP to female SD rats 5 times a week during 2 weeks at doses of 55 

mg/kg (Bernard et al., 1989). Urine collection was carried out before, every 2 days during the 

test and immediately after the last injection. The urinary concentration of albumin and β2-

microglobulin (β2-m) was determined and also the activity of N-acetyl-β-glucosaminidase. 

No significant effects were seen on albumin, β2-m or N-acetyl-β-glucosaminidase urinary 

excretion. In conclusion, no glomerular damages were induced by EGBE treatment. 

4.1.2.6.2 Studies in humans 

Inhalation 

Twenty one male paint formulators with an average age of 41.3 years (27 to 53) exposed to 

various chemicals (IPA, acetone, EA, benzene, toluene, xylene, white spirit) including EGBE, 

at exposure level of 0.09 time Threshold Limit Value of index solvent mixture for 20.2 years 

were studied with a battry of neurobehavioural performance tests including digit span, digit 

symbol, Benton visual retention, finger tapping, grooved peg board and aiming test (Foo et 

al., 1994). A group of 21 male workers unexposed served as controls. Personal exposure 

levels to EGBE ranged from 0.41 ppm to 3.13 with a mean of 1.26 ppm (N=4). The 

performance of the paint formulators was worse than the controls in neurobehavioural tests. 

Statistically significant results were observed in aiming test error score, digit span and 

grooved peg board. These results are not relevant for risk assessment due to the co exposure 

to other solvents. 

Nine floorers occupationally exposed for 8 to 35 years to mixtures of organic solvents were 

subjected to analysis and characterisation of their lymphocyte sub-population (Denkhaus et 

al., 1986). They were matched with 9 healthy volunteers to have control values. Ages ranged 

from 25 to 58 years old. As an indicator of solvent exposure, air and blood samples of 

exposed men were taken by personal monitoring during one shift and one week before 

analysis. In addition to cell typing, the following parameters were obtained: haematological 

status, with differentiation, SGOT, SGPT, γGT, AP, cholesterol and NF. Lymphocytes 

obtained were labelled with monoclonal antibodies and counted for subpopulations: T4 

helpers, T8 suppressor cells, total amount of T11 (all) cells, NK and B lymphocytes. 

Some chemicals were identified by monitoring: butanol, iso-butanol, EGBE, EGEE, 2 

methoxyethynol, toluene, m-xylene, 2-butanone, 2-hexanone. For EGBE, measurements gave 

the following results: 

In air: mean concentration: 24.6 mg/m3

 (maximum concentration: 350 mg/m3) 

In blood: mean concentration: 4.1 μg/dl (maximum concentration: 108,2 μg/dl) 

Between the exposed and the unexposed group there were only negligible differences in Hb 

concentration and erythrocyte number (a very slight decrease). In the relative number of 
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eosinophiles and stab cells (increase), segmented cells (decrease) and lymphocytes (increase), 

these differences were statistically significant. Others measured parameters where within the 

normal ranges. Significant differences in the distribution of the lymphocytes sub-populations 

were seen between exposed and unexposed group. In the exposed workers, T11 cell and T4 

helper were decreased, while NK and B lymphocytes were increased. There were no changes 

in the T8 suppressor cell sub-population. In this study, the authors concluded that the 

observed decrease in helper/suppressor ratio might be an indicator of early haematological 

and/or immunological effect due to chemical exposure, which eventually may induce long 

term disorders of the haematopoetic and/or lymphopoetic system. But they also concluded 

than these results were only quantitative and did not reflect the functional state of each 

subpopulation. Since the workers were exposured to a variety of chemicals, it is not possible 

to draw conclusions for EGBE alone. 

4.1.2.6.3 Mechanistic studies of liver pathology 

Associated with haemolysis in rats and mice exposed to EGBE there were depositions of 

haemosiderin in Kupffer cells and hepatocytes. Haemosiderin is an insoluble product derived 

from iron-rich ferritin. In man, the major pathological effects of chronic hepatic iron overload 

are fibrosis and cirrhosis, porphyria cutanea tarda and hepatocellular carcinoma. Although 

precise pathogenic mechanisms remain unknown, iron probably produces these and other 

toxic effects by increasing oxidative stress and lysosomal lability (Bonkoavsky, 1991). 

Therefore, the development of the oxidative stress that is possible as a result of 

haemosiderosis was studied in male and female mice treated with EGBE by measuring the 

formation of 8-hydroxydeoxyguanosine (OH8dG), as an indicator of DNA damage, and 

malondialdehyde (MDA), as an indicator of lipid peroxidation, and the reduction in αtocopherol concentration, as an indicator of a reduction in antioxidant capacity (Kamendulis 

et al., 1999). It is clear that α-tocopherol is not the only antioxidant in the cells, so studies in 

which only α-tocopherol was measured are not assessing the full antioxidant condition of the 

cells. In a later study, these investigations were continued by comparing EGBE induced 

oxidative stress and altered cell growth in male B6C3F1 mice and male F344 rats (Siesky et 

al., 2002). In addition, the relative sensitivities of male rat and male mouse hepatocytes to 

oxidative damage in vitro were examined (Kamendulis et al., 1999; Park et al., 2002a). 

Groups of male and female B6C3F1 mice were treated orally by gavage with EGBE at dose 

levels of 0, 300, 600 or 900 mg/kg bw for males and 0, 200, 400 or 600 mg/kg bw for females 

then killed by cervical dislocation and the livers removed, snap-frozen in liquid nitrogen and 

stored at -80°C until analysed for OH8dG, MDA and α-tocopherol. The results are presented 

in Table 4.75 as percentages of the control values. 

Table 4.75: Effects of EGBE on indicators of oxidative stress in B6C3F1 mice, mean & standard deviation & 

percentage of the control (100 %) (Kamendulis et al., 1999) 

Dose MDA α-Tocopherol OH8dG 

Males mice 

0 10.54 ±2.74 5.59±0.89 4.74±1.17 

300 122 58 97 

600 143* 27* 162* 
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900 174* 22* 248* 

Female mice 

0 11.5±2.72 7.05±1.20 3.6±1.09 

200 182* 74 123 

400 167* 62* 196* 

600 229* 45* 303* 

* P < 0.05 

units: MDA, nmol/g liver; 

α-tocopherol, nmol/g liver; OH8dG, pmol/μmol dG 

The α-tocopherol concentration was higher in females and was spared to a greater extent than 

in male mice. This could be indicative of a greater protective effect in females than in males. 

The oxidative damage to DNA was, however, very similar in male and female mice (high 

dose results being: males 11.76, females 10.91 pmol OH8dG /μmol dG) (Kamendulis et al., 

1999). 

In the more recent study, EGBE was administered daily by gavage to male B6C3F1 mice at 

doses of 0, 225, 450, or 900 mg/kg bw and to male F344 rats at doses of 0, 225 or 450mg/kg 

bw on 5 days per week for up to 90 days. These doses correlated with those of the 2-year 

inhalation studies (NTP, 2000). After exposure for 7, 14, 28, or 90 days, 15 mice and 5 rats 

from each treatment group were killed by asphyxiation. Seven days prior to sacrifice, an 

osmotic mini-pump filled with BrdU was implanted in each of five animals from each group. 

At sampling, livers were perfused with phosphate-buffered saline, removed, and weighed. A 

portion of liver was fixed in formalin, embedded in paraffin, and sectioned. The remaining 

livers were snap frozen in liquid nitrogen and stored at -80oC. Blood was collected in microcapillary tubes for haematocrit measurements. Liver sections were stained for ferric iron using 

the Perl method (Bugelski, 1985). Positive Perl staining was localized to Kupffer cells and 

approximately 2000 cells were counted for each animal. A “Perl index” was determined (total 

number of labelled Kupffer cells/ total number of cells counted x 100). Immunohistochemical 

detection of BrdU in liver was performed as described (Eldridge et al., 1990). Cells in 15 

random fields were counted (approximately 5000-6000 cells) in each animal. The labeling 

index was determined in both endothelial cells and hepatocytes (total number of labeled 

cells/total number of cells x 100). Endothelial cell identity was confirmed by staining a serial 

section with anti-factor VIII (Sharifi et al., 2000). Apoptosis and mitosis in the liver was 

defined and quantified as described (Bursch et al., 1985). The identification of apoptotic cells 

was confirmed using TUNEL. Cells in 15 random fields were counted (approximately 5000-

6000 cells) in each animal. Apoptotic and mitotic indicies were determined (total number of 

labeled cells/total number of cells x 100). DNA damage expressed as OH8dG was measured 

in DNA isolated by sodium iodide extraction (Wang et al., 1994) and MDA was measured in 

liver as described by Bagchi et al., (1993). 

EGBE decreased haematocrit and increased spleen weights in both rats (225 and 450 mg/kg 

bw dose groups) and mice (450 and 900 mg/kg bw dose groups). Kupffer cell Perl staining 

showed dose and time related increases in the Perl index that also occurred in both rats and 

mice. These observations confirmed that haemolysis and hepatic deposition of iron after 

treatment with EGBE occurred in males of both rats and mice. There was also a reduction in 

antioxidant reserve as a result of this treatment, because hepatic α-tocopherol concentrations 
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were decreased in both mice and rats at 7 and 90 days, although they were not significantly 

reduced at 14 or 28 days. An important difference between mice and rats is that control levels 

of α-tocopherol in this experiment were only about 1.5 nmol/g liver in mice, whereas they 

about 4 nmol/g liver in rats. Even after treatment, the reduced α-tocopherol levels in rats at 

both 7 and 90 days were higher than the control levels in mice. Thus, rats remained 

adequately protected from oxidative damage, whereas mice did not. 

Treatment of mice with EGBE doses of 450 or 900 mg/kg bw resulted in an increase in 

oxidative damage in both DNA (OH8dG) and lipid (MDA) at both the 7 and 90 day treatment 

points, but not at the intermediate times of 14 and 28 days. In contrast, neither OH8dG nor 

MDA concentration was increased in rats at any dose level or at any sampling time. 

DNA synthesis was increased in liver endothelial cells of mice at 7 and 14 days (all dose 

levels) while DNA synthesis in hepatocytes of mice was only increased at 90 days (all dose 

levels). No changes were seen at the other observations times in mice and no changes were 

seen in rats in either endothelial cell or hepatocyte DNA synthesis at any dose level or at any 

time point. No changes in either apoptosis or mitosis were observed in the liver of either 

species at any dose or time point. 

In summary, EGBE caused an increase in oxidative damage and DNA synthesis in mouse cell 

types that correspond to those from which the tumours were observed to arise, while no 

changes were seen in rats, which did not show an increase in either haemangiosarcomas or 

hepatocellular neoplasms (Siesky et al., 2002). 

Hepatocytes were isolated from male B6C3F1 mice and male F344 rats by a two-step in situ

collagenase perfusion, after which the cells were filtered and centrifuged (Kamendulis et al., 

1999; Park et al., 2002a). Viability, determined by trypan blue exclusion, was routinely 

greater than 90 %. The cells were plated and cultured overnight. The medium was changed 

and replaced with EGBE or BAA at concentrations of 1, 5 or 25 mM, or ferrous sulphate at 

concentrations of 1, 5 or 25 μM and incubations continued for 24 hr. Cytotoxicity was 

determined at that time by measurement of lactate dehydrogenase leakage into the medium 

and this compared with total lactate dehydrogenase in lysed cells. This experiment was 

performed on four independent occasions. For each of the substances, the lower two 

concentrations were not cytotoxic to either mouse or rat hepatocytes, whereas the highest 

concentration of each substance increased the concentration of lactate dehydrogenase in the 

medium in the range 1.6-fold to 2.6-fold. 

Using the same exposure conditions as in the previous in vitro study of cytotoxicity, 

measurements were made of OH8dG, MDA and α-tocopherol in three independent 

experiments. The summarised data are shown in Table 4.25. Neither EGBE nor BAA induced 

any increase in oxidative damage expressed as OH8dG or MDA at any concentration in either 

rat or mouse hepatocytes. In contrast, ferrous sulphate induced significant increases in both 

OH8dG and MDA of mouse and rat hepatocytes. A clear species difference in sensitivity of 

the hepatocytes was seen, with mouse hepatocytes being much more sensitive than rat 

hepatocytes. In rat hepatocytes, only the highest concentration of ferrous sulphate induced 

oxidative stress, while in mouse hepatocytes OH8dG was elevated from the lowest 

concentration, 1μM, and cellular MDA was increased and α-tocopherol decreased at 

concentrations of 5 and 25μM ferrous sulphate. MDA in the culture medium reflects the 

amount of lipid oxidation products expelled from the cells during exposure, in contrast to 

cellular MDA that only indicates what remains at the end of exposure. Medium MDA was 

elevated at all ferrous sulphate concentrations in mouse hepatocytes cultures, whereas only 
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the highest concentration of ferrous sulphate elevated medium MDA in the rat hepatocytes 

cultures. 

Further studies in this series demonstrated that there was no interaction between ferrous 

sulphate and either EGBE or BAA in the production of oxidative stress. Furthermore, when 

iron was presented as haemolysed erythrocytes (Table 4.76), there was a dose-dependent 

increase in oxidative stress in mouse hepatocytes, demonstrated as increases in OH8dG and 

MDA and a decrease in α-tocopherol concentration (Park et al., 2002b). 
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Table 4.76: Effects of EGBE, BAA and ferrous sulphate on indicators of oxidative stress in male B6C3F1 mouse and 

male F344 rat hepatocytes, mean & standard deviation & percentage of the control (100 %) (Kamendulis et al., 1999; 

Park et al., 2002a) 

Dose OH8dG α-Tocopherol MDA 

(cells) 

MDA 

(medium) 

Males mouse hepatocytes 

0 2.78±0.26 4.85±0.42 0.70±0.04 3.40±0.39 

EGBE (mM) 1.0 102 97 107 109 

 5.0 147 93 116 115 

 25.0 142 106 118 122 

BAA (mM) 1.0 106 90 98 96 

 5.0 149 86 104 111 

 25.0 132 94 109 112 

FeSO4 (μM) 1.0 151* 96 112 177* 

 5.0 228* 67* 127* 335* 

 25.0 360* 57* 241* 458* 

Male rat hepatocytes 

0 3.63±0.63 6.58±0.59 0.80±0.04 4.04±0.19 

EGBE (mM) 1.0 115 92 97 100 

 5.0 98 92 100 102 

 25.0 91 90 121 118 

BAA (mM) 1.0 153 94 102 103 

 5.0 140 94 106 103 

 25.0 122 91 114 120 

FeSO4 (μM) 1.0 123 93 116 115 

 5.0 141 79 115 108 

 25.0 188* 75 165* 163* 

* P < 0.05 

units:MDA, nmol/106

 cells or nmol/ml medium; α-tocopherol, ng/106 cells; OH8dG, 

pmol/μmol dG. 
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Table 4.77: Effects of haemolysed erythrocytes (RBCs) on oxidative stress parameters in mouse hepatocytes (Park 

et al., 2002a) 

Treatment MDA α-Tocopherol OH8dG 

Water 0.81 ±0.21 4.35±0.37 3.33±0.52 

0.1 x 106 RBCs 0.79±0.15 3.25±0.54 3.41±0.62 

1.0 x 106 RBCs 1.22±0.34* 2.15±0.63* 6.78±0.43* 

10 x 106 RBCs 2.36±0.44* 2.25±0.31* 13.40±1.02* 

units:MDA, nmol/106

 cells or nmol/ml medium; α-tocopherol, ng/106 cells; OH8dG, 

pmol/μmol dG 

* Significantly different from the control (p < 0.05) 

The effect of ferrous sulphate (to mimic the iron deposition in liver resulting from 

haemolysis) on morphological transformation of Syrian hamster embryo (SHE) cells was 

studied (Park et al., 2002b). Significant increases in transformation frequency were observed 

after exposure to concentrations of 2.5 and 5.0 μg/ml for 7 days. The greatest effect was a 

transformation frequency of 0.71 %, compared with a medium control frequency of 0.06 %, at 

2.5 μg/ml. Co-incubation of this ferrous sulphate concentration with antioxidants [25 or 50 

μM (-)-epigallocatechin-3-gallate or 150 μM α-tocopherol] significantly reduced the induced 

cell transformation frequency. Park et al. (2002b) also showed that 1.0 and 2.5 μg/ml ferrous 

sulphate induced DNA strand breaks in SHE cells, as demonstrated with the single-cell gel 

electrophoresis (Comet) assay, as well as the formation of OH8dG and that co-incubation 

with either 25 μM (-)-epigallocatechin-3-gallate or 200 μM α-tocopherol completely 

abolished both of these expressions of ferrous sulphate-induced DNA damage. 

In summary, these studies demonstrate that rat hepatocytes in culture are markedly less 

susceptible to oxidative stress than are mouse hepatocytes. In vivo there appears to be a 

greater antioxidant reserve in rat liver than in mouse liver, so that reduction in α-tocopherol 

levels observed in both species after EGBE treatment resulted in levels in rats that always 

remained higher than even the untreated control level in mice. Also in vivo, male mouse liver 

is marginally more susceptible than female mouse liver to oxidative damage, as demonstrated 

by the higher background rate of hemangiosarcoma lesions. In addition, it has been shown 

that ferrous sulphate can induce DNA damage as well as morphological transformation in 

SHE cells and these effects can be reduced or abolished by antioxidants. 

4.1.2.6.4 Mechanistic studies on forestomach pathology 

Green et al., 2000a, 2001 and 2002 have shown by whole body autoradiography that the 

administration of radiolabelled EGBE to mice by whole body exposure to its vapour results in 

a significant ingestion of the condensed substance during grooming. The autoradiographs 

showed that the fur and skin contained high concentrations of radioactivity even 48 h after the 

end of exposure and there was evidence of selective accumulation of radioactivity in the 

forestomach, the glandular stomach containing only low, background levels of radioactivity. 

Ingestion can, perhaps, also result from condensation of high vapour concentrations in the 

nasopharynx. In this way, EGBE can reach the stomach, where there is the potential to cause 
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the damage observed in the lifetime inhalation exposure study (NTP, 2000). Subsequently, the 

capacity of the stomach to metabolise EGBE was examined and a basis for the differential 

response of mice and rats investigated and comparison made between rodent and human 

stomachs. The study was not conducted to GLP guidelines. 

Although this qualitative study suggests that significant exposure of the forestomach to EGBE 

delivered as a vapour could occur as a result of ingestion of condensed material, this is not 

supported by the results of one quantitative study. In an inhalation experiment in which 

female mice were exposed (whole-body) to 229 ppm EGBE for 6 hr, an average of 205 ± 69 

μg EGBE was recovered from the fur of the animals, i.e., < 10 mg/kg bw, while the 

concentration of EGBE in the blood at the end of the exposure period was 3.0 mg/L and the 

BAA concentration was 235 mg/L (Poet et al., 2003). These blood concentrations were 

similar to those found in the inhalation exposure carcinogenicity assay (Dill et al., 1998). 

Thus, it would appear that some, but not a great deal of EGBE could reach the forestomach as 

a result of grooming. The authors do acknowledge, however, that some material could have 

been lost by evaporation and by the hot water washing procedure, which was adapted from a 

similar process that had been used for ethylene glycol, which is a less volatile material. 

Female B6C3F1 mice were given a single intravenous dose of 2-butoxy[1-14C]ethanol and 

killed at 4, 24 and 48 h after dosing and the distribution of radiolabel examined in the animals. 

High levels of radiolabel were present in the liver, oesophagus and buccal cavity throughout 

the time course of the study. Similar concentrations of radioactivity were found in both the 

forestomach and glandular stomach. The radioactivity in the stomach tissues was derived 

from the systemic circulation and, in addition, there was evidence of ingestion of 

radiolabelled material from the buccal cavity. The origin of the latter is unknown, but it may 

be derived from the salivary and Harderian glands, which contained significant amounts of 

radioactivity (Bennett, 2001). 

Other experiments of Green et al., 2001 and 2002 lend support to other aspects of the 

development of forestomach lesions. Groups of five female B6C3F1 mice were administered 

orally by gavage either EGBE or BAA daily for 10 days at dose levels of 0, 50, 150 or 500 

mg/kg bw per day (Green et al., 2001 and 2002). Seventeen hours after the last dose and 1 hr 

before they were killed, the mice received a subcutaneous injection of bromodeoxyuridine, 15 

mg/kg bw. Stomachs were removed for histology and quantification of cell proliferation, and 

blood samples were taken for haematology. In the top dose group of mice receiving BAA, one 

died as a result of causes unrelated to treatment. Haematological changes typical for these 

compounds were observed in all dose groups administered BAA, but of those receiving 

EGBE, they were largely confined to the 500 mg/kg bw per group. No abnormalities were 

seen in the glandular part of the stomach in any of the groups. No abnormalities were found in 

the forestomach of mice dosed with EGBE at doses of 50 or 150 mg/kg bw per day or with 

BAA at the 50 mg/kg bw dose level. The only abnormality observed in the forestomach was 

hyperkeratosis, which was minimal in two mice given EGBE at 500 mg/kg bw per day and in 

three mice given BAA at 150 mg/kg bw per day. In the 500 mg/kg bw per day group given 

BAA, all four mice that completed the study showed hyperkeratosis, three to a slight degree 

and one to a moderate degree. The cell proliferation part of the study was invalid because of a 

high control group value. Within the groups treated with either compound, there were dose 

related increases in S-phase cells in the forestomach areas. Furthermore, the development of 

hyperkeratosis is, in itself, evidence of increased cellular proliferation. 

As part of the same study, a group of 10 female mice were given oral doses of 2-(1-

[

14C]butoxy)ethanol, 500 mg/kg bw per day for 5 days, then killed 17 hr later. Glandular 

stomach and forestomach areas were removed, washed, separated and homogenised for 
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analysis. Homogenised washed forestomachs and glandular stomachs contained very little 

covalently bound radioactivity and none was identified as associated with particular protein 

bands following SDS-PAGE analysis. Therefore, it appears that covalent binding plays little, 

if any, role in the forestomach toxicity of EGBE. 

Untreated F344 rats and B6C3F1 mice were killed, their stomachs removed and divided into 

forestomach and glandular stomach sections. Pooled samples of the same tissue were 

homogenised, stored at -70° until required and then alcohol and aldehyde dehydrogenase 

enzyme kinetics determined, using EGBE and BAL, respectively, as substrates. Generally, the 

activities were similar in both regions of the stomach for both enzymes and there were no 

major species differences in aldehyde dehydrogenase activity. In the case of alcohol 

dehydrogenase, however, there were major differences between rats and mice, both in affinity 

constants (Km) and maximal rates (Vmax). The Km values were two orders of magnitude 

greater in both regions of the mouse stomach, compared to the rat, and Vmax was up to one 

order of magnitude higher in the stomach of the mouse than in the rat. The absence of any 

marked regional difference in enzyme activity in the homogenates would appear to fail to 

explain the regional differences in toxic and neoplasic response. However, histocytochemical 

localisation of the enzymes showed that, in the forestomach, the dehydrogenase activities are 

strongly localised in the stomach lining, whereas in the glandular stomach they are more 

homogeneously distributed throughout the stomach wall. Estimation of the relative volumes 

of each stomach compartment containing enzyme activity shows an approximately 1:25 ratio 

between forestomach and glandular stomach. Consequently, the enzyme activity is 

concentrated in a much smaller volume in the forestomach, so that, in a whole tissue 

homogenate, the activity is diluted (Green et al., 2001 and 2002). 

Independent experiments on the mode of action in the forestomach have been conducted 

elsewhere. Adult male and female B6C3F1 mice were treated orally by gavage with undiluted 

EGBE at dose levels of 0, 400, 800 or 1200 mg/kg bw for 2 days, followed by 0, 200, 400 or 

600 mg/kg bw for 2 days. The doses were reduced because of excessive mortality caused, 

apparently, but unexpectedly, by the original doses. Survival did not improve at these lower 

doses, so treatment was discontinued. Two other groups of mice were treated by either 

subcutaneous or intraperitoneal injection with EGBE in saline at a dose level of 400 mg/kg 

bw for 5 days. Dose related incidences of forestomach lesions occurred in all male and female 

mice after oral administration of undiluted EGBE. These lesions included focal hyperplasia in 

the low dose group and more diffuse hyperplasia at the higher doses associated with 

significant infiltration by neutrophils and mononuclear inflammatory cells in the submucosa 

and muscularis. There were increases in the proliferating cell nuclear antigen labelling in 

regions surrounding the areas of hyperplasia. Minimal lesions similar to those found in the 

low dose (200 – 400 mg/kg bw) oral administration group were found in 1/6 intraperitoneally 

dosed mice and in 2/6 subcutaneously dosed mice, following 4 days administration of 400 

mg/kg bw (Poet et al., 2003). 

As part of this same study, the tissue dose and pharmacokinetics of EGBE were also 

compared after an oral dose by gavage of 265 mg/kg bw (target dose 250 mg/kg bw) and an 

intraperitoneal injection of 53 mg/kg bw or 261 mg/kg bw (target doses 50 and 250 mg/kg 

bw). Regardless of the dose and the dose route, EGBE disappeared from the circulation within 

1 hr, while BAA was detectable up to 12 hr after a target dose of 250 mg/kg bw by either 

route. EGBE concentrations in the forestomach and glandular stomach tissues were higher 

than in blood and other tissues and EGBE persisted for a longer period in the stomach tissues, 

so that, regardless of the dose route, the t1⁄2 and AUC for EGBE were higher in forestomach 
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tissues than in any other tissues. These data and the corresponding data for BAA are shown in 

Table 4.78. 

In a separate experiment, the concentrations of EGBE and BAA in saliva were measured after 

oral and intraperitoneal dosing. The results are also shown in Table 4.78. Of particular 

significance is the relatively high concentrations of both compounds in the saliva after 

intraperitoneal dosing. 

Table 4.78: Halve lives and AUC in blood, saliva and tissues. (Poet et al., 2003). 

Route, dose in EGBE BAA 

mg/kg bw 

Tissue 

AUC (μg/g/h) t1⁄2 (h) AUC (μg/g/h) t1⁄2 (h) 

Ip (261 ± 34) Blood NA NA 442.8 1.05 

 Liver 4.52 0.66 317.0 1.10 

 Kidney NA NA 507.4 1.44 

 Forestomach 274.2 2.60 468.4 4.62 

 Glandular 

stomach 

34.7 1.86 289.6 1.31 

Oral (265 ± 13) Blood NA NA 1740.7 2.11 

 Liver 16.7 0.57 604.1 2.09 

 Kidney NA NA 847.8 2.71 

 Forestomach 3063.9 3.40 539.3 3.96 

 Glandular 

stomach 

746.5 4.91 186.6 1.57 

Ip (258 ± 66) Blood 43.4 0.16 2194.0 1.42 

 Saliva 66.1 0.17 143.2 0.69 

Oral (264 ± 42) Blood 27.1 0.35 900.6 1.55 

 Saliva 26.3 0.27 66.8 0.85 

Probably as a result of the secretion of EGBE in saliva, significant quantities of the compound 

can reach the forestomach contents, even after intraperitoneal dosing. 

Other, general factors contribute to the preferential toxicity to the forestomach and are 

unrelated to the actual compound being investigated. 

In rodents, including the mouse, rat, and Syrian hamster, the stomach is divided into two parts 

by the mucoepidermoid junction separating squamous from glandular epithelium. The 

proximal part, or forestomach, is non-glandular, continuous with the oesophagus, and lined by 

keratinised, stratified squamous epithelium. The distal part, or glandular stomach, empties 

into the duodenum and is lined by a specialized glandular epithelium. The forestomach is 

separated from the glandular stomach by a grossly visible, elevated fold, the limiting ridge. 

This contrasts with the anatomy of the human stomach, the lining of which is entirely 
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glandular, the human mucoepidermoid junction occurring where the oesophagus joins the 

stomach. There is thus no forestomach in human beings. There is a histological similarity 

between the rodent forestomach and the human oesophagus, but the physiological functions of 

these two organs are quite different. While the forestomach is a storage organ, where ingested 

material may reside for several hours before its transfer to the glandular stomach, the 

oesophagus is not, ingested material passing through in a matter of seconds. Exposure of the 

human (or rodent) oesophagus to ingested material is, therefore, brief and it is to be noted that 

the histology of the rodent oesophagus is not unlike that of the human organ, yet the 

oesophagus of the rats and mice exposed to EGBE in studies described here does not respond 

in the same way as the forestomach. 

4.1.2.6.5 Summary of repeated dose toxicity 

In rats and mice, haemolysis was consistently observed (whichever the route of 

administration) and was sometimes associated with hepatic effects (Kupffer cell pigmentation 

and absolute and relative liver weight increases), effects on body weight gain, hyaline 

degeneration of the olfactive epithelium (by inhalation), effects on the forestomach and 

effects on the WBC sub-populations (T lymphocyte). In these studies and for the inhalation 

route, no NOAEC was identified for mice, whereas a NOAEC value of 25 ppm (121 mg/m3

) 

in rats was identified. In a separate study a LOAEC value of 31 ppm (150 mg/m3

) can be 

established in rats, based on haemolysis and Kupffer cell pigmentation. Due to the closeness 

of the apparent LOAEC and NOAEC, it is considered prudent to take the more conservative 

LOAEC of 31 ppm forward for risk characterisation. However, the likelihood that this figure 

is close to the NOAEC will be taken into account in deriving appropriate assessment factors. 

Slight effects on the immune system were seen in rats, mice and humans on NK cells or T 

lymphocyte sub-population. In the human study, co-exposure to a number of chemicals does 

not allow reliable conclusions to be drawn for EGBE alone. In the rodent studies, however a 

NOAEL of 1000 mg/kg bw in mice by dermal route can be established. The effects seen were 

small. A role of EGBE in the induction of immunotoxicity has not been developed to a point 

where it can be used in risk characterisation. 

For the dermal route, a NOAEL of 150 mg/kg bw/d (the highest dose tested) has been 

determined from a 13-week study in rabbits. 

For the oral route, a LOAEL of 69 and 82 mg/kg/day for male and female rats respectively, 

was found in a 13 -week drinking water study(haemolytical effects). 

As humans are far less sensitive than other species (except Guinea Pig) to the haemolytical 

properties of EGBE, we have tried to assess separately haemolytical effects and related effects 

and other specific toxic effects which could be induced by EGBE. For all the studies, no 

specific relevant toxic effects, other that haemotoxicity, can be identified. 

For the risk characterisation, haemotoxicity will be the end point chosen keeping in mind the 

interspecies differences (human/rodents) to calculate margin of safety. No other lesion has 

been identified which can be specifically attributed to treatment with EGBE. 
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4.1.2.7 Mutagenicity 

The mutagenic properties of ethers of ethylene glycol and their metabolites in general and 

EGBE in particular have been reviewed (Elliott and Ashby, 1997 ; McGregor, 1984 and 1996; 

Tyler, 1982). 

4.1.2.7.1 Studies in vitro 

Bacterial studies

EGBE and its metabolites, BAL and BAA, have been tested for their potential to induce gene 

mutations in bacteria on a number of occasions; these and the results are listed in Table 4.79. 

Two studies in which five different strains of Salmonella typhimurium were used, null results 

were obtained with dose levels up to 20 mg per plate, in one case, and 10 mg per plate in the 

other. The latter was an Ames’ test conducted by the US National Toxicology Program (NTP) 

in which supplementary metabolic activation conditions were provided by rat liver S9 mixes 

at 10 % and 30 % and Syrian hamster liver S9 mixes at 10 % and 30 % (Zeiger et al., 1992). 

Nevertheless, it was concluded in a later study that EGBE was mutagenic as demonstrated by 

its activity in S. typhimurium TA97a, at a minimal dose of 2.2 mg per plate in the absence of 

S9 mix and 9.0 mg per plate in the presence of rat liver S9 mix at 2.5 %. Thus, the activity of 

EGBE was reduced by the presence of the supplementary activation system. The mechanism 

of this reduction was not investigated. The authors imply, however, by referring to the 

absence of mutagenic activity in tests with BAL and BAA, that the reduction in mutagenic 

activity of EGBE was due to its metabolism to nonmutagens (Hoflack et al., 1995). S. 

typhimurium TA97a is very similar to the strain TA97, the difference between the strains 

being in the length of the deletion through the uvrB locus. Consequently, important 

differences in the responsiveness of the strains should not have been expected. Both strains 

carry the hisD6610 mutation that has a run of six cytosines at the mutation site and requires a 

frame shift mutation for its reversion to histidine independence. Furthermore, EGBE is not the 

type of chemical structure that would be anticipated to produce a frameshift mutation. 

Gollapudi et al. (1996) performed a carefully designed study that covered the conditions used 

by Hoflack et al., 1995 and using S. typhimurium TA97a, TA100 and Escerichia coli

WP2uvrA. There was no indication of an increase, significant or not, at any EGBE dose level 

between 0.5 and 10.0 mg per plate. The possible contribution of peroxides to the 

mutagenicity observed in the Hoflack et al., 1995 study was discussed, but dismissed. The 

lack of an effect in strain TA 97a in the Gollapudi et al., 1996 study supports the absence of a 

mutagenic response from the closely related strain TA 97 in the Zeiger et al., 1992 

experiments. Nevertheless, the debate continues, because Hoflacket al. (1997) in a letter to the 

journal editor, reported that the activity in strain TA97a reported earlier was again observed 

upon re-testing in their laboratory. 

Mammalian cell studies

EGBE was tested for gene mutagenic activity in Chinese hamster ovary (CHO) cells at the 

hprt locus (Slesinski and Weil, 1980). Null responses were obtained in this assay, in which 

test compound concentrations used were up to 1 % (v/v), approximately 9 mg/ml, in the 

absence of S9 mix and up to 0.5 % (v/v), approximately 4.5 mg/ml, in the presence of S9 mix. 
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Null responses were also obtained by Chiewchanwit and Au (1995), at the gpt locus in CHOAS52 cells exposed to either EGBE at concentrations up to 0.9 mg/ml (relative survival 79 %) 

or BAL at concentrations up to 0.065 % (v/v) (relative survival 91 %), the next higher dose, 

0.2 %, being too toxic to permit its evaluation for mutagenicity. These cells were derived 

from hprt-deficient CHO cells and contain a single autosomal copy of the xanthine guanine 

phosphoribosyl transferase (gpt) gene, which is the bacterial equivalent of the mammalian 

hprt gene. It had been demonstrated previously in the same laboratory (Ma et al., 1993) that 

methoxyacetaldehyde, a metabolite of EGME, is mutagenic in CHO-AS52 cells, but not in the 

standard CHO cells. Methoxyacetaldehyde was used as a positive control in this study. 

However, Elias et al.,. (1996) reported that both EGBE and BAL induced significant increases 

in 6-thioguanine-resistant phenotypes (i.e., the hprt locus) in Chinese hamster lung V79 cells. 

Unfortunately, the data are inadequately described in this paper, there being no indication of 

variation or of survival. Reading from a graphical presentation (log dose scale), it appears that 

the reported significant response EGBE occurred at a concentration of about 9 mg/ml. This 

description is inadequate for evaluation purposes. 
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Table 4.79: Test for gene mutation induction in bacteria by EGBE and its metabolites 

Resulta Test system Source & purity of chemical 

Without exogenous metabolic system 

With exogenous metabolic system 

Doseb 

(LED

EGBEBacteriophage T4D mutation Not stated - (severe 

toxicity) 

NT 11

Salmonella typhimurium TA100, TA1535, TA1537, TA1538, TA98, reverse mutation 

Dupont de Nemours, USA (purity not stated) 

- - 

20,00

Salmonella typhimurium TA100, TA1535, TA1537, TA97, TA98, reverse mutation

Fisher Scientific, USA > 99 % 

- - 

10,00

Salmonella typhimurium TA100, TA98, TA102 reverse mutation 

Merck, Germany > 99 % 

- - 

14,00

Salmonella typhimurium TA97a, reverse mutation 

Merck, Germany > 99 % + + 2,20

Salmonella typhimurium TA97a, TA100 & Eschichia coli WP2uvrA reverse mutation 

Dow Chemical Co., USA 99.04 % 

- - 

10,00

BALSalmonella typhimurium TA100, TA98, TA97a, TA102 reverse mutation 

Chemical synthesis > 91 % 

- - 

7,00

BAA Salmonella typhimurium TA100, TA98, TA97a, TA102 reverse mutation 

Janssen Chimica, Belgium > 99 % 

- - 

1,00

a +, positive; –, negative; NT, not tested; b LED, lowest effective dose; HID, highest ineffective dose 
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In tests for the induction of sister-chromatid exchanges (SCEs) in CHO cells, it was 

concluded in a US NTP-sponsored study (NTP, 2000) that there was no significant induction 

by EGBE concentrations up to 3.5 mg/ml in the absence of S9 mix and 5.0 mg/ml in the 

presence of rat liver S9 mix, although a trend test was significant (thereby judged to be an 

equivocal result) in the absence of S9 mix in one of two trials, the other being clearly 

negative. Slesinski and Weil (1980) also did not show an increase in SCE frequency at 

concentrations up to 0.25 %. On the other hand, Elias et al. (1996) reported a small induction 

of SCEs in V79 cells and Villalobos-Pietrini et al. (1989) found a significant increase in SCEs 

in cultured human lymphocytes in the absence of S9 mix of approximately 2.5-fold at a 

concentration of 0.5 mg/ml, the lowest tested, rising to approximately 4.3-fold at 3.0 mg/ml, 

the highest tested. Elias et al. (1996) reported a much larger induction of SCEs in V79 cells 

treated with BAL, but no effect of BAA. The data were presented only in a graphical form 

with no indication of variation or survival. It is noted that the reports of significant increases 

in the frequency of SCEs by Villalobos-Pietrini et al. (1989) and Elias et al. (1996) were 

observed at lower concentrations than used in the studies of Slesinski and Weil, 1980 and 

NTP (2000), neither of which reported any significant change in SCE frequency. 

In three independent investigations, chromosomal aberrations were not induced in cultured 

mammalian cells. These included an investigation with CHO cells in which EGBE 

concentrations up to 5.0 mg/ml did not induce aberrations in two of 3 trials in the absence of 

S9 mix or in a single trial in the presence of S9 mix (NTP, 2000); an experiment with V79 

cells for which no experimental details or results were presented (Elias et al., 1996); and two 

experiments with human lymphocytes with concentrations up to 3.0 mg/ml (VillalobosPietrini et al., 1989) and 0.3 mM (Elias et al, 1996). It was reported that chromosomal 

aberrations (not including gaps) increased by BAL in both V79 cells and human lymphocytes 

by Elias et al. (1996). Once again, the data were presented only in a graphical form with no 

indication of variation or survival. A small increase in micronucleated cell frequency was 

observed, however, in V79 cells treated with EGBE at a concentration of least 1.0 mg/ml, 

where there were 1.50 % micronucleated cells compared with 0.89 % ± 0.24 % in the 

controls. A similar very small increase that was, nevertheless, statistically significant was 

observed with BAA (1.71 % at 0.66 mg/ml), while BAL treatment resulted in 1.91 % 

micronucleated cells at about 0.005 mg/ml and 48.13 % micronucleated cells at about 0.020 

mg/ml. Many of these cells contained 3 – 4 nuclei per cell and cells with several micronuclei 

in place of the parental nucleus. These latter observations are suggestive of aneuploidy. 

Investigation of the treated V79 cells showed that the percentage of hyperdiploid cells was 

increased by EGBE and by BAL in particular, whereas BAA had hardly any effect (Elias et 

al., 1996). These in vitro mammalian cell studies are summarised in Table 4.80. 

Other in vitro mammalian cell studies

Inhibition of gap-junctional intercellular communication was studied in a mixed culture of 

hprt+ and hprt- V79 cells. EGBE was active in the assay at non-cytotoxic concentrations, but 

neither BAL nor BAA showed any activity (Elias et al., 1996). 

In a test for morphological transformation of Syrian hamster embryo cells treated for 7 days, 

BAL up to 1.0 mg/ml was not active, but no details of the results were presented. It is not 

clear whether EGBE and BAA also were tested, but found to be inactive (Elias et al., 

1996).The text indicates that BAL was inactive, while a figure shows EGBE to be inactive. In 

a similar assay, but with a 24 hr exposure time, a significant result was recorded for two doses 

(1.0 and 1.25 mg/ml, with relative plating efficiencies of 70 and 53 %, respectively), but not 

for a higher dose (1.5 mg/ml at which the relative plating efficiency was 42 %) (Kerckaert et 
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al., 1996). This result was not repeated in another Syrian hamster embryo cell transformation 

study, in which both EGBE and BAA were tested at concentrations up to 20 mM for 7 days. 

No significant increase in transformation frequency was found with either compound, 

although at 8.0 mM EGBE the transformation frequency was 0.52 when the relative plating 

efficiency was 73.1 %, compared with a control value for the transformation frequency of 

0.27 (Park et al., 2002b). Experiments were also conducted with ferrous sulphate in this 

system. 

Poly(ADP-ribosyl)ation is a requirement for DNA base excision repair and chromosomal 

stability that is synthesised by the NAD+

-dependent enzyme poly(ADP-ribose) polymerase 

(EC 2.4.2.30). The effect of EGBE on the activity of this enzyme was studied in Syrian 

hamster embryo cells treated with methyl methanesulphonate. The glycol ether potentiates 

the clastogenic activity of this alkylating agent, perhaps by consuming NAD+

 when it is 

oxidised to the aldehyde, and the alkylating agent induces poly(ADP-ribose) metabolism. The 

study did not show that competition for NAD+ was responsible for potentiation by EGBE 

(Hoflack et al., 1997). 

In Table 4.80 there is listed a study of unscheduled DNA synthesis (UDS) by Slesinski and 

Weil, 1980. This is included for completeness sake. However, the scintillation counting 

method used to assay for incorporation of tritiated thymidine into DNA has been considered 

unacceptable for many years because of the possibility of a few cells escaping S-phase block 

and thereby distorting the results. Such complications of the test, should they occur, can only 

be recognised and avoided by silver grain counting with the use of a microscope. 
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Table 4.80: Tests for genetic and related effects in cultured mammalian cells by EGBE and its metabolites 

Resulta Test system Source & purity of chemical 

Without exogenous metabolic system 

With exogenous metabolic system 

Doseb 

(LED

EGBEUnscheduled DNA synthesis, rat primary hepatocytes (scintillation counting)*

Union Carbide Corp., USA 99.4 % ? 0.

Gene mutation, Chinese hamster ovary CHO cells, hprt locus 

Union Carbide Corp., USA 99.4 % 

- -

1 

Gene mutation, Chinese hamster ovary CHOAS52 cells, grt locus Aldrich, USA (purity not stated) - 7Gene mutation, Chinese hamster lung V79 cells, hprt locus 

Merck, Germany 99 % + 2

Sister chromatid exchange, Chinese hamster ovary CHO cells in vitro

Union Carbide Corp., USA 99.4 % 

- -

0

Sister chromatid exchange, Chinese hamster ovary CHO cells in vitro

Dow Chemical, USA > 99 % 

- - 

3,50

Sister chromatid exchange, Chinese hamster lung V79 cells in vitro

Merck, Germany 99 % ± 1

Sister chromatid exchange, human lymphocytes in vitro

Not stated 

+ 50

Chromosomal aberrations, Chinese hamster ovary CHO cells in vitro

Dow Chemical, USA > 99 % 

- - 

5,00

Chromosomal aberrations, Chinese hamster lung V79 cells in vitro

Merck, Germany 99 % 

- 

0
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Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and its metabolites Chromosomal aberrations, human lymphocytes in vitro

Merck, Germany 99 % 

- NT 0

Chromosomal aberrations, human lymphocytes in vitro

Not stated 

- NT 3,0

Micronuclei, Chinese hamster lung V79 cells in vitro

Merck, Germany 99 % ± NT 

Aneuploidy, Chinese hamster lung V79 cells in vitro

Merck, Germany 99 % ± NT 16

Cell transformation, Syrian hamster embryo cells, focus assay 

Merck, Germany 99 % 

- NT 20

Cell transformation, Syrian hamster embryo cells, focus assay 

Not stated 

+ NT 

Cell transformation, Syrian hamster embryo cells, focus assay 

Sigma Chemical Co., USA >99 % 

- NT 20 m

Inhibition of gap-junctional intercellular communication, Chinese hamster lung V79 cells in vitro

Merck, Germany 99 % + NT 

BALGene mutation, Chinese hamster ovary CHOAS52 cells, grt locus Aldrich, USA (purity not stated) - NT 7Gene mutation, Chinese hamster lung V79 cells, hprt locus 

Chemical synthesis 91.4 % + NT 2

Sister chromatid exchange, Chinese hamster lung V79 cells in vitro

Chemical synthesis 91.4 % ± NT 1

Chromosomal aberrations, Chinese hamster lung V79 cells in vitro

Chemical synthesis 91.4 % + NT 0

Chromosomal aberrations, human lymphocytes in vitro

Chemical synthesis 91.4 % + NT 0
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Micronuclei, Chinese hamster lung V79 cells in vitro

Chemical synthesis 91.4 % ± NT 

Table 4.80: (continued) Tests for genetic and related effects in cultured mammalian cells by 2-butoxyethanol and its metabolites Aneuploidy, Chinese hamster lung V79 cells in vitro

Chemical synthesis 91.4 % ± NT 0.

Cell transformation, Syrian hamster embryo cells, focus assay 

Chemical synthesis 91.4 % 

- NT 

Inhibition of gap-junctional intercellular communication, Chinese hamster lung V79 cells in vitro

Chemical synthesis 91.4 % 

- NT 

BAA Sister chromatid exchange, Chinese hamster lung V79 cells in vitro

Janssen Chimica, Belgium >99 % 

- NT 0

Chromosomal aberrations, Chinese hamster lung V79 cells in vitro

Janssen Chimica, Belgium >99 % 

- NT 

Micronuclei, Chinese hamster lung V79 cells in vitro

Janssen Chimica, Belgium >99 % + NT 2

Aneuploidy, Chinese hamster lung V79 cells in vitro

Janssen Chimica, Belgium >99 % 

- NT 0.

Cell transformation, Syrian hamster embryo cells, focus assay 

Spectrum Chemicals, USA >99% 

- NT 20 m

Inhibition of gap-junctional intercellular communication, Chinese hamster lung V79 cells in vitro

Janssen Chimica, Belgium >99 % 

- NT 

a +, positive; ±, weak positive –, negative; ? questionable result; NT, not tested; 

b LED, lowest effective dose; HID, highest ineffective dose 

* Scintillation counting is no longer considered a reliable method for measuring unscheduled DNA synthesis.
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4.1.2.7.2 Studies in vivo (table 4.81) 

Sprague-Dawley rats (3/group, sex not specified) weighing approximately 260 g were given a 

single dose (route not stated) of EGBE, 0 or 120 mg/kg bw and killed 24 hr later. The brain, 

testes, liver, spleen and kidney were removed and DNA extracted from each organ. The 

detection of 5-methyldeoxycytosine and hydrophobic DNA adducts was performed using the 

32P-postlabelling technique. No significant differences were found between the groups in the 

levels of methylated deoxycytosine, expressed as percentages of total cytidylic acid found in 

any organs. The percentages ranged from 3.4 % to 4.0 % in the controls and from 3.6 % to 4.0 

% in the treated group. Furthermore, no obvious differences were found in the tlc 

hydrophobic adduct maps of 32P-labelled nucleotides for any of the rat organs examined 

(Keith et al., 1996). 

A micronucleus induction test was performed with male F344/N rats (5/group) at dose levels 

selected on the basis of published toxicity information and the results of a preliminary rangefinding study. The rats were injected intraperitoneally three times at 24 h intervals with EGBE 

dissolved in phosphate-buffered saline at dose levels of 0, 7.03, 14.06, 28.12, 56.25, 112.50, 

225.00 or 450.00 mg/kg bw per day; the total dose volume was 0.4 mL. The positive control 

rats received injections of cyclophosphamide, 7.50 mg/kg bw per day. Rats were killed 24 hr 

after the final injection and smears prepared from femoral bone marrow, which were then 

fixed and stained. Two thousand polychromatic erythrocytes (PCEs) were scored for the 

frequency of micronucleated cells in each rat. Data were tabulated as the pooled mean of each 

exposure group and the standard error of the mean. They were analysed with a one-tailed 

Cochran-Armitage trend test and by pair-wise comparisons between each dosed group and the 

vehicle control group. Statistically significant results were recorded for P ≤ 0.025 with the 

trend test and P ≤ 0.025 divided by the number of dose groups with the paired comparison 

test. A final decision on the biological significance (the “final call”) was made on the basis of 

the statistical analysis, the reproducibility and the magnitude of the effects observed. Two of 

five rats in the 450 mg/kg bw dose group died. The frequency of micronucleated PCEs per 

1000 cells in the vehicle control group was 1.9 ± 0.2 and the range of means in the dose 

groups was from 1.2 ± 0.2 at 225.00 mg/kg bw to 2.2 ± 0.3 at 28.12 mg/kg bw and 2.2 ± 0.6 

mg/kg bw at 450.00 mg/kg bw. The trend test was not significant (P = 0.570). The PCE/NCE 

ratios were not presented in the report (NTP, 2000). 

Male and female F344 rats (treated, 5 – 8, controls 10 - 12/sex/group) were administered 

EGBE by gavage at a dose level of 0 or 250 mg/kg bw per day for 1, 2 or 3 days and then 

killed 24 or 48h after the last dose. Haemoglobinuria was observed. Progressive timedependent macrocytic, hypochromic, regenerative haemolytic anaemia was was observed in 

both males and females treated with EGBE. Additionally, EGBE treatment caused significant 

morphological changes in erythrocytes that were first observed just 24 h after a single dose, 

which included stomatocytosis, macrocytosis with moderate rouleaux formation and 

spherocytosis. These morphological changes became progressively more severe with more 

EGBE doses and included the occasional occurrence of schistocytes and ghost cells, rouleaux 

formation in rats of both sexes and, in female rats, an increased number of reticulocytes 

containing micronuclei. It appeared that the progression of haemolytic anaemia was more 

rapid in female rats than in males. In response to the anaemia, extramedullary haematopoiesis 

was noted in the spleen. The general pathological effects are described in more detail in 

section 4.1.2.2.3.2 (Ghanayem et al., 2001). 
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A micronucleus induction test was performed with CD-1 mice (4/sex/group/observation time) 

at dose levels selected on the basis of published LD50 information. The mice were injected 

intraperitoneally once with EGBE dissolved in Hank’s balanced salts solution at dose levels 

of 0, 150, 300, 600, 800 or 1000 mg/kg bw. The positive control mice received injections of 

cyclophosphamide, 25 mg/kg bw. Mice of the three lower dose groups were killed 24, 48 and 

72 hr after the injection, while mice of all other groups were killed at 24 hr after injection, and 

smears prepared from femoral bone marrow, which were then fixed and stained. A minimum 

number of 1000 polychromatic erythrocytes (PCEs) were scored for the frequency of 

micronucleated cells in each mouse. In addition, the ratios of PCE to mature normochromatic 

erythrocytes (NCEs) were determined among 2000 erythrocytes per mouse. Data were 

tabulated as the pooled mean of each exposure group and the standard deviation. They were 

analysed according to the criteria of Schmid (1976). Three male and three female mice of 

the1000 mg/g bw dose group died, the recorded result at this dose level therefore being based 

on just one mouse of each sex. The frequency of micronucleated PCEs per 1000 cells in the 

vehicle control group was 1.41 ± 0.43 and the range of means in the dose groups was from 

0.36 ± 0.19 at 300 mg/kg bw to 0.97 ± 0.39 at 800 mg/kg bw, both results being in the 24 hr 

samples. The mean result from the two survivors of the toxic, 1000 mg/kg bw dose was 2.46. 

There was no dose related change in the PCE/NCE ratio at either the 48 h or 72 h observation 

times from the common, 24 h control value of 1,53 ± 0. At 24 h, the ration was high at the 

lowest, 150 mg/kg bw dose level (2.19 ± 0.32) and progressively fell to the control level with 

increasing dose (300 mg/kg bw, 2.11 ± 0.25; 600 mg/kg bw, 1.79 ± 0.23; 800 mg/kg bw, 1.54 

± 0.34; and 1000 mg/kg bw, 1.71, mean of two samples). In comparison with other control 

group values in the same publication, none of the PCE/NCE ratios obtained with EGBE was 

outside of the control group range (Elias et al., 1996). 

In this study, mice were also treated with BAA at dose levels of 0, 50, 100 or 200 mg/kg bw, 

but sampling was restricted to 24 hr after the single injection. The frequency of 

micronucleated PCEs per 1000 cells in the vehicle control group was 0.00 for this particular 

experiment (but ranged upwards in other experiments in the series to 1.50 ± 0.46) and the 

range of means in the groups dosed with BAA was from 0.45 ± 0.03 at 50 mg/kg bw to 0.94 ±

0.5 at 200 mg/kg bw. BAA was toxic to bone marrow cells, there being statistically 

significant reductions in the PCE/NCE ratios from 1.79 ± 0.45 in the vehicle control group to 

0.78 ± 0.19 and 0.79 ± 0.13 in the 100 and 200 mg/kg bw dose groups, respectively. The 

authors concluded that BAA did not increase the frequency of micronucleated cells in mouse 

bone marrow (Elias et al., 1996). 

A micronucleus induction test was performed with male B6C3F1 mice (5/group) at dose levels 

selected on the basis of published toxicity information and the results of a preliminary rangefinding study. The mice were injected intraperitoneally three times at 24 hr intervals with 

EGBE dissolved in a total dose volume of 0.4 mL phosphate-buffered saline at dose levels of 

0, 17.19, 34.38, 68.78, 137.50, 275.00, 550.00 or 1100.00 mg/kg bw per day. The positive 

control mice received injections of cyclophosphamide, 10.00 mg/kg bw per day. Mice were 

killed 24 hr after the final injection and smears prepared from femoral bone marrow, which 

were then fixed and stained. Two thousand polychromatic erythrocytes (PCEs) were scored 

for the frequency of micronucleated cells in each mouse. Data were tabulated as the pooled 

mean of each exposure group and the standard error of the mean. They were analysed with a 

one-tailed Cochran-Armitage trend test and by pair-wise comparisons between each dosed 

group and the vehicle control group. Statistically significant results were recorded for P ≤

0.025 with the trend test and P ≤ 0.025 divided by the number of dose groups with the paired 

comparison test. A final decision on the biological significance (the “final call”) was made on 
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the basis of the statistical analysis, the reproducibility and the magnitude of the effects 

observed. All mice in the 1100.00 mg/kg bw dose group died, whereas all mice in the 550 

mg/kg bw dose group survived. The frequency of micronucleated PCEs per 1000 cells in the 

vehicle control group was 2.5 ± 0.2 and the range of means in the dose groups was from 2.3 ±

0.3 at 34.38 mg/kg bw to 3.8 ± 0.8 at 137.50 mg/kg bw. The trend test was not significant (P

= 0.236) and the paired comparison test gave P = 0.05, whereas P ≤ 0.008 was required for 

statistical significance (NTP, 2000). 

4.1.2.7.3 Summary of mutagenicity 

EGBE is not mutagenic in bacteria, not withstanding a significant response according to one 

report in S. typhimurium TA97a. This was not substantiated by another study specifically 

designed to investigate this finding. Neither BAL nor BAA were mutagenic in bacteria. Two 

of three mammalian cell mutation assays did not indicate any mutagenic activity for EGBE 

and a significant result was obtained in an assay using a very high concentration (20 mM) that 

was poorly reported. The same publication reported a significant result at 20 mM with BAL, 

whereas another study found no effect at concentrations up to 7.6 mM. There have been no 

mammalian cell mutation studies with BAA. 

There have been reports of significant activity of EGBE in tests for SCE induction and cell 

transformation, but, again, the results have been inconsistent. Furthermore, the significant 

SCE results could be artefacts due to cell cycle delay. There is also some indication of 

inhibition of gap-junctional intercellular communication in a single study with EGBE and its 

two major metabolites. A single assay for UDS induction used a technique that is now 

considered to be invalid, if a significant response is obtained. 

No evidence for chromosomal aberration induction has been found in a number of 

mammalian cell culture studies with EGBE, or in one with BAL or BAA, whereas weak 

aneugenic effects were obtained in the only available study with EGBE and BAL, but not 

with BAA. Micronuclei found in long exposure in vitro studies with BAL and, to a much 

lesser extent with EGBE itself, but not with BAA appear to be due to aneuploidy, rather than 

chromosomal breakage. 

In vivo, there is no evidence for micronucleus induction in bone marrow cells or interaction 

with DNA in several organs of rats. The possibility of non-disjunction occurring and not 

being detected in these assays appears to be remote, because BAA produced no evidence of 

aneugenicity in vitro. BAA is rapidly formed in vivo and is by far the most prevalent blood 

metabolite of EGBE, so exposure of possible target cells to either EGBE or BAL at high 

concentrations is brief. The balance of the evidence suggests that EGBE do not pose a 

significant mutagenic potential in vivo. 

 
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Table 4.81: In vivo tests in mammals for the genotoxicity of EGBE and its metabolites 

Resulta Test system Source & purity of chemical Doseb 

(LED

EGBEDNA adducts, Sprague-Dawley rat brain, kidney, liver, spleen & testis, in vivo (32Ppost-labelling) 

Merck, Germany 99 % 

- (at 24 h) 120 

or

Methylation level of DNA, Sprague-Dawley rat brain, kidney, liver, spleen & testis in vivo

Merck, Germany 99 % 

-

Methylation level of DNA, FVB/N transgenic mouse brain, kidney, liver, spleen & testis in vivo

Merck, Germany 99 % 

-

Micronucleus test, CD-1 mouse bone-marrow cells in vivo

Merck, Germany 99 % 

-

800 

i

Micronucleus test, B6C3F1 mouse bonemarrow cells in vivo Dow Chemical, USA >99 % - 550 

i

Micronucleus test, male F344/N rat bonemarrow cells in vivo Dow Chemical, USA >99 % - 450 

i

BAL

No data available 

BAA Micronucleus test, CD-1 mouse bone-marrow cells in vivo

Janssen Chimica, Belgium >99 % 

- 

200 

i

a +, positive; –, negative; NT, not tested; b LED, lowest effective dose; HID, highest ineffective dose 
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4.1.2.8 Carcinogenicity 

There appear to have been no epidemiological studies of a relationship between exposure to 

EGBE and human cancer, although there have case reports of testicular cancer and leukaemia 

in occupational circumstances that have involved co-exposure with other chemicals. There are 

only two experiments in rodents available, one in rats and one in mice, in which EGBE was 

administered by the inhalation route. 

4.1.2.8.1 Studies in animals 

Inhalation 

Rat 

The carcinogenic potential of EGBE was studied in an experiment in which it was 

administered by whole-body exposure of groups of 50 male and 50 female F344/N rats to 0, 

31.2, 62.5 or 125 ppm (0, 151, 302 or 604 mg/m3

) EGBE vapour (lot no. QP-921215-26D2, 

Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days per week for 

104 weeks. For haematological and bone marrow analyses, additional groups of 27 male and 

27 female F344/N rats were exposed to 0, 62.5 or 125 ppm for evaluation at 3, 6 and 12 

months and 9 male and 9 female rats were exposed to 31.2 ppm for 3 months (for 

haematological examination only) and 6 months (NTP, 2000). 

Rats were held in the testing laboratory for 18 days and were 7 – 8 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All rats were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study, after which they were 

recorded monthly from week 5 until week 89 and then every two weeks from week 92 until 

the end of the experiment. Clinical observations also were recorded monthly from week 5 

until week 89 and then every two weeks until the end of the experiment. Haematological 

examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 6-months and on 

a Roche COBAS Helios analyser at 12-months. Differential leukocyte and nucleated 

erythrocyte counts were made by microscopic examination of blood films. Bone marrow 

cellularity was made using a Coulter Model ZH counter, while cytological evaluations of bone 

marrow cell morphology and myeloid/erythroid ratios were made microscopically. 

Autopsies and microscopic examinations were made on all of the main study animals that had 

not been used for blood and bone marrow sampling. Organ weights were not measured. At 

autopsy, all organs and tissues were examined for grossly visible lesions and all major tissues 

were fixed and processed for histological examination. A quality assessment pathologist 

evaluated the slides from all tumours and potential target organs, which included bone 

marrow, forestomach, kidney, liver, lung, nose and spleen of males and females and the 

adrenal and clitoral glands of females. The slides and the preliminary quality assessment 

report were then submitted to a National Toxicology Program Working Group of pathologists. 

Survival of exposed male and female rats was similar to the chamber control groups, the mean 

numbers of survivors at the end of the experiment in each of the 0, 31.2, 62.5 and 125 ppm 

groups were 19, 11, 21 and 24 males and 29, 27, 23 and 21 females, respectively. No clinical 
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signs were attributed to EGBE exposure. Body weights of male and female rats in the 31.2 

and 62.5 ppm groups and male rats of the 125 ppm group were generally similar to the 

controls throughout the experiment. Body weights of female rats of the 125 ppm group were 

generally lower than those of the chamber control from week 17 until the end of the 

experiment. 

Non-neoplastic findings are described in section 4.1.2.6.1.3. 

The incidence of benign or malignant phaeochromocytoma (combined) of the adrenal medulla 

in females exposed to 125 ppm EGBE was not significantly increased compared to the 

chamber controls, but it did exceed the historical control range. There was only one malignant 

phaeochromocytoma, which occurred in the 125 ppm group. The data are summarised in 

Table 4.82. 

Table 4.82: Neoplastic and non-neoplastic lesions of the adrenal medulla in female F344/N rats 

 Chamber 

 control 31.2 ppm 62.5 ppm 125 ppm 

No. examined 50 50 49 49 

Hyperplasia (severity) 11 (1.9) 11 (2.3) 8 (2.1) 17 (2.5) 

Benign phaeochromocytoma 3/50 (6 %) 4/50 (8 %) 1/49 (2 %) 7/49 (14 %) 

Benign or malignant 

phaeochromocytoma 

3/50 (6 %) 4/50 (8 %) 1/49 (2 %) 8/49 (16 %) 

It was on the basis of these data that the authors of the report conclude that there was no 

evidence for carcinogenicity in male rats and equivocal evidence for carcinogenicity in female 

rats. The primary criterion used to distinguish phaeochromocytomas from medullary 

hyperplasia was the presence of mild-to-moderate compression of the adjacent tissue. Most of 

the phaeochromocytomas were small and not substantially larger than the more severe grades 

of adrenal medullary hyperplasia. Indeed, there was only one phaeochromocytoma that was 

graded as malignant. Therefore, it is considered that these data from female rats provide only 

the weakest of marginal evidence for carcinogenicity. 

Two nasal tumours were discovered: a chondroma in a 31.2 ppm male and an adenoma in a 

62.5 ppm male. In the absence of any preneoplastic changes, these are considered incidental 

findings. Although the incidence of hyaline degeneration was significantly increased in all 

exposed groups of male rats, the severity of the lesion was minimal and was not affected by 

exposure. 

Mouse 

The carcinogenic potential of EGBE was studied in an experiment in which it was 

administered by whole-body exposure of groups of 50 male and 50 female B6C3F1 mice to 0, 

62.5, 125 or 250 ppm (0, 302, 604 or 1208 mg/m3

) EGBE vapour (lot no. QP-921215-26D2, 

Dow Chemical, Plaquemine, LA, USA, purity > 99 %) for 6 hr per day, 5 days per week for 

104 weeks. For haematological and bone marrow analyses, additional groups of 30 male and 

30 female B6C3F1 mice were exposed to 0, 62.5, 125 or 250 ppm for evaluation at 3, 6 and 12 

months (NTP, 2000). 
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Mice were held in the testing laboratory for 18 days and were 7 weeks old when the study 

began. They were housed individually, water was available ad libitum and feed was available 

ad libitum except during the exposure periods. All mice were observed twice daily throughout 

the study. Body weights were recorded at the beginning of the study, after which they were 

recorded monthly from week 5 until week 93 and then every two weeks from week 94 until 

the end of the experiment. Clinical observations also were recorded monthly from week 5 

until week 93 and then every two weeks from week 94 until the end of the experiment. 

Haematological examinations were performed on an Ortho ELT-8/ds 9000 analyser at 3- and 

6-months and on a Roche COBAS Helios analyser at 12-months. Differential leukocyte and 

nucleated erythrocyte counts were made by microscopic examination of blood films. Bone 

marrow cellularity was made using a Coulter Model ZH counter, while cytological evaluations 

of bone marrow cell morphology and myeloid/erythroid ratios were made microscopically. 

Autopsies and microscopic examinations were made on all of the main study mice that had 

not been used for blood and bone marrow sampling. Organ weights were not measured. At 

autopsy, all organs and tissues were examined for grossly visible lesions and all major tissues 

were fixed and processed for histological examination. A quality assessment pathologist 

evaluated the slides from all tumours and potential target organs, which included bone 

marrow, forestomach, kidney, liver, lung, nose and spleen of males and females and the 

preputial gland, prostate, skin (prepuce), testis and urinary bladder of males. The slides and 

the preliminary quality assessment report were then submitted to a National Toxicology 

Program Working Group of pathologists. 

Survival of male mice exposed to 125 or 250 ppm was significantly less than that of the 

chamber control group, whereas survival in all other treated groups was similar to the 

chamber control group. The mean numbers of survivors at the end of the experiment in each 

of the 0, 62.5, 125 and 250 ppm groups were 39, 39, 27 and 26 males and 29, 31, 33 and 36 

females, respectively. No clinical signs were attributed to exposure to EGBE. Body weights 

of exposed male mice were generally less than those of the chamber control during the last 25 

weeks of the experiment. Body weights of female rats of the 250 ppm group were generally 

lower than those of the chamber control from week 30 until the end of the experiment, the 

difference being about 20 % for much of that time. Body weights of the 62.5 and 125 ppm 

group females were generally lower than the chamber controls from about week 60 until the 

end of the experiment. 

Non-neoplastic findings are described in section 4.1.2.6.1. 

There was a positive trend in the incidences of forestomach squamous cell papilloma and 

squamous cell papilloma or carcinoma combined in female mice. The incidences were 

significantly increased in the 250 ppm group, in which the only squamous cell carcinoma 

occurred (Table 4.83). These incidences exceeded the historical control range for female mice 

(9/973 = 0.9 % ± 1.1 %; range 0 % - 3 %). There was no significant increase in the incidence 

of these neoplasms in male mice, but they did exceed the historical control range for male 

mice (7/973 = 0.7 % ± 1.0 %; range, 0 % - 2 %). Again, there was one squamous cell 

carcinoma, this time in the 125 ppm group (Table 4.83). 
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Table 4.83: Neoplastic and non-neoplastic lesions of the forestomach in male and female B6C3F1 mice 

 Chamber 

 control 62.5 ppm 125 ppm 250 ppm 

Males 

No. examined 50 50 50 50 

Ulcer (severity) 1 (1.0) 2 (1.5) 9** (1.6) 3 (1.7) 

Epithelial hyperplasia (severity) 1 (3.0) 7* (2.3) 16** (1.8) 21** (2.3) 

Squamous cell papilloma or 

carcinoma (percentage) 

1 (2 %) 1 (2 %) 2 (4 %) 2 (4 %) 

Females 

No. examined 50 50 50 50 

Ulcer (severity) 1 (3.0) 7* (1.3) 13** (1.5) 22** (1.4) 

Epithelial hyperplasia (severity) 6 (1.8) 27** (2.0) 43** (2.4) 44** (2.9) 

Squamous cell papilloma or 

carcinoma (percentage) 

0 (0 %) 1 (2 %) 2 (4 %) 6* (12 %) 

* Significantly different from the chamber controls P ≤ 0.05; ** P ≤ 0.01 

In the liver of male mice there was a positive trend in the incidence of haemangiosarcomas 

that led to a significant difference between the 250 ppm group and the chamber controls 

(Table 4.84). The incidence at 250 ppm also exceeded the historical control range for this 

tumour in male mice (14/968 = 1.5 % ± 1.5 %; range, 0 % - 4 %). There was also a positive 

trend in the incidence of hepatocellular carcinomas that led to a significant difference between 

males of the 250 ppm group and the chamber controls. There was, however, no change in the 

incidence of hepatocellular adenomas and carcinomas combined, because of a reduced 

incidence of hepatocellular adenomas in the treated groups (Table 4.84). In female mice there 

was a single haemangiosarcoma, in the 62.5 ppm group, which was considered to be an 

incidental finding. Furthermore, the incidences of hepatocellular adenomas were reduced in 

the 125 and 250 ppm groups of female mice and there was no change in the incidence of 

hepatocellular carcinomas (Table 4.84). 

One possible explanation for the liver neoplasms is infection with Helicobacter hepaticus, 

which had been previously implicated in significant liver disease of mice, including 

hepatocellular neoplasms and haemangiosarcomas (Hailey et al., 1998). Investigation of liver 

samples from this experiment failed to demonstrate the presence of this bacterium, therefore 

the experiment was not confounded by H. hepaticus infection. 

The NOAEC for tumourigenicity in mice is 125 ppm, based on an increased incidence of 

haemangiosarcomas in males and squamous cell papillomas or carcinomas in females at 250 

ppm. 
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Table 4.84: Neoplastic and non-neoplastic lesions of the liver in male and female B6C3F1 mice 

 Chamber 

 control 62.5 ppm 125 ppm 250 ppm 

Males 

No. examined 50 50 49 50 

Kupffer cell, pigmentation, 

haemsiderin (severity) 

0 0 8** (1.0) 30** (1.2) 

Haemangiosarcoma 0/50 (0 %) 1/50 (2 %) 2/49 (4 %) 4/49* (8 %) 

Hepatocellular adenoma 22 18 18 17 

Hepatocellular carcinoma 10 11 16 21** 

Hepatocellular adenoma or 

carcinoma 

30 24 31 30 

Females 

No. examined 50 50 49 50 

Kupffer cell, pigmentation, 

haemsiderin (severity) 

0 5* (1.0) 25** (1.0) 44** (1.0) 

Haemangiosarcoma 0 1 0 0 

Hepatocellular adenoma 16 8* 7* 8* 

Hepatocellular carcinoma 10 12 13 10 

Hepatocellular adenoma or 

carcinoma 

22 16 18 18 

* Significantly different from the chamber controls P ≤ 0.05; ** P ≤ 0.01 

Transgenic mouse models 

A study for increased tumour incidence or decreased tumour latency was conducted in FVB/N 

trangenic mice (Oncomice Neo 01TM), which carry the viral Harvey ras (v-Ha-ras) oncogene 

controlled by the mouse mammary tumour virus (MMTV) promoter. These are mice that 

present a stable, heritable mutation in the Ha-ras gene. The endpoints of the assay can be 

achieved, in principle, by both genotoxic and non-genotoxic agents, but the limitations of the 

assay have not been defined. Two groups of seven-week old mice (25/sex/group) were 

dorsally implanted with subcutaneous minipumps (2002 AlzetTM, Alza Corp.) delivering 

either saline or EGBE continuously at a rate of approximately 120 mg/kg bw perday for two 

weeks. Sub-groups of mice were killed for histological examination on days 5, 10, 15, 60 and 

120 after implantation of the minipumps. Special attention was given to the development of 

mammary gland masses. There was no adverse effect upon either food or water consumption. 

At the 120-day sampling time, the mammary glands of six control female mice and five 

treated mice showed cellular hyperplasia. In addition, a control female mouse had a papillary 

cystic mammary adenoma and a treated mouse had an anaplastic carcinoma of the bladder 

(Keith et al., 1996). The authors conclude that EGBE does not promote initiated cells carrying 
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the v-Ha-ras oncogene. There is some uncertainty about the numbers of mice used in the 

experiment and each sub-group. 

Dermal 

No data were available 

Oral 

No data were available 

4.1.2.8.2 Studies in humans 

No data. 

Mechanism of haemangiosarcomas formation in male mice and its significance for 

human health

Haemangiosarcomas, which arise from the endothelial cell component of the liver (Frith and 

Ward, 1979), have been observed to increase in incidence in male mice treated with EGBE, 

but not in female mice or rats of either sex at high exposure concentrations of EGBE. The 

apparent species and sex specificity of this response may impact upon the human risk 

assessment process so possible mechanisms for the induction of these tumours are addressed 

in this section. The compound-specific experimental data are described in preceding sections 

of this document. 

While some in vitro studies for genotoxicity have reported significant responses to exposure 

to EGBE or its metabolites, others have not and there is no evidence from in vivo studies for 

clastogenic activity or for covalent interactions with DNA. It is considered, therefore, that 

there is a lack of evidence for a role for genotoxicity induced by EGBE or its metabolites in 

the neoplastic process. 

A possible alternative mechanism of haemangiosarcomas induction can be based on the 

haematotoxicity of the major urinary metabolite, BAA. It has been established that BAA is 

the metabolite that induces haemolytic anaemia in mice of both sexes. However, it also 

induces anaemia in rats, this species being slightly more sensitive than mice. For the 

haemolysis hypothesis to be sustained, there should be, therefore, other species and sex 

differences amongst the rodents that are important components of this mechanism. 

Following the haemolysis produced by exposure to EGBE, haemosiderin is deposited in 

several cell types in liver, including Kupffer cells and hepatocytes of mice and rats. In 

addition, it has been recognised that murine endothelial cells have a significant phagocytic 

activity (Steffan et al., 1986), which may, therefore, serve as a mechanism by which insoluble 

iron complexes or senescent erythrocytes can enter these cells. Ferrous iron in haemosiderin 

can undergo redox cycling, the oxidative portion of which produces ferric iron and the highly 

reactive and damaging hydroxyl radical according to the Fenton reaction: 

 Fe(II) + H2O2 → Fe(III) + •

OH + OH-
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The possible development of oxidative stress as a result of these iron deposits was studied in 

hepatocytes of male rats and mice (see Mechanistic studies of liver pathology, above), with 

the conclusion that rat hepatocytes in culture are markedly less susceptible to oxidative stress 

than are mouse hepatocytes. The antioxidant capacity of the endothelial cells of the liver of 

rats is much less than that found in either the hepatocytes or the Kupffer cells (DeLeve, 1998; 

Sporalics, 1999). Therefore, if there should be similar haemosiderin deposition in endothelial 

cells, Kupffer cells and hepatocytes, the endothelial cells would be the least well protected 

and thereby suffer the greatest oxidative damage. An in vivo study (Siesky et al., 2002) has 

demonstrated that treatment of male mice with EGBE at dose levels (orally administered by 

gavage) equivalent to those that were associated with increased incidences of 

haemangiosarcomas in a 2-year inhalation experiment leads to oxidative damage to hepatic 

DNA and lipid and increased DNA synthesis, particularly in endothelial cells, but also in 

hepatocytes. None of these effects occurred in rats. The in vivo and in vitro demonstrations of 

species differences in susceptibility to oxidative stress are, therefore, consistent with each 

other and the hypothesis that oxidative stress is an essential component of the process leading 

to haemangiosarcomas development in male mice treated with EGBE. 

In addition, an in vivo study of possible differences between male and female mice does 

suggest that there is a marginally greater susceptibility of male mouse liver to oxidative 

damage than of female mouse liver. This sex difference does not seem to be large in terms of 

measurements of either acute oxidative DNA damage or lipid peroxidation, but there did 

remain a larger reserve of antioxidant potential in the females than in the males. Accordingly, 

it would not be possible to predict with any assurance that haemangiosarcomas would develop 

in males, but not in females. Indeed, the neoplastic response was not large even in the male 

mice, so it could be that a particular critical level of oxidative damage had been surpassed in 

the males, but had not been attained in the females. Deguchi et al. (1995) showed that the 

presence of female sex hormones imparts higher antioxidant properties than do male sex 

hormones. This same group (Okada, 1996) also found much lower levels of lipid peroxidation 

in female mouse kidney than in male mouse kidney. This result demonstrates that there is a 

higher level of protection in female than in male mouse kidney. It also demonstrates that sexrelated differences in susceptibility to oxidative damage are not restricted to the liver and, 

therefore, the consequences of such damage may show a sex-related incidence pattern in other 

organs of the body. 

In response to BAA, human erythrocytes in culture are clearly much less sensitive than the 

rodent cells by at least an order of magnitude. Furthermore, haemotoxicity was not observed 

in the two available occupationally exposed people or in volunteers under controlled 

conditions to EGBE (see 4.1.1.1). Exceptionally high exposure to EGBE, as encountered in a 

single case of attempted suicide in an adult (estimated dose 4500 mg/kg bw), did show that it 

is possible for human erythrocytes to be damaged as a result of exposure to EGBE, but the 

effects require doses that are close to lethal (they were not seen in other attempted suicide 

cases where the estimated doses were at least 1000 mg/kg bw) and are not encountered in 

occupational circumstances or in normal consumer use (see 4.1.1.2). Consequently, they 

should not be considered in an assessment of risk. In cases of accidental poisonings of 

children, no evidence of haemolytic effects has been found. Therefore, if haemolysis is an 

essential requirement for the induction of haemangiosarcomas by EGBE then man is not a 

susceptible species. 

The data available are consistent with the proposal that haemangiosarcomas observed in male 

mice could arise in mice of both sexes as a result of haemolysis leading to haemosiderin 

deposition. These deposits form nuclei for oxygen radical production that can damage many 
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cellular components, including DNA, unless there is sufficient antioxidant protection. When 

this deposition in the sinusoidal cells of the liver reaches a certain level, the oxidative defence 

mechanisms available to the cells are overwhelmed, creating the conditions for neoplastic 

responses in the endothelial cells of the hepatic blood vessels. Since man is much less 

sensitive to the haemolytic effects of EGBE, damage to blood cells not having been observed 

except in cases of very high exposure found in attempted suicides, the low level of 

haemangiosarcomas induced in male mice, but not in either female mice or in rats of either 

sex might have no significance for human risk assessment. The weakness in this argument is 

that haemosiderin deposition was reported to occur in Kupffer cells, whereas its occurrence or 

otherwise in sinusoidal endothelial cells has not been reported. However, it has been 

demonstrated that active oxygen species can migrate from Kupffer cells to the adjacent 

endothelial cells (Klaunig, 2004), and an endothelial cell response, in the form of 

proliferation, has been demonstrated in male mice, but not male rats (Siesky et al., 2002), at 

dose levels that are associated with an increased incidence of haemangiosarcomas in male 

mouse liver. 

Relevant information from studies not involving EGBE

The process by which haemangiosarcomas incidence is increased in male B6C3F1 mice 

treated with EGBE has been presented as a compound-specific mechanism. Elements of this 

mechanism may be applicable to other chemicals that induce haemangiosarcomas, but it is 

extremely unlikely that generalisations can be made that include all of the steps described. 

This is because of: 

• differences in metabolism and kinetics between species, sexes and routes of 

administration for specific compounds; 

• the interplay of other toxicological processes (e.g., genetic toxicity) that may modify 

fundamentally, or to a lesser degree, the pathway leading to haemangiosarcomas 

development; 

• chance differences in incidence between sexes (particularly when the incidence is 

low); 

• missing information, such as the lack of haematological data (particularly in older 

studies). 

Results have been summarised (Table 4.85) on chemicals tested for carcinogenicity in 

B6C3F1 mice and/or F344/N rats for which evidence of haemosiderin deposition is available 

in relatively complete reports that are in the public domain (specifically, NCI and NTP 

reports). In compiling this list, it was evident that some chemicals did not cause haemosiderin 

deposition in the liver, but did so in other organs. These have not been included. Also, in the 

columns listing the haemangiosarcoma induction response, it is stressed that the incidences 

refer only to the liver. Thus, the emphasis is on the relationships between haematotoxicity, 

haemosiderin deposition and haemangiosarcoma in the liver. In this organ, it was expected on 

the basis of the current hypothesis that, while the reasons for haematotoxicity might be 

different after exposure to different chemicals, the hepatic response to hepatic deposition of 

haemosiderin should be the same for any chemical, including EGBE. In other organs, 

modifying factors may prevail. The spatial relationships between the phagocytic cells and the 

cell populations in which the neoplastic response arises might be different from those found 

between Kupffer cells and hepatic sinusoidal endothelial cells; there may also be differences 
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in protective capacity, e.g., splenic phagocytes have a higher antioxidant capacity than do 

sinusoidal endotheliam cells (DeLeve, 1998). 

The data show that mice are more susceptible to haemangiosarcoma development than are 

rats, and male mice are more sensitive than female mice. None of the chemicals that are listed 

in section C (male mice) of table 4.85 induced haemangiosarcomas in section D (female mice) 

and only those chemicals in section C that demonstrably induced a life-long exposure of the 

liver to Kupffer cell pigmentation also induced increases in hepatic haemangiosarcoma 

incidence. At first glance, the main conclusion to be reached is that most of the data remain 

consistent with the suggested mechanism, in that significant haemosiderin deposition does not 

occur in cases where there is an absence of an increased incidence of haemangiosarcomas. 

However, some results require further investigation. It is not suggested that haemosiderin 

deposition is the only mode of action that can result in haemangiosarcomas. The highest 

incidence of haemangiosarcomas occurred in male mice gavaged with pentachloroanisole. In 

this series of experiments, the Kupffer cell pigmentation observed at 13 weeks did not contain 

iron, bile or PAS-positive material. It appears that special staining techniques were not 

applied to the pigment found in the 2-year study, therefore it is not known if it was 

haemosiderin or the non-iron pigment found in the 13 week study. It is also noted that 

material was more prevalent in hepatocytes than in Kupffer cells. If it were not haemosiderin 

then pentachloroanisole would cease to be of use in looking for support for the mechanism. 

Another case requiring discussion is C.I.Pigment Red 3, which did not induce 

haemangiosarcomas in either mice or rats, but haemosiderin deposition was observed in 

spleen and a “green-brown pigment” occurred in Kupffer cells. The latter was not identified 

as haemosiderin in the report and, if it were not haemosiderin, then C.I.Pigment Red 3 would 

cease to be of use in looking for support for the proposed mechanism. Furthermore, if 

haemolytic anaemia was produced by C.I.Pigment Red 3 in mice, it was transient – unlike in 

rats – as suggested by the haematological changes observed at the end of the preliminary 2-

week study, but not at the end of the 13-week study (p.62 of NTP TR 407). This contrasts 

with the haematotoxic activity of EGBE, which persisted for at least 12 months (the longest 

period examined). This does lend some support to the hypothesis that life-long exposure of 

the liver to Kupffer cell pigmentation is required as a pre-requisite for an increase in liver 

haemangiosarcoma rates in male mice. 

In the case of methyleugenol, haemosiderin deposits were found in the livers of female mice, 

but not of male mice or in rats of either sex. Haemangiosarcomas were not induced, whereas 

there were increases in hepatocellular neoplasms. Although the mice in this study were 

infected with Helicobacter hepaticus, it was argued that this did not compromise the outcome. 

Even in the EGBE study, the increase in haemangiosarcomas incidence in male mice was 

modest and did not occur in female mice; therefore, the absence of these neoplasms in female 

mice of the methyleugenol study does not conflict with their proposed mechanism of 

induction by EGBE. 

The conclusion that can be reached is that, in addition to EGBE, only two compounds support 

the pattern of chronic haemosiderin deposition and increased incidence of 

haemangiosarcomas in male mice but not female mice, these being p-chloroaniline and pnitroaniline. Another group has recently studied the possible association liver hepatic 

haemangiosarcomas and chemically induced haemosiderosis in mice in the US NTP database 

(Nyska et al., 2004) and reached essentially the same conclusion. In addition to the 

qualitative association described here, however, Nyska et al. (2004) also showed that there 

was a very high statistically significant association between hepatic haemangiosarcomas and 
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Kupffer cell pigmentation.(p < 0.001). In all cases, the cause of the haemosiderosis was 

haemolysis due to the chemical. 

There are very few rodent studies with other compounds that are capable of providing support 

for the hypothesis proposed to explain the low level increased incidence of 

haemangiosarcomas in male mice exposed to EGBE. The available evidence is, however, 

consistent with the hypothesis. Currently, the most likely mechanism is that proposed, based 

on metabolism to BAA, followed by rodent erythrocyte-sensitive haemolysis, deposition of 

iron-protein complexes in the liver and the sustained generation of toxic (cytotoxic or DNA 

damaging) radicals from this source. 

Human studies have demonstrated an enormously increased risk for hepatocellular cancer in 

hereditary haemochromatosis (e.g., Niederau et al., 1985 found an increased risk of 219, 

based on 16 cases) that has been attributed to the toxic effects of iron, but there also seems to 

be slightly increased risks for extra-hepatic cancers. Some studies have found genetic 

associations between several cancers and the most common mutation, C282Y, of the HFE 

gene that causes hereditary haemochromatosis. Such associations only occur, however, in the 

presence of a particular allele of the transferrin receptor gene. This suggests that the increased 

cancer risk is due to the effects of iron (Dorak et al., 2002). Additionally and independently of 

the genetic factors, dietary iron appears to be a risk factor for some cancers, e.g., colorectal 

cancer (Nelson, 2001), and dietary iron overload has been associated with about a 10-

foldgreater risk of hepatocellular carcinoma among black Africans (Mandishona et al., 1998). 

Focussing specifically upon liver angiosarcoma, however, the frequently quoted risk factors 

for this rare human cancer (0.5 – 2.5 cases per 107

 people), which include hereditary 

haemochromatosis, contribute to explain no more than about 20 % of the published cases. 

Indeed, apart from occupational exposure to vinyl chloride, the aetiology of this cancer 

remains largely unknown (Zocchetti, 2001). Therefore, it is not possible to conclude whether 

iron plays any role in human angiosarcoma. 
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Table 4.85: Summary of relationship between chemicals inducing haematoxicity and haemangiosarcomas in B6C3F1 mice and F344 ra

and high dose groups, respectively. 

A. Male Rats Chemical Haematology 

indicating toxicity to erythrocytes 

Incidence of Kupffer cell pigmentation (KCP) or hepatic haemosiderin (HS) at 2 years 

Kupffer cell pigmentation (Kor hepatic haemosiderin observed at 3 months 

EGBE Yes, at 14 wks KCP: 23/50, 30/50, 34/50, 42/50 KCP o-Nitroanisole Yes, at 2 & 13 wks KCP: 0/20, 1/20, 18/20 KCP p-Chloroaniline Yes, at 13 wks HS: 1/49, 0/50, 0/50, 26/50 KCP Pentachloroanisole Normal at 13 wks. KCP: 0/50, 1/50, 4/50 None (hypertrophy of Kupffer cPyridine Yes, at 13 wks KCP: 4/50, 11/50, 20/50, 25/50 HS, but cell-type not identified Titanocene dichloride Yes, at 2 wks KCP: 1/60, 39/60, 41/60 None D&C Yellow No. 11 No haematology KCP: 7/50, 15/51, 23/51, 26/54 KCP Butyl benzyl phthalate KCP, 0/59, 1/58, 6/59, 6/58 Cupferron (Nhydroxy-N-nitrosobenzenamine) No haematology. HS: 0/49, 15/48, 28/43 No data No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol, 2,4-diaminophenol.2HCl, CI Pigment Re
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B. Female Rats 

Chemical Haematology 

indicating toxicity to erythrocytes 

Incidence of Kupffer cell pigmentation (KCP) or hepatic haemosiderin (HS) at 2 years 

Kupffer cell pigmentation (Kor hepatic haemosiderin observed at 3 months 

EGBE Yes, at 14 wks KCP: 15/50, 19/50, 36/50, 47/50 KCP o-Nitroanisole Yes, at 2 & 13 wks KCP: 8/20, 2/20, 20/20 KCP Pentachloroanisole Normal at 13 wks. Pigmentation in hepatocytes None (hypertrophy of Kupffer cPyridine Yes, at 13 wks KCP: 6/50, 2/50, 6/50, 17/50 HS, but cell-type not identified Titanocene dichloride Yes, at 2 wks KCP : 3/60, 45/61, 50/60 None CI Pigment Red 3 Yes, at 2 & 13 wks. KCP : 0/50, 3/50, 14/50, 41/50* Pigment (type & cell-type 

identified) 

D&C Yellow No.11 No haematology KCP: 9/50, 11/51, 16/50, 32/51 KCP Butyl benzyl phthalate KCP: 4/60, 1/60, 6/60, 10/60 Cupferron (Nhydroxy-N-nitrosobenzenamine) No haematology HS: 0/48, 9/44, 33/44 No data No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol (anaemia at 14 wks), 2,4-diaminophenol.2
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C. Male Mice 

Chemical Haematology 

indicating toxicity to erythrocytes 

Incidence of Kupffer cell pigmentation (KPC) or hepatic haemosiderin (HS) at 2 years 

Kupffer cell pigmentation (Kor hepatic haemosiderin observed at 3 months 

EGBE Yes, at 14 wks KCP: 0/50, 0/50, 8/50, 30/50 KCP p-Chloroaniline Yes, at 14 wks. KCP/HS: 0/50, 0/49, 0/50, 50/50 KCP Pentachloroanisole No haematology KCP: 1/50, 50/50, 50/50 KCP (but not containing iron) p-Nitroaniline Yes, at 2 & 13 wks KCP: 1/50, 1/50, 8/50, 50/50 KCP o-Nitroanisole Yes, at 13 wks KCP: 0/50, 0/50, 3/50, 16/50 No CI Pigment Red 3 Normal at 13 wks KCP : 0/50, 5/50, 30/50, 41/50* No 2,4-Diaminophenol.2HCl 

No haematology at 13 wks. Yes, at 15 months 

KCP: 0/50, 44/50, 47/50 No 

No significant haemosiderin/pigmentation deposition was observed with: Methyleugenol, Pyridine, Cupferron 
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D. Female Mice 

Chemical Haematology 

indicating toxicity to erythrocytes 

Incidence of Kupffer cell pigmentation (KPC) or hepatic haemosiderin (HS) at 2 years 

Kupffer cell pigmentation (Kor hepatic haemosiderin observed at 3 months 

EGBE Yes, at 14 wks KCP: 0/50, 5/50, 25/50, 44/50 KCP p-Chloroaniline Yes, at 14 wks KCP: 0/50, 0/50, 1/50, 46/50 Pentachloroanisole No haematology KCP: 0/50, 37/50, 48/50 p-Nitroaniline Yes, at 2 & 13 wks KPC: 1/50, 1/50, 4/50, 39/50 Methyleugenol No haematology HS: 0/50, 11/50, 24/50, 19/50* CI Pigment Red 3 Normal at 13 wks KCP: 2/50, 1/50, 1/50, 29/50* 2,4-Diaminophenol.2HCl 

No haematology at 13 wks. Yes, at 15 months 

KCP, 0/50, 31/50, 50/50 

No significant haemosiderin/pigmentation deposition was observed with: Pyridine, Cupferron, o-Nitroanisole *See discussion in the text.  **NTP Technical Report Numbers 
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Mechanism of forestomach tumour formation in mice and its significance for human 

health. 

Squamous cell papilloma and carcinoma of the forestomach have been induced in female 

mice, with some evidence (that does not reach a level of significance) for an increase in male 

mice. In contrast, there is no evidence for the induction of forestomach tumours or even 

preneoplastic effects, such as hyperplasia, in rats of either sex in the 2 year study, although 

hyperplasia was observed in female rats in a 14 week study in which higher exposure 

concentrations were used. 

EGBE is a liquid with low vapour pressure that can therefore deposit on the fur of animals in 

whole body exposure experiments. The inhaled vapour may also condense in the 

nasopharynx. By both this mechanism and as a result of grooming contaminated fur, EGBE 

can achieve a significant exposure by the oral route, even in inhalation experiments. Although 

this is a plausible explanation for the toxic and tumourigenic effects of EGBE on the 

forestomach, only small amounts of EGBE ( < 10 mg/kg) were found on the fur of mice at the 

end of 6 hr, whole body exposures to 250 ppm (the highest concentration used in the NTP 

carcinogenicity experiment). In addition, dosage of mice by either intraperitoneal or 

subcutaneous injection also resulted in forestomach lesions. It is clear, therefore, that EGBE 

accumulates in the forestomach as a result of a combination of processes. In addition to 

grooming of EGBE present on the fur of the mice and, possibly, mice licking the walls of the 

exposure chamber, it is likely that salivary excretion as well as mucociliary transport of 

material deposited in the bronchi, followed by ingestion, are other means by which exposure 

of the stomach could have occurred during the study. 

The stomach of rats and mice consists of a forestomach (not found in man) and a glandular 

portion. The forestomach acts as a storage organ, where ingested material can remain for 

several hours, before it is transferred to the glandular portion in which there is a rapid transit 

and the first stages of digestion occur. Again, this is not the only mechanism that results in 

prolonged exposure of this organ. EGBE and, to a lesser extent, BAA is also eliminated more 

slowly from forestomach tissue than from either blood or other tissues following either oral 

gavage or intraperitoneal injection. Consequently, there is greater potential for damage to be 

induced by toxic substances in the forestomach than in the glandular stomach. 

Study of the stomachs of mice orally administered either EGBE or BAA shows that, at the 

doses used, there was no damage to the glandular stomach by either compound and that 

damage in the form of hyperkeratosis occurred in the forestomach at lower doses of BAA than 

of EGBE. There is significant alcohol and aldehyde dehydrogenase activity in both parts of 

the stomach in rats and mice. While there was little difference in the activity of aldehyde 

dehydrogenase in rats and mice, there were major species differences in alcohol 

dehydrogenase activity, the mouse enzyme having very much higher affinity constants and 

maximal rates than the rat enzyme, when using EGBE as the substrate. Therefore, there is 

greater potential for EGBE to be metabolised to BAA in the forestomach of mice than in the 

forestomach of rats. 

Hyperplasia and hyperkeratosis are histological responses to exposure to a wide range of 

chemicals that also produce papilloma and squamous cell carcinoma of the forestomach. 

Many of these chemicals are devoid of genotoxicity, the neoplasms apparently developing as 

a result of persistent cellular damage and sustained hyperplasia (Kroes and Webster, 1986). 

While there is nothing unique to rodents about this process, the fundamental differences in 

physiology and function between the rodent forestomach, on the one hand, and the human 
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stomach and the rodent glandular stomach, on the other hand, point to the low probability that 

the latter would be targets for neoplasia by this mechanism. This proposal is substantiated by 

the lack of any neoplastic response in the glandular stomach of mice exposed to EGBE under 

conditions that produce forestomach tumours. 
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PROPOSED MECHANISMS OF ACTION ASSESSED WITHIN THE IPCS 

FRAMEWORK 

Introduction 

The experiments that form the bases for these evaluations of mechanism of carcinogenic 

action are summarised in the preceding sections of this document. 

EGBE, when delivered as a vapour, induces tumours of the forestomach in female mice and 

haemangiosarcomas of the liver in male mice. There was also some evidence (which did not 

reach statistical significance) for an increase in forestomach tumours in male mice. Neither 

of these tumours do not occur in the other sex in mice and no significant elevation of tumour 

incidence is found in rats. These findings have not been verified in independent experiments. 

Postulated Mode of Action for the Induction of Tumours of the Forestomach in Female 

Mice 

The proposed mechanism of action for production of tumours in the forestomach is the local 

generation, as well as accumulation of cytoxic metabolite(s) that induce a sustained, 

compensatory cell proliferation, neoplasia arising out of this proliferating cell population. 

The neoplasia was mainly papillomas, a single squamous cell carcinoma arising in the 

highest dose group of female mice. 

Key Events 

The following important steps are involved in the generation of forestomach tumours: 

Enhanced exposure of the forestomach to EGBE and its metabolites from multiple external 

and internal sources. EGBE delivered to female mice by inhalation was distributed throughout 

the internal organs and was present in the stomach contents within 5 min after exposure. It 

was present in the mucosa of the forestomach, the buccal cavity and the oesophagus 24 and 48 

h after exposure, but the high levels observed in the forestomach was in contrast to the much 

lower levels found in the glandular stomach and the duodenum. EGBE was also found on fur 

and in the buccal cavity, oesophagus and stomach contents. EGBE delivered to female mice 

by intravenous injection was selectively concentrated in several tissues, including liver, bone, 

Harderian glands and buccal cavity. The mucosa of the forestomach and glandular stomach 

were also labelled, but to similar extents. After EGBE administration by this route or 

subcutaneous injection, both EGBE and butoxyacetic acid were excreted in saliva and found 

in the stomach periods for prolonged periods. 
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Metabolism to cytotoxic metabolite(s), most probably butoxyacetic acid, if not already 

presented to the forestomach in this form. EGBE is metabolised by preparations from the 

forestomach and glandular stomach of rats as well as mice. The enzymes principally 

responsible for EGBE metabolism are alcohol and aldehyde dehydrogenase (a minor 

oxidative pathway is mediated by cytochromeP450 enzymes). These enzymes are found in 

both the glandular stomach and forestomach of rats and mice, but whereas they are 

concentrated in the stratified squamous epithelium of the forestomach, their distribution is 

diffuse in the glandular stomach of both species. There are no major species differences in 

the tissue average activity of aldehyde dehydrogenase, but the maximum rate of alcohol 

dehydrogenase activity is up to an order of magnitude higher in mice than in rats. The 

forestomach is a target tissue because EGBE entering the stomach is held there and because 

the metabolising enzymes are concentrated in the superficial layers of the forestomach. BAA 

has shown to be a more potent in producing adverse effects on the forestomach than EGBE. 

It may be that there is a species difference in part because the rate of metabolism of EGBE to 

2-butoxyacetaldehyde is slower in rats than in mice, hence it is probable that either the 

generation of the more cytotoxic butoxyacetic acid is slower, or its maximum concentration is 

lower in rats than in mice. 

Cytotoxicity and cell proliferation, as indicated by epithelial hyperplasia, was often 

accompanied by ulceration in a two-year inhalation experiment in mice, especially in females, 

but these responses were not reported in rats. Administration of undiluted EGBE to male and 

female mice by gavage induced dose-related irritation of the forestomach; the compensatory 

cell proliferation was confirmed by immunochemical staining. Similar forestomach lesions 

could be induced by EGBE administered by intraperitoneal or subcutaneous injection and by 

butoxyacetic acid given orally. This metabolite was more potent than EGBE itself. 

Squamous cell papillomas or carcinomas were significantly increased only at the highest 

concentration in female mice. It also appears that mice may be more susceptible to the 

induction of forestomach tumours than rats, the background incidence being higher in mice. 

3⁄4 Dose-Response Relationship. In female mice, the incidence and severity of 

epithelial hyperplasia of the forestomach increased with rising dose in female mice, 

as did the incidence of ulceration of the forestomach. Ulceration occurred in male 

mice also, but the incidences were lower and not clearly concentration related.

3⁄4 Temporal Relationship. In a 14 week inhalation study in mice, epithelial 

hyperplasia of the forestomach was observed at 125 ppm and above and had 

progressed at 250 ppm or 500 ppm (the highest concentration) to inflammation, 

necrosis and ulceration. Thus, preneoplastic events preceded neoplasia, which was 

only observed in the two-year study.

3⁄4 Strength, Consistency and Specificity of Association of the Tumour Response 

with Key Events. There has not been any investigation of the association of key 

events with a neoplastic outcome at the level of individual female mice. In broad 

terms, however, it is clear from the dose-response and the temporal relationships that 

there is consistency in these events. The induction of forestomach tumours has been 

observed only in a single experiment. The entire chain of events has not, therefore, 

been verified by independent study. Such verification should be more readily 

achieved by oral administration of EGBE directly into the forestomach, by gavage; 
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however, such an experiment would have little applicability to occupational or 

consumer exposures. 

3⁄4 Biological Plausibility and Coherence. As generalisations, chemicals that do not 

induce carcinogenesis through mutagenesis do so by modes of action that include an 

increase in cell proliferation, either by mitogenesis or by stimulating reparative cell 

proliferation provoked by cytotoxicity. Based on the evidence available, the latter 

mode of action is biologically plausible for the induction of forestomach tumours in 

female mice by EGBE.

3⁄4 Other Modes of Action. While no other mode of action has been proposed, genetic 

toxicity has been considered and rejected. Other possibilities could include aberrant 

control of gene expression because of alkylation of regulatory proteins (e.g., in 

chromatin), with ensuing loss of genetic stability. No studies have been conducted to 

investigate such a mechanism. 

3⁄4 Assessment of the Postulated Mode of Action. The data available are fully 

consistent with the proposed mechanism for the induction of forestomach tumours in 

female mice. The apparent female sex specificity is likely to be due to chance.

3⁄4 Uncertainties, Inconsistencies and Data Gaps. The experimental observations are 

consistent with the general hypothesis proposed; however, there are two areas that 

require more specific reasoning. These are: the species difference in response and the 

progression from hyperplasia to neoplasia. The proposed reason for the difference in 

response between rats and mice is the differences in kinetic properties of the rat and 

mouse alcohol dehydrogenase. Whether this actually leads to a difference in 

concentration or quantity of butoxyacetic acid in the forestomach is unknown. As an 

alternative hypothesis, mice may be more susceptible than rats to the induction of 

forestomach tumours. This suggestion is based on the observed higher incidence of 

forestomach tumours in untreated mice. It is not known how proliferation of 

apparently normal cells becomes transformed into neoplasia. This criticism can be 

levelled at any non-genotoxic mechanism of carcinogenesis (but see 1.3.5). There is 

consistency within the database as currently known. It would be expected, however, 

that exposure of male mice by inhalation to somewhat higher concentrations of EGBE 

should also lead to forestomach neoplasia. Similarly, exposure of female and male 

rats to higher concentrations could also lead to neoplasia if such concentrations could 

be tolerated. Since the lower concentrations used in the two-year inhalation 

experiment with rats were selected on the basis of carefully executed shorter term 

studies to identify the maximum tolerated dose, this hypothetical experiment would 

be unlikely to be successful.

Postulated Mode of Action for the Induction of Hamangiosarcomas of the Liver in Male Mice 

The proposed mechanism of action for production of haemangiosarcomas of the liver is the 

deposition of haemosiderin in relevant cell-types, possibly including endothelial cells from 

which haemangiosarcomas arise, and the generation of cytotoxic reactive oxygen species that 

either induce genetic changes by this secondary mechanism or sustained cell proliferation 

within the endothelial target tissue, neoplasia arising out of this proliferating cell population. 
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Key Events 

The following important steps are involved in the generation of haemangiosarcomas: 

Metabolism to haemolytic metabolite(s), most probably butoxyacetic acid. EGBE is 

principally metabolised by alcohol dehydrogenase and aldehyde dehydrogenase to 2-

butoxyacetaldehyde and butoxyacetic, respectively. Butoxyacetic acid is a more potent 

haemolytic agent than either of its precursors; furthermore, these precursors appear not to be 

haemolytic unless they are metabolised. Inhibition of aldehyde dehydrogenase reduced the 

haemolytic activity of 2-butoxyacetaldehyde. At least in vitro, butoxyacetic acid causes 

haemolysis at much lower concentrations in rat erythrocytes than in human erythrocytes. 

Haemolysis caused by butoxyacetic acid and followed by persistent, dose related anaemia 

results in haemosiderin deposition in the liver. This material has been observed in Kupffer 

cells and hepatocytes of both mice and rats, but there is currently no evidence for 

haemosiderin deposition in endothelial cells. Since haemolysis occurs in mice and rats of 

both sexes, it is necessary to provide a rationale for the specificity of the neoplastic response 

to male mice; however, this could be difficult in view of the low (although statistically 

significant) tumourigenic response in male mice. 

Cell toxicity resulting from the iron-mediated generation of reactive oxygen species from 

haemosiderin. Compared to rats and female mice, male mice have a reduced antioxidant 

capacity in the liver, causing them to be more susceptible to oxidative damage; however, it is 

not clear that this difference is important, given the greater inherent susceptibility of male 

mice to the neoplasm of interest. Oral administration of EGBE to mice at doses up to 600 

mg/kg bw/day for up to 90 days produced increased DNA synthesis in endothelial cells during 

the first 14 days of exposure and in hepatocytes after 90 days. Increased oxidative damage 

was also observed mice in this experiment, while no change was observed in rats. 

Haemangiosarcomas arise from the endothelial target cells and are significantly increased in 

male mice to 8% in the 250 ppm group. It also appears that male B6C3F1 mice may be more 

susceptible to haemangiosarcomas than are female mice (control incidence rates in NTP 

experiments being about 2.5% in males and 0.9% in females) and mice are more susceptible 

than F344 rats (none having been reported in control groups in NTP experiments). Thus, the 

apparent sex and species specificity could merely be a reflection of inherent sensitivity. 

3⁄4 Dose-Response Relationship. Haemolysis and persistent anaemia have been shown 

to occur in a dose related manner in a number of experiments. In two-year inhalation 

experiments, Kupffer cell pigmentation was significantly increased over that seen in 

control animals for male and female rats exposed at 62.5 or 125 ppm, male mice at 

125 or 250 ppm and female mice at 62.5, 125 or 250 ppm. The increases observed in 

the male and female mice were dose related, but the incidence of Kupffer cell 

pigmentation was greater in female mice than in male mice. A significant increase in 

the incidence of haemangiosarcomas occurred only in male mice and only at the 

highest exposure concentration of EGBE.

3⁄4 Temporal Relationship. The experiments show that haemolysis is an acute response 

and anaemia is a sustained response to repeated exposure to EGBE. Haemosiderin 

deposition is observed in short-term experiments and therefore it is present for a 
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considerable period before the emergence of the haemangiosarcomas, which occurred 

at increased incidence only in a two-year inhalation exposure experiment. 

3⁄4 Strength, Consistency and Specificity of Association of the Tumour Response 

with Key Events. Data from experiments with other chemicals show that mice are 

more susceptible to haemangiosarcoma development than are rats, and male mice are 

more sensitive than female mice, but the database in support of the hypothesis that 

life-long exposure of the liver to Kupffer cell pigmentation is a pre-requisite for an 

increase in liver haemangiosarcoma rates in male mice is small, after exclusion of 

doubtful examples.

3⁄4 Biological Plausibility and Coherence. It has long been proposed (e.g., for 

peroxisome proliferators) that increased, persistent generation of reactive oxygen 

species can result in neoplasia by an indirect genotoxic mechanism. It has been 

shown that EGBE does increase oxidative damage in cells, as demonstrated by the 

generation of malondialdehyde, a deposition product of lipids, and 8-

hydroxydeoxyguanosine, an oxygen adduct of DNA, and that differences in reduced 

antioxidant concentrations within cells may be responsible for sex and species 

differences in response. Nevertheless, no substance has been shown unequivocally to 

induce tumours through this mechanism. 

3⁄4 Other Modes of Action. While no other mode of action has been proposed, genetic 

toxicity has been considered and rejected. Other possibilities could include aberrant 

control of gene expression because of alkylation of regulatory proteins, as suggested 

for forestomach tumours, but there is no evidence to support this suggestion either for 

the forestomach mucosa or endothelial cells of the hepatic sinusoids. Kinetic studies 

with liver slices suggest that concentrations of 2-butoxyacetaldehyde that might 

induce genetic damage are unlikely to be reached under the conditions of the longterm inhalation experiments.

3⁄4 Assessment of the Postulated Mode of Action. Haemangiosarcomas are uncommon 

and their incidence in this experiment was low (4/49) although out with the historical 

control range, at the highest dose. Because this is a low increase in incidence, and 

because it has not been confirmed, it is difficult to assign any mechanism. With the 

rejection of genotoxicity as a possible mechanism and the strong evidence for a 

potential source of reactive oxygen species within the liver, it is reasonable to 

presume that these may play a definitive role in neoplasia. 

Uncertainties, Inconsistencies and Data Gaps. By their nature, reactive oxygen species are 

difficult to localise in the target tissue. In addition, the generator of these cytotoxic species, 

haemosiderin, has not been demonstrated in endothelial cells, from which 

haemangiosarcomas arise. Haemosiderin was demonstrated in Kupffer cells, so reactive 

oxygen species could arise in these cells, and the recent demonstration in one study of the 

migration of reactive oxygen species from Kupffer cells to endothelial cells does suggest a 

manner in which the latter might be damage. The involvement of cytokines secreted by 

Kupffer cells has been suggested in the case of peroxisome proliferators, but the target in 

those cases is the hepatocytes. A similar interplay of cytokines between Kupffer cells and 

endothelial cells may be involved in the case of haemosiderin deposition, but this remains to 

be demonstrated and may not be necessary in view of the alternative suggestion. A 

fundamental major uncertainty is that it is unknown if the observation in a single experiment 
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of a low, barely significant incidence of haemangiosarcomas in male mice is reproducible or 

whether it might also be observable in female mice, should the experiment be repeated. 

However, there is reason to expect that female mice would be less susceptible to this 

lesion.Summary of carcinogenicity 

EGBE is carcinogenic in male mice, where it causes a low incidence of haemangiosarcomas, 

and female mice, where is causes an increased incidence of forestomach tumours. It is not 

carcinogenic in rats. Genotoxicity is not an important toxicological property of this chemical 

and it is unlikely that the low, variable and uncertainly defined genotoxic activity can be the 

cause of the carcinogenic responses. Hypotheses have been proposed and supported by 

experiment data in an attempt to explain the carcinogenic responses. These have been 

described above. In the case of forestomach tumours, the argument that they arise in a tissue 

subject to sustained abuse and consequent repair is clear. It is likely that this finding is in 

reality not sex specific but merely due to chance that the low level incidence in females rose 

above the level of statistical significance but it did not do so in males. In the case of the 

haemangiosarcomas, data from experiments with other chemicals show that mice are more 

susceptible to haemangiosarcoma development than are rats, and male mice are more 

sensitive than female mice, but the database in support of the hypothesis that life-long 

exposure of the liver to Kupffer cell pigmentation is a pre-requisite for an increase in liver 

haemangiosarcoma rates in male mice is small, after exclusion of doubtful examples. With 

the rejection of genotoxicity as a possible mechanism, strong evidence for a potential source 

of reactive oxygen species within the liver, and a mode of action where each step has at least 

some supporting data, it is reasonable to presume that these may play a role in the neoplasia. 

With regard to human relevance, the mechanism proposed for the induction of 

haemangiosarcomas strongly suggests that EGBE is not likely to be a carcinogenic hazard 

under conditions of normal handling and use, because human erythrocytes are demonstrably 

more resistant to haemolysis than are rodent erythrocytes. The mechanism proposed for the 

induction of forestomach tumours would also point to a lack of human relevance under 

conditions of normal handling and use. As stated recently (IARC, 2003), while people do not 

possess forestomachs, they do have comparable squamous epithelial tissues in the oral cavity 

and the upper two-thirds of the oesophagus. Thus, in principle, carcinogens targeting the 

forestomach squamous epithelium in rodents are relevant for man. However, the relevance for 

man is probably low for agents that have no demonstrable genotoxicity and that are solely 

carcinogenic for the forestomach squamous epithelium in rodents after oral administration. 

Consequently, for these agents, the mode of carcinogenic action could be specific to the 

experimental animals (IARC, 2003). EGBE satisfies only some of these conditions. On the 

other hand, there are proposed mechanisms that are supported by experimental evidence to 

show how this chemical, even when inhaled, can accumulate in the forestomach contents, 

where it can remain for many hours to cause damage directly or after its metabolism to BAA. 

In conclusion, given the species and sex specificity of the neoplastic responses and the current 

evidence supporting the hypothesis that the more likely mechanism of action is based on 

haematotoxicity, then EGBE is unlikely to be a human carcinogen.Therefore, an appropriate 

classification for “No classification” is proposed for carcinogenicity. This classification 

proposal was agreed by the C&L working group. Moreover, the last IARC evaluation (2004 

and 2006) has classified EGBE in the list 3 of carcinogens : not classifiable as to their 

carcinogenicity to humans (Group 3) on the basis of limited evidence in experimental animals 

and inadequate evidence in humans. 
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Since the only carcinogenic effects can be considered secondary to haemolysis, and 

haemolysis is the key end point for repeat dose toxicity, no separate risk characterisation is 

necessary for the cancer end point. If there are no concerns for repeat dose toxicity, it can be 

considered that there will be no concerns for cancer either.

4.1.2.9 Toxicity for reproduction 

4.1.2.9.1 Effects on fertility 

Oral route 

Repeat Dose Toxicity Studies 

Two of the repeated dose toxicity studies reported effects of EGBE on the testis in male 

rodents. The first of these studies was from the Mellon Institute of Industrial Research 

(1963), wherein rats were given diets containing 0, 0.01, 0.05, 0.25 or 1.25% EGBE for 93 

days. These levels of EGBE were approximately equivalent to 0, 7, 38, 188 and 919 

mg/kg/day. At 1.25% EGBE, male rats had decreased appetite, decreased body weight gain 

(50% less than control animals), increased liver and kidney weights and testicular atrophy. 

Rats given 0.25% EGBE also exhibited decreased appetite and body weight gain, as well as 

testicular atrophy. There were no apparent effects at 0.05% EGBE. Note that the purity of 

the EGBE used in this study is not given, nor does there appear to be analytical verification of 

EGBE doses, homogeneity and stability. The results of this study are difficult to interpret 

given that testicular atrophy is not reported in other oral, repeated-dose studies even at higher 

doses of EGBE greater than 0.25% (e.g., 14-week drinking water study in rats and mice from 

NTP, 1993; continuous breeding study reported in Morrissey et al., 1989). The weight of 

evidence indicates that EGBE does not affect the testis at doses below those causing 

significant systemic toxicity. 

In the 14-week inhalation study, B6C3F1 mice (10/sex/group) were exposed to 0, 31, 62.5, 

125, 250 and 500 ppm EGBE vapor (6 hr/day, 5 days per week). During the first 2 weeks of 

study, 2 male and 2 female mice died and an additional 2 males and 2 females were killed 

moribund. These mice had numerous clinical signs of toxicity, including abnormal breathing, 

red urine stains, and lethargy. Histopathologically, male mice had ulcerations and necrosis of 

the forestomach and female mice had forestomach necrosis. Forestomach ulcerations and 

necrosis were accompanied by inflammation, which in some cases included suppurative 

inflammation. These animals also exhibited atrophy of the spleen, thymus, testis and lymph 

nodes, liver and epididymal necrosis, and renal tubule degeneration. With 8 of 20 animals 

succumbing to toxicity at 500 ppm, it is apparent that the maximum tolerated dose MTD) had 

been exceeded. Mice exposed to 500 ppm that survived to scheduled necropsy had effects on 

the spleen liver, forestomach and kidney, but did not exhibit testicular or epidiymal lesions. 

There were no effects on the testis or epididymis at lower concentrations, nor were there 

effects on the testis/epididymis in the 14-week rat study, the 2-year mouse chronic study, or 

the 2-year rat chronic study. Thus, effects on the testis and epididymis at 500 ppm EGBE 

were not specific to the reproductive organs as numerous tissues were affected and systemic 

toxicity was lethal. 

As mentioned previously, the most sensitive endpoint in the 14-week study was haematology 

changes that were seen in males at >125 ppm and in females at all concentrations of EGBE.
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Continuous breeding 

Male and female Swiss CD-1 mice received 0 0.5, 1.0 or 2.0 % EGBE (purity > 99 %), 

equivalent to daily intakes of 0, 720, 1340 and 2050 mg/kg bw, in their drinking water during 

a continuous breeding phase (CBP) with a 7-day pre-mating period and a 98-day cohabitation 

period (Morrissey et al., 1988 and 1989 ; Heindel et al., 1990). The study was performed 

according to GLP. During the cohabitation period, deaths occurred in the female mice: 13/20 

in the 2 % group, 6/20 in the 1 % dose group, 1/20 in the 0.5 % dose group and 1/40 in the 

control group. The average body weights in the female 2 % dose group were consistently 

lower than the controls. In the male mice, no deaths occurred but weight loss (1-2 % of initial 

body weight) in the two highest doses and reduced weight gain were noted. Reduced fluid 

consumption was observed at all dose levels in both sexes (22 %, 18 % and 36 % reduction 

relative to controls at 0.5 %, 1.0 % and 2.0 %, respectively after 14 weeks of dosing). The 

numbers of fertile pairs from the surviving pairs were 38/39, 19/19, 13/14 and 5/7 at 0, 0.5, 

1.0 and 2.0 % dose levels, respectively. Significant reduction in reproductive performance 

occurred at 1 and 2 % dose levels as indicated by dose-related decrease in number of litters 

per fertile pair, litter sizes, pup viability and live pup weight. A small but significant reduction 

(by 5 %) of live pup weight was also observed in the 0.5 % dose group without other 

significant reproductive effects. 

At the completion of the continuous breeding phase, the F0 breeding pairs were separated and 

housed individually and exposure to EGBE continued. When the last litter was weaned F0 

males and females from the 1 % dose group were mated with male and female control animals 

in a one-week crossover mating study to determine any sex-related reproductive effects of 

EGBE. Exposure to EGBE was discontinued during the one-week mating period and then 

reintroduced at 1 % dose level (estimated daily intake 1830 mg/kg bw). Control males and 

females were also mated for comparative purposes. The proportion of successful copulations 

from the breeding pairs was similar in all groups. However, the number of fertile females was 

significantly reduced in the group where treated females were mated with control males. 

When evaluated over the 7 day period prior to necropsy, proportionally more females (7/13) 

in this 1 % treated group than controls (9/38) had oestrus cycles longer than 7 days. At 

necropsy, male and female mice from this 1 % dose group had significantly lower body 

weights and increased relative kidney weights,and female also had significant increases in 

relative liver weight. No significant differences were observed between the control and treated 

animals for the weights of reproductive organs, sperm motility, morphology or 

averageoestrous cycle length and frequency. In the only histopathological examination carried 

out on the treated females, no treatment related kidneys lesions were observed. The crossbreeding results suggest that the fertility effects were only due to effects on the female mice. 

Furthermore, these effects may have been an indirect consequence of the severe general 

systemic toxicity rather than a direct effect of EGBE on the reproductive organs. 

A final phase was conducted to assess the fertility and reproductive effects of EGBE in 

second generation (F1) pups. The pups selected were those born after the CBP and when the 

maternal animals were individually housed. As there were insufficient pups in the 1 and 2 % 

dose groups, only the pups from the 0.5 % dose group were used. The F1 generation pups 

were nursed, weaned and reared to sexual maturity. After weaning, the mice received 0.5 % 2-

butoxyethanol in their drinking water (estimated daily intake 950 mg/kg bw). At 74 ± 10 days 

of age, the F1 animals from different litters were mated. The animals were necropsied after 

delivery. No significant fertility and reproductive effects were observed in the F1 animals as 

indicated by the proportions of successful copulation and fertile females, litter size, pup 

viability and live pup weights. Similarly, no treatment-related changes in the weights of 
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reproductive organs, sperm motility, morphology and the oestrous cycle length and frequency 

were noted. However, a significant increase in relative kidney weight in the females and a 

significant increase in relative liver weight in both the males and females were observed. 

In summary, significant adverse reproductive effects were observed in the females at very 

high dose levels (1340 mg/kg and above) which also caused severe toxicity, including death. 

Under the conditions of the study, the NOAEL for reproductive toxicity of EGBE (fertility) 

can be set as 720 mg/kg/day. For developmental toxicity, no NOAEL can be derived. A 

conservative LOAEL of 720 mg/kg/day can be taken as only a very slight decrease in pup 

weight was observed at this dose. No NOAEL or LOAEL can be determined for systemic 

parental toxicity because this kind of study is not designed to assess systemic toxicity 

although it is of note that there were effects (reduced fluid consumption) even at the lowest 

dosage of 720 mg/kg/day. 

Other studies: 

Groups of five male JCL-ICR mice received doses of 500, 1000, or 2000 mg/kg bw/d EGBE 

(purity and GLP status unknown) by gavage for 5 days/week for 5 weeks (Nagano et al., 

1984). The mice were killed on day 6 of week 5 and the effects on the testis and associated 

structures were observed. Controls received water. At the highest dose level all the mice died 

and dose-related haematotoxicity (reduced red blood cell count) was observed at the other 

doses. There were no significant effects on the absolute or relative weights of the testis or 

seminal vesicle and coagulating gland at any dose tested. An earlier publication (Nagano, 

1979) mentioned testicular atrophy in one of five mice dosed with EGBE (1000 mg/kg). This 

observation was not considered significant by Nagano and his co-authors in their later 

publication (Nagano et al., 1984). 

In 13 week duration toxicity studies in F344/N rats and B6C3F1 mice conducted by the 

National Toxicology Program (1993), EGBE (purity 99 %) was administered in drinking 

water at concentrations of 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 male and 10 

female rats and at 0, 750, 1500, 3000, 4500 or 6000 ppm to groups of 10 male and 10 female 

mice. These concentrations provided target dose levels of 0, 100, 150, 250, 400 or 650 mg/kg 

bw per day (NTP, 1993). In addition to conventional toxicological endpoints, vaginal 

cytology and sperm morphology evaluations were performed. Vaginal cytology was assessed 

over the 7 days prior to sacrifice and sperm morphology and motility were estimated from 

epididymal sperm samples taken at necropsy. In the rat, haematotoxicity was evident in 

males at doses of and above 3000 ppm and in all female groups. Terminal body weight was 

decreased by 9 and 14% in males given 4500 and 6000 ppm EGBE. In females, body weights 

were decreased by 14 and 22% at these concentrations. There was no treatment effect on testis 

weights but there was a reduction in the size of the uterus in females at 4500 and 6000 ppm. 

Changes in uterine weight were considered by the authors to be secondary to the reduction in 

body weight gain rather than a direct effect of EGBE. The only spermatozoal measurement 

that showed significant change relative to the control group was sperm concentration which 

was slightly decreased in all groups of treated males; however, this effect was not doserelated. There were no significant differences from the control group in oestrous cycle length 

for treated females, although females treated at 4500 and 6000 ppm spent more time in 

dioestrous than the other groups. This correlates with the smaller uterine size, which was 

attributed to a secondary consequence of reduced body weight gain. In mice there were no 

chemical-related gross or microscopic lesions and no biological significant changes in any of 

the reproductive parameters evaluated. The small differences in the rat reproductive 
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endpoints with EGBE were in marked contrast to the significant effects of EGME and EGEE 

in both species, where concurrent studies demonstrated treatment-related testicular 

degeneration/ atrophy and, for EGEE, extended oestrous cycle length. 

Table 4.85 bis: Data on estrous cycling for rats exposed to EGBE for 13 weeks in drinking water. 

Study parameters 0 ppm 3000 ppm 4500 ppm 6000 ppm 

n 7 6 7 6 

Necropsy body weight (g)1 186 + 4 172 + 2 160 + 2 145 + 2 

Estrous cycle length (days) 6.50 + 0.70 6.83 + 0.95 7.57 + 0.53 5.83 + 0.70 

Estrous stages (% of cycle) 

Diestrus 28.9 45.6 52.8 67.5 

Proestrus 8.8 11.4 13.9 7.0 

Estrus 57.9 38.6 20.4 17.5 

Metestrus 4.4 4.4 13.0 7.9 

1

 Based on n = 10. 

With respect to oestrous cycle evaluations, normally cycling Fischer 344 females have 5-6 

day oestrous cycles. While there were 10 females per dose group, 3-4 females per group had 

estrous cycles longer than 12 days or cycles that were unclear, including the control group. 

These animals were excluded from the analysis, meaning that only 6-7 females per group 

were evaluated. Furthermore, given the approximate times in the various stages of estrous 

(see table below from Zarrow et al., 1964), it appears that the control animals spent an 

excessive amount of time in estrus. Typically, estrus would comprise approximately 30% of 

the estrous cycle, rather than the 57.9% reported for the control females. Using Zarrow’s 

table, if one assumes 2 days for estrus and minimal durations for the other estrous stages, 

estrus would only comprise 44% of the cycle (2 days out of 4.5 days total estrous cycle). This 

value is well below the 57.9% reported in the control animals in the NTP study. Vaginal 

smears were collected only once per day. With only 6-7 rats per group and stages of the cycle 

that are less than one day in duration, some phases could have gone undetected. 

Table 4.85 ter: Vaginal cytology during the estrous cycle of the rat1

Time (days) Stage Vaginal smear 

0.5-1 Proestrus (P) Many rounded epithelial cells, few leukocytes 

1-2 Estrus (E) Cornified epithelial cells, no leukocytes 

0.5-1 Metestrus (M) Maximum number of leukocytes, often in clumps surrounding 

epithelial cells. For our purposes this will be classified as part of 

Diestrus. 

1.5-2 Diestrus (D) Few epithelial cells; few to many leukocytes. Sometimes divided into 

Diestrus I and II 

1 Modified from Zarrow et al. (1964). 
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These points, coupled with the lack of effect on overall estrous cycle length and absence of 

histopathologic lesions, suggest that the effects on estrous cycle length are unlikely to indicate 

a specific treatment-related reproductive effect. 

Thus, the effects on male and female rat reproductive endpoints are not indications of 

selective reproductive system toxicity. These effects were minor in nature, only present in 

one species and only occurred in the presence of other systemic toxicity (i.e., decreased body 

weights, haematotoxicity). There were no histopathological lesions in reproductive organ 

Inhalation route 

In an inhalation study (Doe, 1984), male Alpk/Ap Wistar-derived rats were exposed whole 

body to 800 ppm (4 mg/l – saturated vapour) EGBE (purity unknown) for 3 hours (no GLP). 

This study was performed to compare the effects of EGBE and EGME in rats. The rats were 

then observed throughout the next 14 days. The animals were sacrificed on day 15 and subject 

to gross macroscopic post-mortem examination, which included the weighing of the testes. 

Clinical observation revealed haematuria in treated animals. EGBE had no effect on testicular 

weights. 

In the 90 day toxicity study of EGBE (purity 99.4 %) administered by the inhalation route 

(whole body) to Fisher 344 rats at doses of 5, 25 or 75 ppm for 6 hours/day, 5 days/week for 

approximately 13 weeks (16 males and 16 females/group) (Bushy Run Research Center, 

1981b), there were no adverse effects on testicular weights and no reported pathological 

changes in the reproductive organs. 

Studies on metabolites : 

In vitro studies 

Primary testicular cell cultures were treated with a series of GE and Alkoxyacetic acids (Gray 

et al., 1985). In contrast to the action of methoxy-acetic acid (MAA) and ethoxy-acetic acid 

(EAA), neither n-propoxy-acetic acid (PAA) nor n-butoxyacetic acid (BAA) was overtly toxic 

at a concentration of 10 mM for 72 hours. Under these conditions BAA produced no 

discernible effects. At 20 mM, BAA was still without appreciable effects. 

To explore the development of quantitative indices of toxicity in the cell cultures, 

measurements were made of Carnitine AcetylTransferase (CAT) and lactate dehydrogenase-X 

(LDH-X), two enzymes whose activity is higher in the pachytene spermatocytes than in the 

other cell types present. When the 4 alkoxyacetic acids were compared, there was a parallel 

between their effects on CAT and LDH-X activity and the severity of the morphological 

changes. BAA produced a decrease in CAT activity (10 mM) in the attached germ cell 

fraction less marked than those of others alkoxyacetic acids, although statistically significant 

(88 nmol/mim/culture vs 121.7 nmol/mim/culture in controls). When expressed as specific 

activities, the result was still significant (54.8 nmol/mim/mg cell protein vs 68 nmol/mim/mg 

cell protein). No effect was seen with BAA treatment in LDH-X activity. 

Mixed cultures of Sertoli and germ cells were incubated during 24 hours in the presence of 

BAA at doses ranging from 0 to 10 mM (Foster et al., 1987). Cultures were also made with 

MAA and EAA. BAA did not produce any changes when tested in Sertoli-germ cell cultures 

at 5mM in contrast with effects seen with MAA and EAA (to a lesser extent) for which an 
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enhancement of germ cell loss (pachytene spermatocytes) were seen at this dose level. Some 

non specific cell loss was observed at 10 mM..

In vivo studies 

BAA was administered orally by gavage to young SD rats (4 weeks old) for 4 days 

concurrently with 3 other alkoxyacetic acids at equimolar doses to characterise their relative 

testicular toxicity (Gray et al., 1985). The rats were killed 24 hr after the last dose. With PAA 

(776 mg/kg) and BAA (868 mg/kg) no morphological changes in the testis were observed at 

the dose studied. Only MAA produced testicular toxicity at the dose of 592 mg/kg. 

In male Wistar rats (Foster et al., 1987), the administration by gavage of a single dose of 

BAA did not result in any testicular damages at the highest dose tested of 868 mg/kg (the 

animals were terminated 14 days after dosage). At the dose of 868 mg/kg, animals showed 

evidence of haematuria. No other systemic nor specific effects on testes were seen at doses of 

174 and 434 mg/kg. This study was concurrently performed with MAA and EAA and 

confirmed the testicular toxicity of these other alkoxyacids. 

In all sub-acute, sub-chronic and chronic studies no direct effects were seen on male or female 

reproductive organs at all doses tested (even sub-lethal doses). Metabolism of EGBE leads to 

the formation of a majority of BAA, which is not a male reproductive toxic substance, unlike 

MAA or EAA (main metabolites of EGME and EGEE respectively). 

In the continuous breeding study, effects were seen on fertility only at doses which were 

severly toxic to the animals (1340 and 2050 mg/kg bw/d). A NOAEL of 720 mg/kg bw/day 

can be fixed for fertility effects by oral route in mice. 

4.1.2.9.2 Developmental toxicity 

Studies in animals

Oral route 

Studies in rats

The teratogenicity of EGBE (> 99.0 % purity) was evaluated using Fisher 344 rats (Sleet et 

al., 1991; Schwetz and Harris, 1993 ; Research triangle institute, 1988 as part of the 

National Toxicology Programme (NTP)). The purpose of the study was to determine whether 

the exposure of pregnant rats to EGBE during critical periods of cardiovascular development 

adversely affected the structure of the foetal heart and great vessels. Other developmental 

toxicity was also recorded. EGBE (in distilled water) was administered via gavage to a total 

of 298 rats; 104 rats served as controls. The distribution of doses for the 184 animals dosed on 

gestation days (GD) 9 through 11 was as follows: 46 rats (0 mg/kg/day), 46 rats (30 

mg/kg/day), 47 rats (100 mg/kg/day), and 45 rats (200 mg/kg/day). A total of 93 rats (all 

dose groups included) were killed on GD 12 of gestation, and the remaining 91 rats, on GD 

20. For the 220 rats dosed on GD 11 through 13, the distribution of doses was as follows: 58 

rats (0 mg/kg/day), 52 rats (30 mg/kg/day), 59 rats (100 mg/kg/day), and 51 rats (300 

mg/kg/day). A total of 104 rats (all dose groups included) were killed on GD 14, and the 

remaining 116 rats, on GD 20. Also, on GD 20, foetuses were killed and examined. 
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The maternal effects of EGBE given from GD 9 - 11 or from GD11 - 13 at doses of and 

greater than 100 mg/kg/day included marked reductions in body weight and/or weight gain, 

increases in organ weights (kidney and spleen) and severe haematotoxicity. In particular, 

dramatic reductions in circulating red blood cells, haematocrit and haemoglobin resulted by 

24 hours after treatment. By GD20 the haematotoxic effects were nearly reversed. The 

changes observed in haematological parameters and organ weights in this study are typical 

of haemolytic anaemia and the compensatory haematopoietic response associated with 

recovery. The maternal NOAEL is 30 mg/kg/day based on the effects describes before. 

Embryo/foetal effects were: increased resorptions, nonlive implants, and adversely affected 

implants per litter in the 200 mg/kg/day group dosed on GD 9 - 11, and decreased platelet 

count but no embryolethality in the 300 mg/kg/day group dosed on GD 11 - 13. 

EGBE exposure during gestation did not increase the incidence of foetal malformations; 

particularly, no cardiovascular malformations were observed. When 200 mg/kg were given 

from day 9 to 11, an increased foetal lethality without malformations was noted. When 300 

mg/kg were given from day 11 to 13, a decrease platelet count was seen in foetuses. For 

developmental toxicity the NOAEL is 100 mg/kg when EGBE is administered from GD 9 - 

11 and the conservative NOAEL is 100 mg/kg when EGBE is administered GD11 – 13 

(although the NTP conclusion was for a NOAEL of equal to or greater than 300 mg/kg/day). 

Studies in mice 

In a gavage probe study in pregnant CD-mice (Exxon, 1985 ; Wier et al., 1987), EGBE 

(purity 97 %)was administered in distilled water at 0 – 350 – 650 – 1000 – 1500 and 2000 

mg/kg bw/day (6 animals per group) during gestational days (GD) 8-14(Note GD0 = vaginal 

sperm plug and sacrificed GD18). Haemolytic effects in the dams were observed from 650 

mg/kg/day. At 1500 mg/kg/day the maternal mortality rate was 3/6 and at 2000 mg/kg/day 

6/6. Increased resorption rates and numerically reduced number of viable foetuses were 

observed at 1000 and 1500 mg/kg/day. 4/43 foetus (in one litter) at 1000 mg/kg/day and 1/25 

at 1500 mg/kg/day had cleft palates. For this study, the NOAEL for maternal toxicity is 350 

mg/kg/day and the NOAEL for developmental toxicity is 650 mg/kg/day. 

In a subsequent GLP reproduction study (following a Chernoff modification study design 

with treatment of the pregnant mice (20 per group) at 650 or 1000 mg/kg/day between GD 8-

14, the animals were allowed to give birth and the offspring were observed till Post natal Day 

(PND)22). No significant effects on pup growth or survival resulted. No adverse 

developmental effects were reported. Signs of haemolytic effects and bw gain reduction were 

seen at 1000 mg/kg in dams. The NOAEL for maternal toxicity is 650 mg/kg/day in this 

study, and as no effects were seen in pups, the NOAEL for developmental toxicity can be 

considered to be greater than 1000 mg/kg/day. 

In another Chernoff assay, fifty mated CD1 mice were orally administered EGBE (99 % 

purity) by gavage at 1180 mg/kg/day (calculated LD10 based on a non-pregnant mouse pilot 

study) in corn oil from GD7-14 (GD1=vaginal sperm plug), then allowed to litter and to rear 

pups to PND3. 20 % of the dams died, maternal weight gain was reduced and, of 31 surviving 

pregnant females, there were only 24 viable litters (77 %) compared with 97 % control litter 

viability. No external malformations were seen, pup survival to PND was unaffected and no 

other indication of specific developmental toxicity was found (Schuler et al., 1984). A 

maternal NOAEL could not be calculated and, although pup development was unaffected, the 
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observation of reduced numbers of live litters precludes a calculation of the developmental 

NOAEL. 

Summary oral route : 

One study is available in rats and 2 studies in mice. The lowest foetal NOAEL is 100 

mg/kg/day in the Research triangle institute (1988) rat study. It is based on effects seen at 200 

mg/kg/day: increase foetal lethality without malformations. These effects were seen with 

maternal toxicity (haemolytic anaemia) and retarded body weight gain evidenced from 100 

mg/kg/day. The maternal NOAEL is 30 mg/kg/day in this study. 

Inhalation route 

Studies in rats 

In an initial GLP study, mated female Fischer 344 rats (30 per group) were exposed whole 

body to 0, 100, 200 or 300 ppm (0, 483, 966 or 1449mg/m3

) EGBE vapour for 6 hours/day on 

GD6-15 (GD0 = sperm plug positive) (Tyl et al., 1984 ; Bushy Run Research Center, 1984). 

Maternal weight loss was observed at 300 ppm and 200 ppm and retarded weight gain at 100 

ppm. During exposure, females were hypoactive, with a dose-related increase in the 

frequency of signs indicative of haematuria and with dose dependent reductions in food 

consumption at all levels and in water consumption at 200 ppm and 300 ppm. At 300 ppm 

one animal died. In blood samples taken from females at necropsy (six days after the last 

exposure), red blood counts were reduced at 300 ppm and 200 ppm. At 300 ppm, 16 of 23 

pregnant dams showed 100 % resorptions. There was no increase in resorptions at the lower 

doses, but low pregnancy incidences in all groups, including controls, resulted in only 7, 15, 

21 and 21 litters being evaluated at 0, 100, 200 and 300 ppm, respectively. The authors 

considered this number of litters too few for definitive interpretation of results at higher doses. 

Interpretation of the results was complicated by maternal toxicity, which included decreased 

body weight gain at 100 ppm, and body weight losses at 200 and 300 ppm, a condition that 

causes marked maternal stress. Contributing to this stress was decreased food consumption at 

all concentrations, and decreased water consumption at 200 and 300 ppm. Dams were 

hypoactive with signs of haematuria, indicating maternal haemolysis. Red blood cell counts 

were reduced at necropsy in dams exposed to 200 and 300 ppm. Two foetuses in 2/7 litters at 

300 ppm and 1/1 litter at 100 ppm had cardiovascular (ventricular septum) defects. The 

relevance of these findings as an indication of selective toxicity to the foetus was considered 

unlikely by the authors, although it did trigger the conduct of a developmental phase staged 

oral study described above (Sleet et al., 1991; Schwetz and Harris, 1993 ; Research Triangle 

Institute, 1988 as part of the National Toxicology Program (NTP)) which confirmed the lack 

of teratogenicity. In increased incidences of foetuses at 300 ppm in the inhalation study, the 

development of the inominate artery was affected (shortened or missing), the lateral brain 

ventricles were enlarged, ossification was retarded and extra ribs were present. Foetal 

ossification was also retarded at 200 ppm and enlargement of the lateral brain ventricles was 

observed at increased incidences at 200 ppm and 100 ppm. These variations were considered 

indicative of embryo/foetal toxicity at maternally toxic dose levels. To investigate these 

potential effects further, a second inhalation study was performed in rats together with a study 

in rabbits. It should be noted that the effects seen on lateral ventricle size were not reproduced 

in the subsequent rat and rabbit studies, despite exposure over the same sensitive period with 

previously effective concentrations of EGBE (100 and 200 ppm). In contrast, other effects 
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(e.g., delayed ossification) were reproducible across studies. Thus, the finding of increased 

incidence of enlarged lateral ventricles cannot be considered significant. 

In a GLP study, mated female Fischer 344 rats (36 per group) were exposed whole body to 0, 

25, 50, 100 or 200 ppm (0, 121, 242, 483 or 966 mg/m3) EGBE vapour (purity 99.6 %) for 

6hr/day on GD 6-15 (Tyl et al., 1984 ; Bushy Run Research Center, 1984)(GD0 = sperm plug 

positive). No adverse reproductive or developmental effects were observed in animals 

exposed to 25 ppm or 50 ppm. Maternal toxicity was observed in a dose-related incidence 

during the exposure period, included evidence of haematuria from 100 ppm and pale, cold 

extremities with necrosis of the tail tip at 200 ppm. Dose-dependent maternal weight loss was 

observed at 200 ppm and 100 ppm, associated with significant reductions of food 

consumption (for both groups) and water consumption (for the 200 ppm group). 

Haematological findings from blood samples taken at necropsy on GD21 (six days after the 

last exposure) evidenced signs of haemolytic anaemia for treatment with 100 ppm EGBE and 

higher. 

At sacrifice, maternal gravide uterine weight was significantly reduced and the absolute and 

relative spleen and relative kidney weight were elevated compared to controls at 200 ppm. 

Exposure to 200 ppm was associated with a statistically significant increase in the number of 

totally resorbed litters and a reduction in the number of viable implants and in percentage of 

live foetuses per litter. However, there were no statistical significant increases in incidences of 

external, visceral, skeletal, or total malformations associated with exposure to EGBE. 

Exposure to 200 ppm was also associated with a significant increase in the number of litters 

with one or more foetuses with unossified skeletal elements and poorly ossified skeletal 

elements (anterior arch of atlas, cervical centra 5 and 6 and forelimb proximal phalanges) and 

poorly ossified skeletal elements (cervical arches and sternebrae 1, 3, 4 and 6). Primarily 

because the skeletal elements were poorly ossified there was a decreased incidence of bilobed 

cervical centra 5 and 6 and bilobed thoracic centra 9 and 13. There was also a decreased 

incidence of poorly ossified hindlimb proximal phalanges. At 100 ppm, the number of litters 

with one or more foetuses with unossified cervical centrum 6 was significantly increased and, 

because fewer also showed ossification of cervical centrum 5, the number bilobed was 

significantly decreased. No significant differences were observed for other aspects of skeletal 

ossification. The occurrence of unossified skeletal elements was considered by the authors to 

be an indication of delayed development in animals exposed to EGBE under maternally toxic 

concentrations. For this study the NOAEL is 50 ppm for maternal toxicity based on 

haemotoxicity and bw and food consumption retardation at 100 ppm. A LOAEL of 100 ppm 

for developmental toxicity is due to the small increase in skeletal ossification retardation, but 

without any other foetal effects, variant, observed at this dose level, this leads to a NOAEL of 

50 ppm for developmental toxicity. 

In another inhalation study, the teratogenicity of EGBE (98 to 99.5 % purity) was evaluated 

using female SD rats (Nelson et al., 1984). The animals were exposed whole body to EGBE 

vapour on GD 7 – 15 (GD0 = sperm positive vaginal smear) for 7hr/day (GLP status 

unknown). Sixteen and fifteen rats were exposed to 150 ppm and 200 ppm, respectively. The 

untreated control group consisted of 34 dams. Higher dose levels (250 - 500 ppm) in a 

preliminary study were associated with dose-related maternal deaths, haematuria and tail 

necrosis. 

On the first day of exposure, haematuria was observed in the dams in 150 and 200 ppm 

groups. This was the only effect observed in dams. There were no significant differences in 
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the number of resorptions foetal weights and the incidence of malformations or variations 

between the group exposed to 200 ppm EGBE and the control group. A statistically 

significant decrease in foetal weights was observed in the 150 ppm exposure group. Since 

differences in foetal weights between experimental and control groups were slight, and, also, 

that significant differences were not observed in the 200 ppm exposure group, these 

differences were not thought to have been of biological significance. For this study a very 

conservative LOAEL of 150 ppm can be determined for dams for haematuria seen in the first 

day of treatment. For developmental toxicity, the NOAEL is 200 ppm. 

In a briefly reported study (Barilyak, 1989 in Russian), mated female rats were exposed to 

EGBE at concentrations of 77, 12 and 2 mg/m3

, 4 hours a day during GD1 - GD19. At GD20, 

animals were killed and pups removed by laparoscopy. Number of Corpora Lutea and, normal 

and abnormal pups were determined. A complete identification of teratogenic effects was 

performed in abnormal animals. In this study, EGME was also tested concurrently with 

Glycols. 

For EGBE, at the two high dose tested, a significant increase of total losses was seen 

compared to controls. These increases were due to an increase in pre-implantation loss for the 

high dose group (77 mg/m3) and due to a post-implantation loss for the mid-dose group (12 

mg/m3). No abnormalities were seen in any treated group but 32.5 % of the pups of the high 

dose group exhibited a delayed development. 

This publication is not detailed enough to consider it usefull for risk assessment. 

Studies in rabbits 

In a GLP study, mated New Zealand white rabbits (24 per group) were exposed whole body to 

0, 25, 50, 100 or 200 ppm (0, 121, 242, 483 or 966 mg/m3) EGBE vapour (purity 99.6 %) for 

6hr/day on GD 6-18 (GD0 = day of copulation) (Tyl et al., 1984 ; Bushy Run Research 

Center, 1984). No adverse reproductive or developmental effects were observed in animals 

exposed to 25 ppm or 50 ppm. At 200 ppm four females died or were sacrificed by the third 

day after the onset of dosing and four aborted. The blood samples taken at necropsy on GD29 

(eleven days after the last exposure) showed increased haemoglobin concentration and 

haematocrit at 100 and 200 ppm, but statistical significance was only evident at 100 ppm.. 

Maternal body weight loss was observed in all groups including controls during exposure, but 

the difference was greatest at 200 ppm and, by GD15, the actual body weight at 200 ppm was 

significantly lower.. Exposure to 200 ppm produced a significant reduction in maternal body 

weight, gravide uterine weight and number of total implants and viable implants. There were 

no significant increases in the number of foetuses or litters with one or more affected foetuses 

with pooled external, visceral, skeletal (total) malformations in any treatment group. For 

individual malformations, there was a significant increase relative to controls in the number of 

litters at 100 ppm with one or more foetuses exhibiting fusion of papillary muscles in the left 

ventricle. Five foetuses from 4/19 litters were affected. However, since this malformation was 

not observed at the higher or other concentrations it was considered by the authors, the 

reviewers and the rapporteur as coincidental and not treatment-related. The incidences of 

skeletal variations did not indicate an adverse treatment effect on ossification. There were 

significant decreases in the incidences of litters at 200 ppm with unossified sternebra 6 and 

with rudimentary ribs on lumbar vertebra 1. However the former observation was indicative 

of better ossification and the latter observation was because more ribs at 200 ppm were full 

(extra) rather than rudimentary and there were actually no intergroup differences in 

supernumerary rib numbers. For this study, the NOAEL for maternal toxicity is 50 ppm and 
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the NOAEL for developmental toxicity is 100 ppm based on fewer viable implants seen at 

200 ppm. 

Summary inhalation route: 

In both Tyl studies (1984), the developmental NOAEL of 50 ppm can be taken into account 

for the risk assessment (rats). This value is based on variations seen at 100 ppm. These effects 

were seen in presence of maternal toxicity (haemolytic anaemia) which was seen at 100 ppm 

and higher. The maternal NOAEL for this effect was 50 ppm. In one study performed on rats, 

no effects were seen at the highest tested dose (200 ppm). 

Dermal route: 

EGBE was applied to the skin (unoccluded) of 9 pregnant SD rats to investigate its potential 

for developmental toxicity (Hardin et al., 1984). Four doses each 2.5 hours apart of 106 mg 

EGBE (total daily dose of 424 mg, 1760 mg/kg/day) were applied daily to shaved 

interscapular skin of rats on GD 7 – 16 (GD0 = sperm positive). A previous replicate of 

females dosed at a concentration three times higher had been terminated because of marked 

toxic effects (haematuria, tail necrosis, death after 3-7 days treatment). No maternal, 

embryotoxic, fetotoxic, or teratogenic effects were detected with EGBE treatment at the lower 

concentration of approximately 1760 mg/kg/day. 

No clear conclusion can be drawn from the findings of this study since EGBE was applied to 

the skin without occlusion which would potentially enable evaporative loss from the site of 

application. 

Other routes : 

Studies in rats

Groups of rats were injected subcutaneously with aqueous solutions of EGBE to determine 

the teratogenicity of the chemical (Unilever, 1976). In the first of two preliminary studies to 

set the dose range, non-pregnant rats received a single injection with 225-1350 mg/kg; 80% of 

the high dose animals died, haemoglobinuria was observed and general poor condition of the 

rats resulted. In the second preliminary study, no effects were observed when a similar group 

of rats received repeated injections at 23-90 mg/kg/day. Experimental details for the 

preliminary studies were not available. 

In the main study, conducted in accordance with OECD test guideline 414, groups of 20 

pregnant CD rats received sub-cutaneous injections of 0. 45, 90 or 180 mg/kg/day as an 

aqueous solution on GD6 - 15. No mortality resulted but haemoglobinuria and body weight 

loss were observed in the medium and high dose dams after the first two injections only. No 

significant pathological findings were noted at necropsy. Pre-implantation loss increased in 

the medium (13.8 %) and high dose (11.9 %) dams compared with the controls (6.9 %), but 

the values were within the laboratory's normal range. Other parameters were normal. In the 

foetuses, a slight increase in rib effects (wavy/thickened) and a dose-dependent increase in 

incomplete ossification of cranial bones were observed, but such changes are considered as 

variations and not malformations. 

Under the conditions of the study, pre-implantation loss occurred in dams at maternally toxic 

doses and there were no treatment related signs of teratogenicity. In this study, the NOAEL 
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for dams is 45 mg/kg/day and the NOAEL for developmental toxicity is 90 mg/kg/day based 

on the variations observed at 180 mg/kg/day. 

In vitro studies

EGBE and other glycol ethers were tested in 2 series of Hydra Regeneration Assays (Bowden 

et al., 1995 ; Johnson et al., 1984 and 1988). The Hydra Regeneration Assay compares the 

toxicity of a test compound to an adult hydra polyp with its ability to inhibit the regeneration 

of an isolated digestive region (Bowden et al., 1995) or the regeneration of pellets prepared 

from non-specific dissociated adult cells (Johnson et al., 1984 and 1988). In the Bowden 

study (1995), EGBE was administered at doses ranging from 0.09 mg/ml to 0.92 mg/ml to 

groups of 10 polyps and 10 regenerates. The mean morphological scores for polyps were 10, 

8.6, 9.8, 9.0, 4.4 and 4.1 and for regenerates were 8.1, 7.2, 6.6, 6.6, 3.0 and 1.9 for the control 

and concentrations of 0.09, 0.18, 0.37, 0.74 and 0.92mg/ml, respectively. Comparing the two 

polyp versus regenerate scores, the estimated T50 (corresponding to the concentration 50 % 

toxic to the polyps) was 0.69 mg/ml and the I50 (corresponding to the concentration which 

cause an inhibition of 50 % of the regeneration) was 0.54 mg/ml. However, if the treated 

concentrations versus their respective controls are compared, this gives equivalent T50/I50

concentrations of approximately 0.6mg/ml . The T50/I50 ratio is considered equivalent to the 

mammalian A/D ratio (adult to developmental toxicity) and is used as a predictor for 

developmental hazard. In this study the T50/ I50 ratio was 1.3 for the first calculation and 

approximately 1 for the second, indicating that the adult and developmental stages were 

similarly sensitive. If this ratio is compared to other glycol ethers, the developmental hazard 

was less than with EGEE and EGME and higher or approximately equivalent to DGEE. 

These results were not consistent with the Johnson study in which the “A/D” ratio was 4.4 for 

EGBE, 5 for EGEE and 1.3 for EGME, indicating that EGBE could be more toxic to the 

“developmental” stage than EGME. However, the developmental stages were different 

between the studies so a direct comparison is inappropriate. In any event, and assaid by the 

authors, these in vitro studies cannot be use for quantitative risk assessment. 

EGBE and other glycol ethers were tested in the Rat Whole Embryo Culture (WEC) assay 

(Bowden et al., 1995). The WEC system used mated Sprague-Dawley CD rats (GD1 = sperm 

positive). Animals were paired on a one-to-one basis. The morning following pairing was 

designated day 1 of gestation. On GD 10, dams were sacrificed and embryos dissected out. 

Embryos were cultured in a special medium with S9 metabolic activating system. EGBE in 

DMSO solution was diluted to 10 % (v/v) with sterile water and added to the culture medium. 

EGBE was tested at concentrations of 0, 0.3, 0.5, 0.75 and 1 mg/ml. Culture were terminated 

after approximately 48 hours. To estimate growth, the yolk sac diameter, crown-rump length 

and head length were measured and the number of somites counted. Each embryo was 

examined for dysmorphogeneses and the morphological score was assessed. The rate of 

dysmorphogeneses was calculated as: Malformation index = n° of observed malformations / 

n° of embryos in the group. Results were compared with other glycol ethers: EGME – EGEE 

– DGEE – EGTA – EGS. 

EGBE produced marginal reductions in growth and developmental parameters at 0.3 mg/ml; 

these effects being greatly increased at 0.5 mg/ml and above. Increased incidences of 

malformations were observed at 0.3 mg/ml and above, including poor yolk sac circulation, 

thin allantois, twisted flexions, incomplete fusion and/or irregular formation of the caudal 

neural tube and brains, irregular posterior neuropore and growth-retarded forelimb buds. At 1 

mg/ml, 5/10 embryos were dead. For this test, results obtained with glycol ethers are not 
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suitable for effect assessment because, in vivo, they have a very low half life in the circulating 

blood (< 10 min for EGBE) and the high doses at which glycol ethers produced effects in 

vitro are totally unrealistic in vivo. Although a S9 metabolic activation system was included in 

the embyo culture medium, the results might therefore have been expected to show the 

toxicity profile of the metabolite. However, the alcohol / aldehyde dehydrogenase system is 

not present in the S9, this may explain the discrepancy between embryo culture results and in 

vivo data. For these reasons in addition to the general difficulties in extrapolating in vitro data, 

the results obtained in this study cannot be taken into account for the risk characterisation. 

In another WEC assay (Jakobsen, 1995), rats embryos were cultured in a medium containing 

Glycol ethers (EGME, EGEE, EGIPE and EGBE) and their alkoxy acids (MAA, EAA, IPAA 

and BAA). The doses tested were 2, 4, 8 and 16 mM for alkoxy acids and 10 20, 40 and 80 

mM for the glycol ethers. After 48 hours of incubation, effects on development were assessed. 

The results obtained are summarized in the following tables: 
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Table 4.86: effects of EGBE on the development of rat embryos explanted on day of gestation and cultured for 48h. 

*** p < 0.001 ** p < 0.01 * p < 0.05

Concentration Control EGBE 5 mM EGBE 10 mM EGBE 20 mM EGBE 40 mM 

Nb of embryos 

evaluated 

123 20 19 10 16 

Development 

Yolk sac ∅

(mm) 

3.5 ± 0.4 3.5 ± 0.3 2.9 ± 0.3 *** 1.6 ± 0.5 *** 1.0 ± 0.3 *** 

Crown rump 

lengh (mm) 

2.8 ± 0.5 2.4 ± 0.3 *** 1.9 ± 0.2 *** 1.3 ± 0.2 *** - 

Nb of somites 29.2 ± 2.2 29.6 ± 2.3 25.0 ± 4.4 - - 

Morphological 

score 

36.3 ± 2.1 35.5 ± 1.6 29.0 ± 5.2 *** 11.7 ± 10.1 *** 0.0 *** 

% abnormalities 

Grossly 

abnormal 

embryos 

0 0 36.8 *** 100.0 *** 100.0 *** 

Cranial 

neuroporus, open 

0 0 57.9 *** 100.0 *** 100.0 *** 

Caudal 

neuroporus, open 

9.8 15.0 10.5 20.0 - 

Brancial arches, 

abnormal 

0 0 0 16.7 *** - 

Vesicles 0 15.0 *** 5.3 * - - 

Tail, abnormal 0.8 0 5.3 - - 

Flexion, 

ventrally convex 

2.4 5.0 10.5 60.0 *** - 

Somites irregular 0 0 0 - - 

Yolk sac, 

abnormal 

0 0 0 0 0 

Yolk sac, none 

circulation 

30.9 100.0 *** 100.0 *** 100.0 *** 100.0 *** 
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Table 4.87: Effects of BAA on the development of rat embryos explanted on day 9 of gestation and cultured for 

48 hr. 

*** p < 0.001 ** p < 0.01 * p < 0.05 

With EGME and EGEE, specific embryotoxic effects are found following exposure to very 

low levels of their alkoxyacetic acids whereas no specific effects were found when embryos 

were exposed to comparable concentrations of EGBE and BAA. The reproductive effects 

observed were due to non specific toxic damage to the embryos at comparable concentration. 

Concentration Control BAA 2 mM BAA 4 mM BAA 8 mM BAA 16 mM 

Nb of embryos 

evaluated 

123 15 19 20 18 

Development 

Yolk sac ∅

(mm) 

3.5 ± 0.4 3.6 ± 0.4 3.5 ± 0.3 3.4 ± 0.4 2.7 ± 0.2 *** 

Crown rump 

lengh (mm) 

2.8 ± 0.5 2.8 ± 0.5 2.7 ± 0.3 2.2 ± 0.3 *** 1.4 ± 0.4 *** 

Nb of somites 29.2 ± 2.2 29.4 ± 2.1 30.5 ± 1.6 * 25.8 ± 4.3 *** 21.8 ± 3.0 *** 

Morphological 

score 

36.3 ± 2.1 33.3 ± 9.5 35.3 ± 2.3 30.9 ± 3.1 *** 19.6 ± 5.3 *** 

% abnormalities 

Grossly 

abnormal 

embryos 

0 6.7 ** 0 0 16.7 *** 

Cranial 

neuroporus, open 

0 6.7 ** 0 25.0 *** 100.0 *** 

Caudal 

neuroporus, open 

9.8 40.0 *** 68.4 *** 45.0 *** 0 

Brancial arches, 

abnormal 

0 0 0 10.0 *** 0 

Vesicles 0 0 0 0 0 

Tail, abnormal 0.8 0 0 15.0 *** 50.0 *** 

Flexion, 

ventrally convex 

2.4 0 0 0 22.2 *** 

Somites irregular 0 0 0 0 0 

Yolk sac, 

abnormal 

0 0 0 0 0 

Yolk sac, none 

circulation 

30.9 46.7 57.9 * 90.0 *** 100.0 *** 
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This study demonstrates that embryotoxicity of glycol ethers increases with the increasing 

chain lengh of the alkyl group and also that the developmental toxicity of the alkoxyacetic 

acids decrease with the increasing chain lengh of the alkyl group. 

In another Rat Whole Embryo Culture assay (Giavini et al., 1993) EGBE, Butoxy acetic acid 

(BAA) and 2 other glycol ethers and their corresponding alkoxyacids were tested. EGBE was 

tested at 3.12, 6.25, 12.5 and 25mM and BAA (adjusted to pH6 with NaOH solution) at 0.4, 

0.8, 1.6 and 3.2mM. General embryotoxic effects, as shown by a reduction of the somite 

number and of total protein/embryo, were seen with EGBE concentrations of and greater than 

6.25 mM. Embryonic development was blocked at the EGBE concentrations of 25 mM. 

Exposure of embryos to 12.5 mM resulted in severe dysmorphogenic effects (inhibition of 

rotation, severe reduction of telecephalic vesicles). A dose dependant increasing percentage of 

abnormal development was seen with BAA at concentrations of 0.8, 1.6 or 3.2 mM. For this 

test, results obtained with glycol ethers are not suitable for effect assessment because, in vivo, 

they have a very low half life in the circulating blood (< 10 min for EGBE) and the high doses 

at which glycol ethers produced effects in vitro are totally unrealistic compared to the in vivo

situation. 

For BAA, effects were seen from 0.8 mM (40 % for 0.8 mM, 44.4 % for 1.6 mM and 60 % 

for 3.2 mM vs 22.2 % for controls and 20 % for 0.4 mM). Total proteins and somites numbers 

were also decreased for the 0.8 mM dose. This could suggest effects due to general toxicity 

rather than specific toxicity but as usual developmental parameters were not assessed, this 

could not be fully confirmed (Saillenfait, pers. com.). The malformative effects affecting 

prosencephale in this study are common effects seen with such a test. 

In Whole Embryo Cultures of rat BAA (in the form of sodium salt) and 6 other alkoxy acids 

were investigated (Rawlings et al., 1985). At a concentration of 5 mM, BAA adversely 

affected the development of conceptuses both in growth and the incidence of abnormal 

embryos. The most frequent abnormality produced was an irregularity of the neural suture 

line. No effect was observed at 2mM. In comparison with other alkoxy acids (MAA, EAA, 

nPAA, 3MPA and 4MBA), it was apparent that the potency of the effects decreased with 

increasing length of the alkoxy chain. Dysmorphogenesis was least affected with BAA, 

nPAA, 3MPA and 4MBA. The authors state that the lack of teratogenicity of butoxyethanol in 

vivo is a reflection of the limited teratogenic potential of its alkoxy acid metabolite. 

EGBE was examined for its ability to block junction-mediated intercellular communication 

(Welsch and Stedman, 1984 ; Loch-Caruso et al., 1984). The blockage of a specific type of 

intercellular communication, that is mediated by gap junctions, could be a possible 

mechanism of action of some teratogens. Interruption of intercellular communication was 

measured in vitro by an assay that depends on the transfer of metabolites via gap junctions, 

i.e., metabolic cooperation (V79 cell assay). EGBE was an effective blocker of intercellular 

communication at non cytotoxic doses in this assay. Results obtained with other glycol ethers 

demonstrated that as the length of the aliphatic chain increased so did the cytotoxicity. 

Summary in vitro studies: 

In rat whole embryo culture assays EGBE and BAA seems to cause developmental 

abnormalities in conjuction with effects on growth. The NOAEL for these effects are 3.12 

mM for EGBE and 0.4 mM for BAA (Giavini et al., 1993). These studies have demonstrated 

that the glycol ether embryotoxicity increased with increasing chain length 

(EGBE>EGEE>EGME). In contrast, the alkoxyacid embryotoxicity decreased with 
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increasing alkoxy chain length (MAA>EAA>BAA). The in vitro studies performed with 

EGBE (and glycol ethers in general) exibit effects which are not relevant in vivo (according to 

the kinetic of glycol ethers in vivo and the great doses needed to produce effects) and 

therefore are not directly usable in the risk assessment. 

Studies in humans

For developmental toxicity, no studies are available for assessing the risk of exposure to 

EGBE alone. 

In an case-control study, the risk of congenital malformations related to glycol ether exposure 

during pregnancy have been evaluated (Ha et al., 1996). Cases were any infant, born alive or 

stillborn, and foetuses of any gestational age aborted because of a malformation. 

Malformations of known origin were excluded. Control were normal neonates born within 

one week of the case in the same place (maternity or locality). This study involved 991 cases 

and 1144 controls. Mothers were submitted to a standard questionnaire including occupational 

history, socio-demographic characteristics, pregnancy history and hobbies. Father’s work was 

also determined. Occupational exposure to glycol ethers was assessed. Glycol ethers were 

classified in four group: 

- group 1: EGME, EGEE and their acetates and some polyethylenic compounds 

- group 2: EGBE, EGPE, and their acetates 

- group 3: PGME, Propylene Glycol Methyl Ether Acetate (PGMA) and some 

polyethylenic and propylenic compounds 

- group 4: other glycol ethers 

 In this study, there was a significant excess of mothers occupationally exposed to glycol 

ethers among the cases (27 % versus 20 % of the controls). When types of malformations 

were studied, a significant excess of oral clefts and central nervous system malformations was 

found in glycol ether exposed mothers compared to controls (OR = 2, 95 % CI = 1.1 – 4.1 and 

OR = 1.8, 95 % CI = 1.1 – 3.3 respectively). An excess of musculoskeletal malformations in 

exposed mothers was also seen although it was on the borderline of significance. 

In an case-control study (same data and same study than the study described before ?), the risk 

of congenital malformations related to glycol ether exposure during pregnancy have been 

evaluated (Cordier et al., 1997). The cases were women with any product of conception with 

a major congenital malformation diagnosed prenatally of during the perinatal period. The 

study period was between 1989 and 1992 and was conducted on 6 European registries (2 in 

France, one in UK, 2 in Italie and one in The Netherlands. Each case was match with a control 

chosen with the same birth date and born in the same town. Mothers was submitted a 

standardized questionnaire to assess their socio-economic status, medical and obstetric 

history, consumption of alcohol – tobacco – drugs, hobbies and occupation, both before and 

during each trimester of the pregnancy. Concerning oral clefts, a distinction was made 

between cleft lip with or without cleft palate [CL(P)] and cleft palate only (CP) due to their 

etiological difference. 

Between 1989 and 1992, a total of 984 cases of malformation were reported. The overall odds 

ratio (OD) of congenital malformation associated with glycol ether exposure was 1.44. after 

adjustment for several potential confounders. The association with exposure to glycol ethers 
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appeared particularly strong in 3 malformation subgroups: neural tube defects (OR=1.94, 95 

% CI = 1.16 – 3.24), multiples anomalies (OR=2.00, 95 % CI = 1.24 – 3.23) and cleft lip 

(with or without cleft palate) (OR=2.03, 95 % CI = 1.11- - 3.73). In this last subgroup, risk, 

especially of an isolated defect, tended to increase with level of exposure. 

In the same study, the risk of oral cleft formation related to maternal occupation was studied 

in depth (Lorente et al., 2000). 

Upon the including 984 cases, 161 cases were oral clefts. Differences between working 

mothers concerned only the place of residence (mothers of CL(P) cases came from less urban 

background than their referent) and mother’s age (older for CL(P) cases). 

The analysis of occupational exposures was restricted to women who worked during 

pregnancy (100 cases and 75 referents). For occupational, the odds ratios were estimated for 

each type of cleft and were highest for service occupation. Within this group the ORs were 

higher for CP than for CL(P) and significant for CL(P) for only 2 occupations: housekeepers 

and hairdressers. For production workers, the elevated ORs concerned CL(P). For chemical 

exposure, Ors adjusted were estimated. For glycol ethers Ors significantly different for 1 were 

observed: 

Exposure CL(P) 

n=64 

CP 

n=36 

Referents 

n=751 

OR 

CL(P) 

95 % CI 

CL(P) 

OR 

CP 

95 % CI 

CP 

Glycol 

ethers 

23 11 137 2.1 1.14-3.88 1.82 0.82-4.03 

For CL(P) exposure to glycol ethers was associated with an excess risk. A more detailed 

analysis of the exposure variables of the level, frequency, and reliability of exposure could be 

performed only when enough subjects were exposed to each product. This analysis showed 

that, for glycol ethers among isolated CL(P) cases, the risk increased with the level and 

frequency. 

In a case-control study, the risk of Neural Tube Defects related to maternal occupation al and 

hobby chemical exposure was assessed (Shaw et al., 1999). 653 Neural Tube Defects cases 

(anencephaly, spina bifida cystica, craniorachischisis or iniencephaly) were extracted from a 

cohort of 708129 births and foetal death between June 1989 and May 1991. An interview of 

538 cases and 539 controls was performed. Information about paternal occupation were also 

checked. Using information about occupation and hobbies, exposures to 74 chemical agents 

were classified as likely, maybe or not exposed. A classification was also made regarding 

end-use of chemicals in 9 group. 

Compared to control mothers, case mothers were more likely to be Hispanic, less than 25 

years old and to have a household income under 10000 dollars. They also were less likely to 

have graduated from college, to have used multivitamins in the 3 months before conception, 

to have smoked cigarettes in the first trimester or to have been employed in the 

periconceptional period. 

Exposure to house keeping cleaner give an OD of 0.97. Overall there was no increase risk for 

solvent exposure based on combined occupational and hobby/household exposure or for 

occupational exposure along among employed women. 
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For glycol ethers and derivatives: 

NTD cases Controls Odds ratio 95 % confidence interval 

75 80 0.93 0.66-1.3 

 The risk of NTD was not increased for glycol ethers exposures. 

A case control study similar than those already performed was made in Slovakia with data 

collection ranging from 1995 to 1996 (Cordier et al., 2001). 196 case of live or stillborn 

babies with a major malformation or foetuses from therapeutic abortion were included in the 

study. Assessment of various chemical exposure (including glycol ethers) was performed. 

The distribution of congenital malformations in each broad group of anomalies was very 

similar to those observed in the previous study. Odds ratio for glycol ethers exposure during 

the first trimester of pregnancy were estimated among working mothers for each subgroup of 

abnormalities, adjusted for maternal age, rural residence and mother’s socio-economic status. 

The overall risk of congenital abnormalities was elevated (OR = 2.3; 95 % CI = 0.7-7). 

Among 15 potentially exposed women, 10 (7 cases, 3 controls) were exposed to the highest 

exposure category; in this group, the OR for all malformations was slightly higher (OR = 2.7; 

95 % CI = 0.7-11). Malformation-specific Ors were increased with high exposure for all 

except cardiac anomalies (unchanged) and urinary anomalies (decreased). Caution should be 

given to this study due to the very small size of the studied population. 

In a case-control study, the risk of Neural Tube Defect related to parental occupation was 

assessed among Mexican Americans (Brender et al, 2002). Case women were Mexican 

Americans whom had a product of conception (live or stillborn infant or spontaneous or 

induced abortion) with an NTD during the period of June 1995 to May 2000. NTD included 

were: anencephaly, spina bifida and encephalocele. A complete questionnaire was submitted 

to each case and control woman to determine their maternal health and reproductive history, 

family demographic data and medical history, and the use of medications, nutritional 

supplement, tobacco, alcohol, and drugs. Environmental and occupational exposures were 

also assessed. 

Of the 225 cases, 184 completed the interview (82 %). For the controls, only 225 of the 378 

selected (60 %). Case women tended to be slightly younger, less educated and significantly 

more case than controls where obese. For occupation, case women are more likely to work as 

cleaners and in health case. They are also more likely to have work exposures to glycol ethers 

(OR ∞, 95 % CI 1.8 - ∞). Women who reported activities associated with solvent exposure 

outside the workplace also were likely to have NTD-affected pregnancies. For paternal work: 

risk estimates were close to the null for exposure to glycol ethers. 

Based on these findings, maternal work exposure to solvents or glycol ether and maternal 

occupations in health care or cleaning may be risk factors for NTDs in the studied population. 

Through a review of the epidemiological studies available, Maldonado et al. (2003) 

demonstrated that the results available all suffers from methodological deficiencies which 

weaken the conclusions of these studies. 
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Other relevant information 

In a study by Wilson (1953), dams were bled to induce haemorrhagic anaemia at different 

stages of pregnancy. Dams were bled at 3 critical periods during development: (1) on gd 7-9 

to cause anaemia when the mesoderm is forming and organogenesis is beginning on gd 9-10; 

(2) on gd 9-11 or gd 11-13 to produce anaemia during the most active period of organogenesis 

on gd 11-14; and (3) on gd 13-15 during late organogenesis and early histogenesis, which 

occurs on gd 15-16. Blood loss by dams was 5 cc or more per 100 g body weight, which 

caused a marked drop in haematocrit values. For dams bled on gd 7- 9, removal of 5 to 5.9 cc 

blood per 100 g body weight resulted in a 52 % decrease in haematocrit (relative to controls) 

and totally resorbed litters for 33 % of pregnancies (3 of 9). Haemorrhages in excess of 6 cc 

caused a 63 % decrease in haematocrit and early termination of 13 out of 15 pregnancies due 

to either maternal death (8 dams) and total resorbed litters (5 dams). For dams bled on gd 9-

11, 25 % of dams losing 5-5.9 cc per 100 g body weight exhibited a 54 % decrease in 

haematocrit and totally resorbed litters with 1 of 8 term litters (gd 20) exhibiting retarded 

development. For dams with more severe anaemia (in excess of 6 cc with a 59 % decrease in 

haematocrit), one third exhibited total resorbed litters. Of the 13 pregnancies with fetuses 

surviving to term (gd 20), the mean rate of prenatal survival was decreased (18 % of fetuses 

resorbed). Four litters exhibited growth retardation (delay). In general, the authors concluded 

that “although an occasional abnormality may be produced at this time, maternal anaemia 

usually causes either early termination of pregnancy or permits continuation of pregnancy 

without detriment to the young.” Bleeding the dams on the 11-13 or 13-15 days of pregnancy 

caused a higher rate of maternal death (45 % of dams). In some cases, death of the fetuses 

preceded death of the dam. Total litter loss due to resorption was not seen at these time points. 

Some delayed development was seen. Bleeding that was initiated on gd 9 had the greatest 

likelihood of resulting in malformation, growth retardation (4 of 13 litters), or increased rate 

of resorption. 

Similar results have been reported for pregnant rabbits in the presence of maternal anaemia. In 

studies with diflunisal, an antiinflammatory drug, pregnant does given a single oral dose of 

diflunisal on gd 5 exhibited anaemia that persisted through gd 15, despite clearance of the 

drug from maternal blood by gd 9 (Clark et al., 1984). Signs of anaemia in pregnant does 

included haemoglobinuria, and decreased erythrocyte counts (a 74 to 46 % decrease relative 

to controls on gd 9 to 15) and decreased haemoglobin concentrations (a 63 to 21 % decrease 

relative to controls on gd 9 to 15). Haematotoxicity was the most severe on gd 9, the first day 

of data collection, with some recovery by gd 15. These does showed an increase in 

preimplantation loss (10 of 25 inseminated does showed no evidence of implantations and 

there was an increase in preimplantation loss in other does), as well as an increase in totally 

resorbed litters (11 of 25 litters). The percentage of resorptions and dead fetuses (mean of 

litter values) was 10.6 % in control does compared with 74.3 % in diflunisal-treated does, 

despite clearance of the test material from maternal blood by gd 9. These effects were 

attributed to maternal anaemia rather than a direct effect of diflunisal, because diflunisal is 

highly protein bound in maternal blood (greater than 98 %); thus, there are only low 

concentrations of diflunisal in rabbit embryos. Furthermore, rats, which are resistant to 

diflunisal-induced maternal anaemia, do not demonstrate embryo/fetal effects, despite 

reaching approximately equivalent doses of diflunisal in rat embryos. Furthermore, there is 

little accumulation of diflunisal in embryos, which is likely a reflection of its low placental 

transfer. This study provides strong evidence that the embryo/fetal effects (similar to those 

seen with EGBE) were secondary to maternal anaemia. 
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4.1.2.9.3 Summary of toxicity for reproduction 

Unlike EGME and EGEE, EGBE seems to have no specific effects on fertility (no effects 

were seen in the continuous breeding study and neither macroscopic nor microscopic effects 

on reproductive organs in the repeated dose toxicity studies at doses which does not exhibit 

severe general toxicity.) A NOAEL of 720 mg/kg was derived from the continuous breeding 

study for fertility effects (it should be noted that effects seen at the higher dose tested are 

certainly due to general toxicity) 

For developmental toxicity, studies performed on animals via various administration routes 

did not demonstrate any teratogenic potential, but foetotoxicity and embryotoxicity (lethality 

and resorptions) were often observed in relation with maternal toxicity (regenerative 

haemolytic anaemia). Other effects seen on foetuses were an increase in the incidence of 

skeletal variations which are generally described as ossification delays. In vitro studies 

showed some adverse effects on development with EGBE and its metabolite BAA, but only in 

conjuction with growth effects. Effects seen in foetuses are certainly related to maternal 

toxicity. Some studies have previously shown a relationship between maternal haemotoxicity 

and effets seen with EGBE (resorption, growth retardation and variations). 

Haemotoxicity described in § 4.1.2.2.3 generally occured at low doses of EGBE whatever the 

route of administration used. In theses studies, data on haemolysis were often observed with 

acute dosing. Developmental toxicity studies would require daily dosing with test material, 

which may produce more marked effects on haematopoietic parameters. In addition, female 

mice and rats were more affected by EGBE haemolysis than males. Thus, these data 

demonstrate that the concentrations of EGBE used in these developmental toxicity studies 

were adequate to produce severe maternal anaemia of the magnitude sufficient to cause 

effects on embryo/fetal survival. These data give plausible support to the hypothesis that the 

effects seen in developmental toxicity studies with EGBE were due to haemolysis and 

subsequent maternal anaemia. 

In human, all the epidemiological studies, except one, studying glycol ethers, showed an 

increased risk of malformation (cleft lip, neural tube defect). For EGBE, these studies did not 

allow to draw any conclusion about its potential effects on human because no studies are able 

to distinguish clearly an unique source of glycol ether, usually studies described co-exposure 

to various glycol ethers, including known developmental toxins such as EGME and other 

chemicals as well. 

Overall, it is not possible to obtain a suitable NOAEL for developmental toxicity relevant for 

humans and based on animals studies. Regarding kinetic properties and SAR with other 

glycol ethers, it can be assumed that developmental toxicity due to EGBE in humans could 

not be expected without maternal toxicity. Consequently, there is no concern for this endpoint and no need for risk characterisation. 
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4.1.3 Risk characterisation 5

4.1.3.1 General aspects 

The human population may be exposed to EGBE at the workplace, both from use of consumer 

products and indirectly via the environment (see 4.1.1.1, 4.1.1.2 and 4.1.1.3) 

From the oral absorption studies it is concluded that oral absorption is complete. For risk 

characterisation 100 % oral absorption should be assumed. 

From dermal absorption studies, a wide range of absorption values were observed depending 

on the species (rats having a greater dermal penetration than humans), the dilution of EGBE 

(40 % or 80 % water solutions of EGBE being absorbed at twice the rate compared to lower 

dilutions or undiluted EGBE), physical state of EGBE and occlusion status of administration. 

In general, dermal absorption of liquid EGBE varies between 20 to 30 % of applied dose in 

rats. Variations are seen in the reported values for individuals humans for the rate of 

absorption (ranging from 0.064 mg/cm2

/hr to 1.66 mg/cm2/hr in vitro and from 0.826 to 11.3 

μg/cm2

/hr in vivo). For dermal absorption of vapour EGBE, studies on volunteers have shown 

a percentage of internal dose due to dermal absorption of 11 to 39 % (depending on the 

conditions of exposure). According to the PbPk model, for a worst case exposure (100 % of 

the body exposure with no cloths), internal dose of EGBE due to the percutaneous uptake 

when a subject is exposed to EGBE as vapour would be 15 to 27 % (this range being not 

negligible compared to the dose due to inhalation). For risk characterisation, dermal 

absorption of liquid EGBE can be assumed to be 30 % of applied dose. For dermal 

absorption of vapour a value of 39 % of the internal dose due to dermal absorption can be 

taken into account but keeping in mind that this value has been demonstrated only during 

extreme exposure conditions (high temperature, high humidity and overalls clothing) which 

can be considered to be the worst case of exposure. 

From human volunteers inhalation studies, an absorption of 55 % to 60 % has been measured. 

These values varies from the theoretical absorption value of 80 %, calculated from various 

studies, due to a wash in / wash out mechanism on the surface of the respiratory tract. For risk 

characterisation, 60 % inhalation exposure should be assumed (highest measured value). 

Table 4.88: absorption coefficients taken into account for the calculations of internal doses 

 Oral Inhalation Dermal route 

% of absorption 100 % 60 % EGBE liquid EGBE vapour 

30 % 1 39 % of the 

internal dose 1

1 Maximum worst case percentage, can be reassessed for each scenario in a case by case basis 

if needed . 

 

5 Conclusion (i) There is a need for further information and/or testing. 

Conclusion (ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond those 

which are being applied already. 

Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into 

account.
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For interspecies extrapolation, PBPK models exist for the rat, mouse and human. These 

enable the internal dose of EGBE to be estimated with some precision. Of the models 

available, that from Corley et al. 1994 and 1997 is considered the most complete and 

appropriate for potential use in the derivation of an interspecies extrapolation factor for oral 

exposure because it has been experimentally validated, covers relevant routes of exposure, 

and addresses both the distribution and excretion of the metabolite BAA which is invariably 

the key substance of interest. For the inhalation route, the model of Lee et al. (1998) was 

used to estimate BAA blood concentrations in female rats following inhalation exposure to 

EGBE because it is a recent extension of the Corley et al. (1994 and 1997) model for 

inhalation exposures and includes added parameters for female rats. 

The PBPK model allows the concentration of the proximate toxicant (BAA) to be predicted 

following either inhalation or oral exposure to EGBE. The following four steps are carried 

out to obtain a human equivalent concentration (HEC), following a similar approach to that 

adopted by the US EPA (US EPA, 1999): (1) calculate the internal dose metric (Cmax BAA in 

blood) corresponding to the female rat LOAEL, using the actual experimental exposure 

pattern (6hrs/day, 5 days/week) in the model simulation; (2) verify that steady state was 

achieved (e.g., no change in BAA Cmax as a result of extending the exposure; (3) simulate the 

internal dose metric (Cmax BAA in blood) for humans inhaling EGBE and 4) calculate the 

HEC (mg/m3

) that results in this internal dose Cmax BAA. The relationship between EGBE 

exposure and resultant BAA for humans and female rats as used in step 3 is shown below: 

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 50 100 150 200 250 300

Concentration of EGBE in air (ppm)

CmaxBAA in blood (μM)

Note: Data for rats is the upper line, data for humans the lower line

For acute toxicity, animal studies have given a LD50 of 1000 mg/kg by oral route in reliable 

mice and rats studies. By dermal route, depending on the application (occlusive or not) the 

LD50 was 500 mg/kg or > 2000 mg/kg respectively. By inhalation route, mortality was 

evidenced in reliable studies from about 500 ppm concentration (the LC50 being of about the 

same level). The effects seen in acute toxicity studies in animals were mainly acute 

haemolysis accompanied with non specific signs of toxicity (laboured breathing, lethargy, 

ataxia). Some hepatic and renal pathology was also seen in these studies. 
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In humans cases of massive ingestions the main toxic effect was a metabolic acidosis with 

sometimes haematotoxicity. For haematotoxicity, humans are much less sensitive than rodents 

and according to human studies, it appears that there is not a great intraspecies sensitivity (no 

influence of age or haematological status). Moreover, this end point was not the most 

sensitive end point for acute toxicity in human because in case of massive ingestions 

haemotoxicity was not observed in all cases. But metabolic acidosis was observed in all case. 

The lowest dose leading to metabolic acidosis in human was 400 mg/kg (LOAEL). 

As human data is preferred if exists, this LOAEL will be taken into account for the risk 

characterisation. As no data exists in human by dermal or inhalation route, an extrapolation of 

this LOAEL will be done for risk characterisation for these routes using appropriate route to 

route extrapolation factors. 

EGBE liquid is classified as irritating to the skin and to the eyes. For risk characterisation of 

EGBE vapour, a human NOEL of 50 ppm is used based on recent volunteer studies. No 

sensitisation properties liked directly to EGBE were seen from the available data on humans 

and animals. 

For repeated dose toxicity, it has been shown that rodents are much more sensitive than 

human to the haemolytical effects of EGBE, therefore if a NOAEL based on these effects is 

used for risk characterisation, the MOS used should reflect the sensitivity of rodents. Kupffer 

cell pigmentation is secondary to the haemolytic effects. Effects on spleen (including spleen 

fibrosis) can also be related also to haemolysis. This effect usually occur following 

administration of substances which is able to cause iron accumulation in the splenic red pulp 

(Goodman et al., 1984 ; Weinberger et al., 1985). Effects on the forestomach of rodents do 

not appear to be relevant for humans. With regard to the increased incidence of hyaline 

degeneration of the olfactory epithelium observed in rodents, this appears to be an adaptive 

response, the severity of the lesion being unaffected by increasing exposure concentrations. 

In other species than rodents, old studies are available. In dogs and monkeys, haemolysis was 

also described at relatively low doses. In guinea pigs, a species which is resistant to EGBE 

induced haemolysis, mortality was seen from 375 ppm and higher. Due to the limitations of 

these old studies these results cannot be taken into account for the risk assessment. The most 

reliable inhalation data is the LOAEL of 31 ppm derived from a a 6 month satellite group in a 

two-year study in rats. For oral route, a LOAEL of 69 mg/kg bw/d in male rats was derived 

from a 14 week oral study in rats. For dermal exposure, a NOAEL of 150mg/kg/day can be 

used. 

Since all key effects are induced by haemolysis in rodents, a NOAEL based on haemotoxicity 

will be used in the risk characterisation. The selection of an appropriate interspecies chemical 

safety assessment factor (CSAF) must take into account the lower sensitivity of humans to 

BAA than rats (or mice). IPCS have proposed splitting the CSAF two components 

representing the toxokinetic and toxodynamic adjustment factors (AKAF and ADAF

respectively). The toxicokinetic factor is taken account of by use of the PBPK model 

described above. The ADAF factor needs to be set to an appropriate value to reflect the lower 

sensitivity of humans to haemolysis. The data available on the most sensitive measure (prehemolytic changes) suggests that a value of 0.01 would be realistic. However, a more 

cautious and conservative initial approach would be to propose a value of 0.1. 

For mutagenicity, it has been demonstrated that EGBE is not a mutagenic substance in vivo. 
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For reproductive toxicity, no specific effects were seen for fertility. In the continuous 

breeding study a NOAEL of 720 mg/kg was set based on non specific effects observed at the 

higher doses tested. For developmental studies, embryonic and foetal effects seen in animals 

were related to maternal toxicity (haemolysis). 

For carcinogenicity, some effects were seen in mice in the 2-year studies. As the mechanism 

of haemangiosarcomas in male mice is related to haemotoxicity, the risk characterisation 

made for repeated dose toxicity is considered sufficient to also assess for carcinogenicity. The 

other tumours (mouse forestomach) are considered not relevant to humans; no risk 

characterisation is needed for them. 

4.1.3.2 Workers 

Assuming that oral exposure is prevented by personal hygienic measures, the risk 

characterisation for workers is limited to the dermal and the inhalation routes of exposure. 

Three sets of values have been utilised for dermal exposure: the EASE estimates, the data 

measured on DEGBE, and biomonitoring data from EGBE use in actual situations. As 

previously discussed in 4.1.1.1.2.2 and in 4.1.1.2.4, it is proposed to take the available 

RISKOFDERM data as a basis and to adapt them using EGBE’s biomonitoring data when 

applicable. Summary of proposed reasonable worst case exposure is indicated below. MOS 

are calculated and presented in the relevant part of the risk characterisation. 

Table 4.89: Summary of proposed reasonable worst case exposures (see also table 4.29 in & 4.1.1.2.4) 

Scenario Inhalation 

(mg/m3) 

External dermal 

exposure (mg/day) 

1 - Manufacture 12 42 

2 - Formulation 15.7 2,000 

3 - Use of products 

3.1 Coating/Paintinga

- industrial 

- Spraying 

- Other works 

- decorative 

3.2 Printing 

- silk screening 

- general printing 

3.3 Cleaning 

- spraying 

- wiping 

58.1 

30.4 

30.4 

20 

5 

49 

49 

2000

430 

70

23 

168

250

1,040 
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4.1.3.2.1 Acute toxicity 

According acute toxicity data in human (suicide attempts cases) a conservative LOAEL of 

400 mg/kg by oral route can be taken into account. As limited data exist in humans by dermal 

or inhalation route an extrapolation route to route of this LOAEL is done. The MOSs obtained 

are compared with minimal MOS calculated as follows: 

Table 4.90: Assessment factors applied for the calculation of minimal MOS for acute toxicity (for inhalation and 

dermal route) 

Interspecies differences 1 (human data) 

Intraspecies differences 3 (workers: homogen population) 

Type of effect 1 

Extrapolation LOAEL to NOAEL 5 

Confidence of the database 1 

Minimal MOS 15 for inhalation and dermal routes 

Inhalation route: 

Oral LOAEL of 400 mg/kg has to be extrapolate to a inhalation NOAEL to compare with 

inhalation exposure. 

The dose of 400 mg/kg by oral route correspond to 400 mg/kg of internal dose (100 % 

absorption by oral route) 

To obtain this internal dose by inhalation, it is necessary to add internal dose due to inhalation 

of vapours and internal dose due to percutaneous penetration of vapours. The ratio between 

these two doses has been calculated 61% / 39 % respectively. Leading to: 

400 x 0.61 = 244 mg/kg of internal dose due only to inhalation and 

400 x 0.39 = 156 mg/kg of internal dose due only to percutaneous absortion of vapours. 

- To obtain 244 mg/kg of internal dose in one day, a worker of 70 kg with a respiratory 

volume of 10 m3

/worday, withan absorption factor of 60 % for inhalation uptake Should be 

exposed to 

244 mg/kg * 70 kg / 10 m3

 / 0.60 = 2846 mg/m3. 

This value is the extrapolated LOAEC and should be compared with exposure levels. 

The MOSs between the LOAEL and the inhalation exposure levels are mentioned in table 

4.91. The MOSs are evaluated by comparison with the minimal MOS (table 4.90). The 

conclusions are given in the table 4.91. Based on the risk assessment for inhalation exposure, 

it is concluded that toxicity due to acute exposure are not expected. 

Conclusion ii is reached for all occupational scenarios. 
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Table 4.91: Occupational risk assessment of EGBE for acute toxicity 

Risk assessment for inhalation 

exposure

Risk assessment for dermal exposure 

to liquid EGBE

Estimated Skin 

exposure 

mg/day 

(mg/kg bw/d

Scenario

8-hour 

TWA 

inhalation 

(mg/m3

)

MOS1 Conclusion MOS Conclusion

1 - Manufacture 12 237 ii 42 

(0.6) 

2220 ii 

2 - Formulation 15.7 181 ii 2,000 

(28.57) 

47 ii 

3.1 Coating/Painting

3.11 –Industrial

-spraying 58.1 49 ii 2000 

(28.57) 

47 ii 

-other works 30.4 94 ii 430 

(6.14) 

217 ii 

3.12 - decorative 30.4 94 ii 70 

(1) 

1333 ii 

3.2 Printing

3.21 Silk 

screening

20 142 ii 23 

(0.33)

4039 ii 

3.22 General 

printing 

5 569 ii 168 

(2.4)

555 ii 

3.3 Cleaning 

- spraying 49 58 ii 250 

(3.57) 373 

ii 

3 - Use of 

end 

products

- wiping 49 58 ii 1040 

(14.86) 

90 ii 

a: non dispersive use, b: wide dispersive use. 
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1- calculation based on a respiratory volume of 10 m3/workday, a worker bw of 70 kg, an absorption 

factor of 60 %, an internal dose due to inhalation uptake (61 %) and dermal uptake of vapour (39 %) 

and an oral LOAEL of 400 mg/kg bw. 

2- calculation based on a worker bw of 70 kg, an absorption factor of 30 % and an oral LOAEL of 400 

mg/kg bw. Worst case.

Dermal route:

Oral LOAEL of 400 mg/kg has to be extrapolate to a dermal LOAEL to compare with dermal 

exposure. 

Oral dose of 400 mg/kg would give an internal dose of 400 mg/kg. To reach this dose with a 

dermal exposure to liquid EGBE, the external dose should be : 

400 / 0.3 = 1333 mg/kg bw 

because 30 is assumed to be the worst case percentage of absorption of liquid EGBE by 

dermal route. 

This LOAEL of 1333 mg/kg bw should be compared with estimated skin exposures. The 

MOSs between the LOAEL and the dermal exposure levels are mentioned in table 4.91. The 

MOSs are evaluated by comparison with the minimal MOS (table 4.90). The conclusions are 

given in the table 4.91. Based on the risk assessment for dermal exposure, it is concluded that 

toxicity due to acute exposure are not expected. 

Conclusion ii is reached for all occupational scenarios. 

Combined exposure: 

For the combined exposures the estimated internal doses are calculated from the biological 

exposure data. The oral LOAEL of 400 mg/mg would lead to an internal doses of 400 mg/kg 

because an absorption factor of 100 is taken into account for oral route. This LOAEL should 

be compared with internal doses calculated from exposures in each scenario (inhalation + 

dermal). The internal doses are calculated as follow : 

Inhalation exposure will give internal dose of: 

X (value of the 8-hour TWA inhalation (mg/m3

)) x 10 m3 (inhaled air during a workday) x 0.6 

(percentage of absorption by inhalation) / 70 (mean bw of a worker) = Y (inhalation internal dose). 

This value does not take into account the possible dermal absorption of vapour during the 8hr 

TWA. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % 

of the internal dose of EGBE. To take into account this value (which is not negligible) the 

value of internal dose due to dermal exposure to vapours (Z) should be added to the former 

value (Y). Z represent 39 % of the total internal dose and can be calculated as follow : 

Z = 0.39/0.61 x Y = 0.64 Y 

The total internal dose due to inhalation exposure (inhalation output + dermal vapour penetration 

output) is Y + Z = 1.64 Y 

For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of 

absorption of 30 % (liquid EGBE, worst case). 
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Table 4.91 bis: Occupational combined risk assessment for acute toxicity

Scenario 

Internal dose 

after exposure 

to 8-hour 

TWA (mg/kg 

bw) 

Y+Z 

Internal dose after 

Dermal exposure 

to liquid EGBE 

(mg/kg bw) 

worst case (based 

on maximal dose)

Total internal 

dose 

(inhalation + 

dermal 

exposure) 

MOS Conclusion 

1 - Manufacture 1.69 0.18 1.87 213 ii 

2 - Formulation 2.2 8.58 10.78 37 ii 

3 - Use of products 

3.1 

Coating/Painting 

- industrial 

- Spraying 

- Other 

works 

- decorative 

3.2 Printing 

- silk screening 

- general 

printing 

3.3 Cleaning 

- spraying 

- wiping 

8.17 

4.28 

4.28 

2.81 

0.70 

6.89 

6.89 

8.58 

1.84 

0.30 

0.10 

0.72 

1.07 

4.46 

16.75 

6.12 

4.58 

2.91 

1.42 

7.96 

11.35 

24 

65 

87 

137 

281 

50 

35 

ii 

ii 

ii 

ii 

ii 

ii 

ii 

The calculated MOS should be compared with minimal MOS. To be in the worst case, the 

highest minimal MOS should be chosen for the comparison: 15. 

For acute toxicity a conclusion ii is reached for all scenarios for combined exposure. 

4.1.3.2.2 Irritation and corrosivity 

Skin

EGBE is classified as a skin irritant, and the use of appropriate PPE is recommended. This 

endpoint is of concern, but that this concern is managed by the information given by the 

classification and labelling of the substance so that no further concern remain. (conclusion ii). 
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Eye

Eye irritation (liquid) : 

The substance is classified as a eye irritant, and the use of appropriate PPE is recommended 

This endpoint is of concern, but that this concern is managed by the information given by the 

classification and labelling of the substance so that no further concern remain. (conclusion ii). 

Eye and respiratory tract irritation (vapours): 

Since the NOEL for respiratory tract irritation is derived from humans, the only assessment 

factor needed is that to allow for possible intraspecies variation. This is particularly true since 

the effects are not physiological but more discomfort in nature. A factor of 3, and therefore 

minimal MOS of 3 is considered sufficient for this end point. The MOSs between the NOAEL 

and the inhalation exposure levels and the conclusions of the risk assessment are mentioned in 

table 4.92. 

Table 4.92: Occupational risk assessment of EGBE for eye and respiratory irritation by vapour exposure. 

Scenario Risk assessment for eye and respiratory irritation by vapour exposure

8-hour TWA inhalation 

(mg/m3

)

MOS1 Conclusion

1 - Manufacture 12 20 ii 

2 - Formulation 15.7 16 ii 

3.1 Coating/Painting

3.11 –Industrial

-spraying 58.1 4.2 ii 

-other works 30.4 8 ii 

3.12 - decorative 30.4 8 ii 

3.2 Printing

3.21 Silk 

screening

20 12 ii 

3.22 General 

printing 

5 50 ii 

3 - Use of 

end 

products

3.3 Cleaning 49 5 ii 

For eye and respiratory tract irritation due to the exposure of EGBE in vapour form, 

conclusion ii is reached for all occupational scenarios. 
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4.1.3.2.3 Sensitisation 

Given the results from the dermal sensitisation studies it is concluded that EGBE is of no 

concern for workers with regard to skin sensitisation (conclusion ii). 

There are neither data from human experience nor other indications for respiratory 

sensitisation but this toxicological end-point is not expected for EGBE (conclusion ii).

4.1.3.2.4 Repeated dose toxicity 

It is clear from the available data that haemolysis is the primary and critical response elicited 

in the main animal test models (rats and mice) following inhalation, oral or dermal exposure 

to EGBE. Blood from humans, pigs, dogs, cats, and guinea pigs is less sensitive to 

haemolysis by BAA. In sensitive species, EGBE produces a characteristic toxicity that is 

revealed clinically by the appearance of haemoglobinuria and pathologically by changes in a 

variety of blood parameters. A 100-fold greater concentration of BAA (10mM) is required 

for human erythrocytes to develop pre-hemolytic changes consistent with those seen in the rat 

(0.1mM). Such in vivo blood concentrations are unlikely to occur under normal conditions of 

human exposure to BE. Haemolysis did not occur with any blood from any individual, even 

sensitive sub-populations, when exposed in vitro to BAA(10 mM). Studies have also shown 

that potentially sensitive human sub-populations, including the children, the elderly and those 

with sickle cell anaemia are also equally resistant to the effect. 

PBPK models exist for the rat, mouse and human. 

These can be applied to derived the appropriate toxokinetic element of the interspecies 

assessment factor . The use of the model is described in the General Aspects section and is 

applied here as follows: 

Step 1: Calculate Cmax for BAA in blood corresponding to female rat LOAEL 

Female rat (critical species and sex) LOAEL = 31 ppm. 

Resultant Cmax BAA from such an exposure= 285 μM. 

Step 2: Verify steady state. 

There were no changes in the Cmax of BAA in blood during any 24-hour simulation 

period using a 6 hours/day, 5 days/week exposure regime at the female rat LOAEL, 

indicating that steady state was achieved. 

Steps 3 and 4: Calculate the Cmax for BAA in blood for humans continuously exposed to 

EGBE vapour 

and calculate the LOAELHEC for EGBE for human exposures producing the same Cmax

of BAA in blood as that that produces effects in rats. 

For aBAA concentration of 285 μM, the HEC = 98 ppm (474mg/m3). 
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Table 4.92a: Equivalent human doses for LOAEL(C) / NOAEL(C) 

 Oral Inhalation Dermal 

End point LOAEL LOAEC NOAEL 

Value 69mg/kg/day (male 

rat) 

31ppm (152mg/m3) 150mg/kg/day 

Equivalent BAA Cmax

concentration 

129 μM 285μM 

Equivalent human dose 9.5 mg/kg/day 97ppm (474mg/m3) 

A Risk Characterisation not using PbPk model is also proposed in Appendix B. 

The risk characterisation for repeat dose then becomes as follows: 

4.92b: Assessment factors for repeated dose toxicity 

Interspecies differences 0.1 (toxicodynamic factor) 

Inhalation: toxicocinetic element taken into account by 

PBPK model 

Dermal: Additional factor of 4 to account for allometric 

scaling of rats to humans 

Intraspecies differences 5 (default for workers) 

Duration of study No factor required. The critical study is 6 months in 

duration and the effects for this end-point (haemolysis) 

would not be expected to be more severe compared to 

lifetime exposure. (haemolysis due to EGBE is 

considered to be an acute or sub-acute effect on rodents, 

moreover in some studies animals tended to “recover” 

(red blood cells being less sensitive ) with long times of 

exposure. 

Type of effect 1 

Extrapolation LOAEL to NOAEL 3 for oral and inhalation. The effects were mild at this 

dose and there is evidence to show that the LOAEL is 

near the threshold level for effects of concern (eg 

NOAECs from other studies) 

Factor of 1 for dermal exposure 

Confidence of the database 1 

Minimal MOS 1.5 for oral and inhalation 

2.0 for dermal exposure 
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Note that the critical LOAEC used for the inhalation risk characterisation is derived from a 

whole body inhalation exposure study (as are all similar studies). These animals will have 

been subjected to exposure by both the dermal and inhalation routes. The dermal absorption 

rates in rats and mice are also higher than in humans. Therefore it is not necessary to make a 

correction for additional uptake from dermal absorption of vapours.

Risk characterisation 

Oral exposure 

No oral exposure has been identified for workers, therefore there are no concerns for this 

route of exposure 

Inhalation 

Table 4.92c: Risk assessment for inhalation and dermal exposure for RDT 

Risk assessment for inhalation 

exposure

Risk assessment for dermal exposure 

to liquid EGBE

Scenario

8-hour 

TWA 

inhalation 

(mg/m3

)

MOS1 Conclu

sion

Estimated 

Skin exposure 

mg/day 

(mg/kg bw/d

MOS2 Conclusion

1 - Manufacture 12 39 ii 42 

(0.6) 

250 ii 

2 - Formulation 15.7 30 ii 2000 

(28.57) 

5 ii 

3.1 Coating/Painting

3.11 –Industrial

-spraying 58.1 8 ii 2000 

(28.57) 

5 ii 

-other works 30.4 16 ii 430 

(6.14) 

24 ii 

3.12 - decorative 30.4 16 ii 70 

(1) 

150 ii 

3.2 Printing

3 - Use of 

end 

products

3.21 Silk 

screening

20 24 ii 23 

(0.33) 

454 ii 
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3.22 General 

printing 

5 95 ii 168 

(2.4)

62 ii 

3.3 Cleaning 

spraying 49 10 ii 250 

(3.57) 

42 

wiping 49 10 ii 1040 

14.86) 

10 ii 

For exposure via the inhalation route, conclusion (ii) is drawn for all scenarios. 

Dermal exposure: 

Exposure values are compared with the NOAEL of 150 mg/kg. 

It is not possible to use the PBPK approach for the dermal assessment and a more 

conventional approach is therefore used. 

See previous table and table showing derivation of MOSs 

For exposure via the dermal route, conclusion (ii) is drawn for all scenarios. 

Combined exposure: 

For the combined exposures the estimated internal doses are calculated from the biological 

exposure data. The inhalation LOAEC is chosen for comparison as this is lower than the 

equivalent for the dermal route. The LOAEC of 474mg/m3 would lead to an internal doses of 

474 mg/m3

 x 10m3/day x 0.6/70kg = 40.6 mg/kg/day. This internal dose should be compared 

with internal dose calculated. This LOAEC should be compared with internal doses calculated 

from exposures in each scenario (inhalation + dermal). The internal doses are calculated as 

follow: 

Inhalation exposure will give internal dose of: 

X (value of the 8-hour TWA inhalation (mg/m3)) x 10 m3 (inhaled air during a workday) x 0.6 

(percentage of absorption by inhalation) / 70 (mean bw of a worker) = Y (inhalation internal 

dose). 

This value does not take into account the possible dermal absorption of vapour during the 8hr 

TWA. It has been demonstrated that dermal absorption of vapour EGBE could count for 39 % 

of the internal dose of EGBE. To take into account this value (which is not negligible) the 

value of internal dose due to dermal exposure to vapours (Z) should by added to the former 

value (Y). Z represent 39 % of the total internal dose and can be calculated as follow: 

Z = 0.39/0.61 x Y = 0.64 Y 

The total internal dose due to inhalation exposure (inhalation output + dermal vapour 

penetration output) is Y + Z = 1.64 Y 
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For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of 

absorption of 30 % (liquid EGBE, worst case)

The values are summarized in the following table: 

Table 4.92d: Risk characterisation for combined exposure and for RDT 

Scenario 

Internal dose 

after exposure 

to 8-hour 

TWA (mg/kg 

bw) 

Y+Z 

Internal dose after 

Dermal exposure 

to liquid EGBE 

(mg/kg bw) 

worst case (based 

on maximal dose)

Total internal 

dose 

(inhalation + 

dermal 

exposure) 

MOS Ccl 

1 - Manufacture 1.69 0.18 1.87 22 ii 

2 - Formulation 2.2 8.58 10.78 3.8 ii 

3 - Use of products 

3.1 

Coating/Painting 

- industrial 

- Spraying 

- Other 

works 

- decorative 

3.2 Printing 

- silk screening 

- general 

printing 

3.3 Cleaning 

- spraying 

-

- wiping 

8.17 

4.28 

4.28 

2.81 

0.70 

6.89 

6.89 

8.58 

1.84 

0.30 

0.10 

0.72 

1.07 

4.46 

16.75 

6.12 

4.58 

2.91 

1.42 

7.96 

11.35 

2.4 

6.6 

8.8 

14 

29 

5.1 

3.6 

ii 

ii 

ii 

ii 

ii 

ii 

ii 

1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m3

 a workday, a 

worker of 70 kg bw and 60 % of absorption. 

The calculated MOS should be compared with minimal MOS. To be in the worst case, the 

highest minimal MOS should be chosen for the comparison: 2. 

Conclusion ii is reached for all scenario for combined exposure. There are no concerns for 

repeat dose exposure for all scenarios. (conclusion ii). 
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4.1.3.2.5 Mutagenicity 

Given the results from the mutagenicity studies it is concluded that EGBE is of no concern for 

workers with regard to mutagenicity (conclusion ii). 

4.1.3.2.6 Carcinogenicity 

Inhalation exposure:

As the mechanism of haemangiosarcomas in male mice is related to haemotoxicity, the risk 

characterisation made for repeated dose toxicity (RDT) is also relevant for carcinogenicity. 

The other tumours seen in the animal studies being not relevant to humans no risk 

characterisation is needed for them. 

See RDT risk characterisation section 

Results: 

Exposure cannot be excluded for: 

There are no concerns for carcinogenicity for all scenarios. (conclusion ii). 

4.1.3.2.7 Toxicity for reproduction 

It is possible to derive a no effect level of 720mg/kg/day for fertility effects based on a 

continuous breeding study in mice. The MOSs obtained are compared with minimal MOS 

calculated as follows: 

Table 4.93: Assessment factors applied for the calculation of minimal MOS fertility effects. 

Interspecies differences 10 

Intraspecies differences 5 (workers: homogen population) 

Type of effect 1 

Confidence of the database 1 

Minimal MOS 50 

Internal dose due to 720 mg/kg bw (NOAEL) would be 720 mg/kg bw (100 % oral 

absorption). This value should be compared with estimated internal doses due to exposure 

(see RDT section for calculation). 

The following table summarise risk characterisation for inhalation exposure, dermal exposure 

and combined exposure. 

It is necessary in this case to correct the inhalation value for the dermal absorption of vapour 

during the 8hr TWA as it is being compared to a NOAEL derived from an oral study. It has 

been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the 

internal dose of EGBE. To take into account this value (which is not negligible) the value of 
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internal dose due to dermal exposure to vapours (Z) should by added to the former value (Y). 

Z represent 39 % of the total internal dose and can be calculated as follow : 

Z = 0.39/0.61 x Y = 0.64 Y 

The total internal dose due to inhalation exposure (inhalation output + dermal vapour 

penetration output) is Y + Z = 1.64 Y. The values for Y are shown in the table used for the 

repeat dose combined route assessment. 
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Table 4.93bis: Risk characterisation for reprotoxicity 

Scenario 

Internal dose 

after exposure 

to 8-hour 

TWA (mg/kg 

bw) 

Y+Z 1

MOS 

Inhalation 

only 

Internal dose after 

Dermal exposure to 

liquid EGBE 

(mg/kg bw) 

worst case (based 

on maximal dose) 

MOS 

dermal only

Total internal 

dose 

(inhalation + 

dermal 

exposure) 

1 - Manufacture 1.69 426 0.18 4000 1.87 2 - Formulation 2.2 327 8.58 84 10.78 3 - Use of products 3.1 Coating/Painting - industrial 

- Spraying - Other works - decorative 

3.2 Printing - silk screening - general printing 3.3 Cleaning - spraying 

- wiping 

8.17 4.28 4.28 2.81 0.70 6.89 6.89 

88 

168 168 256 

1029 

104 104 

8.58 

1.84 0.30 

0.1 

0.72 1.07 4.46 

84 

381 

2286 6957 1000 

640 154 

16.75 

6.12 4.58 2.91 1.42 7.96 

11.35 

1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m3 a workday, a worker of 70 k

percutaneous absorption of vapour EGBE (39 % of the total internal dose). 
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The calculated MOS should be compared with minimal MOS. To be in the worst case, the 

highest minimal MOS should be chosen for the comparison: 50. 

Conclusion ii for all scenarios 3.1 industrial spraying. 

For scenario 3.1. The calculated MOS is closed to the minimal MOS. Given the uncertainties 

of the dermal exposure assessment and the non specificity of the effects observed in the 

continuous breeding study, it can be considered that there is no concern for this end-point. 

Conclusion ii is reached for all scenarios. 

The developmental toxicity studies for EGBE clearly indicate that the developmental effects 

observed are a consequence of and secondary to maternal toxicity. Any formal risk 

characterisation for humans using this data would not be meaningful. The available data does 

not suggest that EGBE shares the developmental toxicity properties of certain other glycol 

ethers. There is no other data which indicates potential developmental toxicity concerns. 

Therefore the conclusion is that there is no concern for this end point, conclusion (ii). 

(conclusion ii). 

4.1.3.2.8 Summary of risk characterisation for workers 

Conclusion (ii) applies for all scenarios concerning each endpoint. 

4.1.3.3 Consumers 

Assuming that oral exposure could only be accidental by ingestion of a product, the risk 

characterisation for consumers is limited to the dermal and the inhalation routes of exposure. 

For risk characterisation, a value of 30 % for dermal absorption and a value of 60 % for 

inhalation exposure can be taken into account. 

Table 4.94: Internal dose exposure depending on scenarios 

Scenario Inhalation 

(mg/kg/d)

Skin 

(mg/kg/d)

Sum of exposures 

(mg/kg/d) 

1 - Household surface cleaners 0.054 2.1 2.154 

2 – Indoor air 0.06 0.06 

3 – Painting 6 1.3 7.3 

4 - Household surface cleaners + indoor 

air 

0.114 2.1 2.214 

5 - Painting + indoor air 6.06 1.3 7.4 

4.1.3.3.1 Acute toxicity 

According to acute toxicity data in human (suicide attempts cases), a LOAEL of 400 mg/kg 

can be taken into account. As limited data exist in humans by dermal or inhalation route an 

extrapolation route to route of this LOAEL is done. The MOSs obtained are compared with 

minimal MOS calculated as follows: 
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Table 4.95: Assessment factors applied for the calculation of minimal MOS for acute toxicity and for inhalation and 

dermal route. 

Interspecies differences 1 (human data) 

Intraspecies differences 10 

Type of effect 1 

Extrapolation LOAEL to NOAEL 5 

Confidence of the database 1 

Minimal MOS 50 

The MOSs between the LOAEL and the inhalation and skin exposures and for the sum of 

exposures are mentioned in table 4.96: 

Table 4.96: MOSs and conclusion for acute toxicity 

Scenario Inhalation Dermal Sum of exposures 

 MOS Conclusion MOS Conclusion MOS Conclusion 

1 – Household 

surface cleaners 

7407 ii 190 ii 185 ii 

2 – Indoor air 6667 ii 6667 ii 

3 – Painting 67 ii 308 ii 55 ii 

4 – Household 

surface cleaners 

+ indoor air 

3508 ii 190 ii 180 ii 

5 – Painting + 

indoor air 

66 ii 308 ii 54 ii 

Based on the risk assessment for the various scenarios, it is concluded that the toxicity due to 

acute exposure is not expected. So conclusion ii is reached for all scenarios. 

4.1.3.3.2 Irritation and corrosivity 

Skin

EGBE is classified as a skin irritant, and the use of gloves is recommended. This concern is 

managed by the information given by the classification and labelling of the substance so that 

no further concern remains (conclusion ii). 
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Eye

Eye irritation (liquid): 

The substance is classified as an eye irritant but in normal use, liquid should not be in contact 

with eyes. This concern is managed by the information given by the classification and 

labelling of the substance so that no further concern remains (conclusion ii). 

Eye and respiratory tract irritation (vapours): 

Scenarios of concern are those for which an inhalation exposure by vapours of EGBE has 

been evaluated. 

The NOAEL is 50 ppm that can be converted in mg/m3. So the NOAEL for this effect is : 

50 x 4.9 = 245 mg/m3

. 

Table 4.97: Consumer risk assessment of EGBE for eye and respiratory irritation by vapour exposure. 

Scenario Inhalation 

(mg/m3) 

MOS Conclusion 

1 – Household surface cleaners 0.27 906 ii 

2 – Indoor air 0.3 816 ii 

3 – Painting 30 8 ii 

4 – Household surface cleaners + indoor air 0.57 430 ii 

5 - -Painting + indoor air 30.3 8 ii 

For eye and respiratory tract irritation due to the exposure of EGBE in vapour form, 

conclusion ii is reached for all consumer scenarios. 

4.1.3.3.3 Sensitisation 

Skin

Given the results from the dermal sensitisation studies it is concluded that EGBE is of no 

concern for consumers with regard to skin sensitisation (conclusion ii). 

Respiratory tract

There are neither data from human experience nor other indications for respiratory 

sensitisation. 

4.1.3.3.4 Repeated dose toxicity 

It is clear from the available data that haemolysis is the primary and critical response elicited 

in the main animal test models (rats and mice) following inhalation, oral or dermal exposure 
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to EGBE. Blood from humans, pigs, dogs, cats, and guinea pigs is insensitive to haemolysis 

by BAA. In sensitive species, EGBE produces a characteristic toxicity that is revealed 

clinically by the appearance of haemoglobinuria and pathologically by changes in a variety of 

blood parameters. A 100-fold greater concentration of BAA (10mM) is required for human 

erythrocytes to develop pre-hemolytic changes consistent with those seen in the rat (0.1mM). 

Such in vivo blood concentrations are unlikely to occur under normal conditions of human 

exposure to BE. Haemolysis did not occur with any blood from any individual, even sensitive 

sub-populations, when exposed in vitro to BAA(10 mM). Studies have also shown that 

potentially sensitive human sub-populations, including the children, the elderly and those with 

sickle cell anaemia are also equally resistant to the effect. 

The same models will be used for consumers than for workers to calculate the equivalent 

human EGBE concentration that would produce this dose, and finally extrapolate this on the 

assumption. The NOAEL(C)/LOAEL(C) were calculated using the PBPK model in humans. 

Table 4.97 bis: Equivalent human doses for NOAEL(C) / LOAEL(C) 

 Oral Inhalation Dermal 

End point LOAEL LOAEC NOAEL 

Value 69 mg/kg/day 

(female rat)* 

31 ppm (152mg/m3) 150mg/kg/day 

Equivalent BAA Cmax

concentration 

129 μM 285 μM 

Equivalent human dose 9.5 mg/kg/day** 97 ppm (474mg/m3) 

* as quoted by US EPA. **assuming 70kg person drinking 2 litres water/day 

To select an appropriate interspecies chemical safety assessment factor (CSAF), we use the 

same method than in the workers’ risk characterisation leading to an interspecies factor of 0.1 

(see Table 4.97 ter). 

The risk characterisation for repeat dose then becomes as follows: 

Table 4.97 ter: Assessment factors for oral exposure 

Interspecies differences 0.1 (toxicodynamic factor based on the difference in 

sensitivity between rodents and humans as described in 

the risk characterisation for workers part) 

Inhalation: toxicokinetic element taken into account by 

PBPK model 

Dermal: Additional factor of 4 to account for allometric 

scaling of rats to humans 

Intraspecies differences 10 (default for consumers) 
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Duration of study No factor required. The critical study is 6 months in 

duration and the effects for this end-point (haemolysis) 

would not be expected to be more severe compared to 

lifetime exposure. (haemolysis due to EGBE is 

considered to be an acute or sub-acute effect on rodents, 

moreover in some studies animals tended to “recover” 

(red blood cells being more resistant) with long times of 

Type of effect 1 

Extrapolation LOAEL to NOAEL 3 for oral and inhalation The effects were mild at this 

dose and there is evidence to show that the LOAEL is 

near the threshold level for effects of concern (eg 

NOAECs from other studies) 

Confidence of the database 1 

Minimal MOS 3 for oral and inhalation 

4 for dermal exposure 

Risk characterisation 

For repeated dose toxicity, daily exposure level has to be averaged over a year. So the internal 

exposure dose used for risk characterisation will be : 

internal dose x number of events over a year 

365 

Table 4.98: Internal dose exposure depending on scenarios average over a year 

Scenario Number of events Inhalation 

(mg/kg/d)

Skin 

(mg/kg/d) 

Sum of 

exposures 

(mg/kg/d) 

1 – Household surface cleaners 1.8-4.8 

events/week (1) = 

94-250 events/year

0.036 1.44 1.476 

2 – Indoor air Each day 0.06 0.06 

3 – Painting 10 events/year 0.16 0.036 0.196 

4 – Household surface cleaners + indoor 

air 

1.8-4.8 

events/week (1) = 

94-250 events/year

0.096 1.44 1.536 

5 – Painting + indoor air 10 events/year 0.22 0.036 0.256 

(1) Data from the Technical Guidance Document 

The NOAEL is 474 mg/m3

 by inhalation and 150 mg/kg/day for dermal exposure. There is no 

oral exposure in normal use of products containing EGBE. 
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Internal NOAELs have to be calculated and to be compared with the internal daily exposures 

by skin and by inhalation and to the sum of internal exposures. 

For combined exposure, the NOAEL concerning the dermal exposure will be chosen as it is 

more protective for consumers. 

The respiratory volume for an adult is 20 m3

 and the mean bodyweight is 60 kg. The 

absorption of EGBE by inhalation is 60 %. So the internal NOAEL by inhalation will be : 

474 x 20 x 0.6 

60 

= 94.8 mg/kg/d 

The dermal absorption factor is 30%. So the internal NOAEL by dermal route will be : 

150 x 0.3 = 45 mg/kg/d 

Table 4.99: MOSs and conclusion for repeated dose toxicity 

Inhalation Dermal Sum of exposures 

Scenario 

MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 2633 ii 31 ii 30 ii 

2 – Indoor air 1580 ii 750 ii 

3 – Painting 592 ii 1250 ii 230 ii 

4 – Household surface cleaners 

+ indoor air 

987 ii 31 ii 29 ii 

5 – Painting + indoor air 431 ii 1250 ii 176 ii 

Conclusion ii is reached for all scenarios. 

4.1.3.3.5 Mutagenicity 

Given the results from the mutagenicity studies it is concluded that EGBE is of no concern for 

consumers with regard to mutagenicity (conclusion ii). 

4.1.3.3.6 Carcinogenicity 

As the mechanism of haemangiosarcomas in male mice is related to haemotoxicity, the risk 

characterisation made for repeated dose toxicity can also be used for carcinogenicity. The 

other tumours are not relevant to humans so no risk characterisation is needed for them. 

For carcinogenicity, daily exposure level has to be averaged over a year as for repeated dose 

toxicity. So the same values of internal exposures will be compared to the NOAEL defined 

for carcinogenicity. 

See RDT risk characterisation. 
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The values are summarised in the following table: 

Table 4.99 bis: Risk characterisation for carcinogenicity 

Inhalation Dermal Sum of exposures 

Scenario 

MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 2633 ii 31 ii 30 ii 

2 – Indoor air 1580 ii 750 ii 

3 – Painting 592 ii 1250 ii 230 ii 

4 – Household surface cleaners 

+ indoor air 

987 ii 31 ii 29 ii 

5 – Painting + indoor air 431 ii 1250 ii 176 ii 

Conclusion ii is reached for all scenarios. 

4.1.3.3.7 Toxicity for reproduction 

It is possible to derive a no effect level of 720 mg/kg/day for fertility effects based on a 

continuous breeding study in mice. Daily exposure level has been averaged over a year as 

reported in Table 4.98. 

The MOSs obtained are compared with minimal MOS calculated as follows: 

Table 4.100: Assessment factors applied for the calculation of minimal MOS fertility effects. 

Interspecies differences 10 

Intraspecies differences 10 

Type of effect 1 

Confidence of the database 1 

Minimal MOS 100 

Risk characterisation

Table 4.101: MOSs and conclusion for reproduction 

Inhalation Dermal Sum of exposures 

Scenario 

MOS Conclusion MOS Conclusion MOS Conclusion

1 – Household surface cleaners 20000 ii 500 ii 488 ii 

2 – Indoor air 12000 ii 12000 ii 
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3 – Painting 4500 ii 20000 ii 3673 ii 

4 – Household surface cleaners 

+ indoor air 

7500 ii 500 ii 469 ii 

5 – Painting + indoor 

3273 

ii 20000 ii 2813 ii 

Conclusion ii is reached for all scenarios: 

The developmental toxicity studies for EGBE clearly indicate that the developmental effects 

observed are a consequence of and secondary to maternal toxicity. Any formal risk 

characterisation for humans using this data would not be meaningful. The available data does 

not suggest that EGBE shares the developmental toxicity properties of certain other glycol 

ethers. There is no other data which indicates potential developmental toxicity concerns. 

Therefore the conclusion is that there is no concern for this end point, conclusion (ii). 

(conclusion ii). 

4.1.3.4 Humans exposed via the environment 

The key health effect is repeated dose toxicity. Irritation (via dermal or ocular routes)) is of 

low concern where exposure is dissipated throughout the environment. Comparison of the 

total internal dose of 94.8 mg.kg-1 (corresponding to the LOAEL of 31 ppm for RDT via 

inhalation route corrected with PbPk modelling to obain human internal dose see also 

calculation of internal NOAEL by inhalation in the consumer part in chapter 4.1.3.3.4.) with 

the highest estimated exposure at regional (3.22.10-4 mg.kg-1.day-1) and local (3.73.10-2

mg.kg-1.day-1) levels leads to margins of safety of, respectively, 2.9.105

 and 2.5.103 which do 

not lead to concern (compared to the minimal MOS of 3 calculated for consumers). 

4.1.3.4.1 Summary of risk characterisation for exposure via the environment 

(ii) There is at present no need for further information and/or testing and or risk 

reduction measures beyond those applied already.

This conclusion applies for all endpoints in relation to local and regional exposure. 

4.2 HUMAN HEALTH (PHYSICO-CHEMICAL PROPERTIES) 

EGBE has a low vapour pressure and is moderately flammable ( flash point is 60°C). It has no 

explosive or oxidising properties. However, it is noted that oxidation by air may involve 

peroxidation of the substance, which may increase explosive properties. A general warning to 

this effect is recommended. Use of antioxidants reduces the potential to peroxidation. 

It can be concluded that there is no concern for human health with regard physicochemical properties (conclusion ii). 
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5 RESULTS 6

5.1 INTRODUCTION 

5.2 ENVIRONMENT 

5.3 HUMAN HEALTH 

5.3.1 Human health (toxicity) 

5.3.1.1 Workers 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

5.3.1.2 Consumers 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

Conclusion (ii) applies to all end points and all scenarios: 

5.3.1.3 Humans exposed via the environment 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 

 

6 Conclusion (i) There is a need for further information and/or testing. 

Conclusion (ii) There is at present no need for further information and/or testing and no need for risk reduction measures beyond those 

which are being applied already. 

Conclusion (iii) There is a need for limiting the risks; risk reduction measures which are already being applied shall be taken into 

account.
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5.3.2 Human health (risks from physico-chemical properties) 

Conclusion (ii) There is at present no need for further information and/or testing and no 

need for risk reduction measures beyond those which are being applied 

already. 
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EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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ABBREVIATIONS 

ADH Alcohol Dehydrogenase 

ADI Acceptable Daily Intake 

AF Assessment Factor 

ALP Alcaline Phosphatase 

ARDS Adult Respiratory Distress Syndrome 

ASTM American Society for Testing and Materials 

ATP Adaptation to Technical Progress 

ATSDR Agency for toxic substances and disease registry (USA) 

AUC Area Under The Curve 

B Bioaccumulation 

β2-m β2-microglobulin 

BAA Butoxy Acetic Acid 

BAL Buthoxy aldehyde 

BBA Biologische Bundesanstalt für Land- und Forstwirtschaft 

BCF Bioconcentration Factor 

BEG Glucuronide conjugate of EGBE 

BEI Biological Exposure Indice 

BES Sulfo conjugate of EGBE 

BMC Benchmark Concentration 

BMD Benchmark Dose 

BMF Biomagnification Factor 

BSA Bovine Serum Albumin 

bw body weight / Bw, b.w.

C Corrosive (Symbols and indications of danger for dangerous substances and preparations 

according to Annex III of Directive 67/548/EEC) 

CA Chromosome Aberration 

CA Competent Authority 

CAS Chemical Abstract Services 

CAT Carnitine Acetyl Transferase 

CBC Complete Blood Count 

CEC Commission of the European Communities 

CEN European Standards Organisation / European Committee for Normalisation 

CEPE European council of the paint, printing ink and artists’ colours industry 

CI Clearance 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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CMNI Maximal Concentration with No Irritation 

CMR Carcinogenic, Mutagenic and toxic to Reproduction 

CNS Central Nervous System 

COD Chemical Oxygen Demand 

CSTEE Scientific Committee for Toxicity, Ecotoxicity and the Environment (DG SANCO) 

CT50 Clearance Time, elimination or depuration expressed as half-life 

d.wt dry weight / dw 

dfi daily food intake 

DG Directorate General 

DIN Deutsche Industrie Norm (German norm) 

DNA DeoxyriboNucleic Acid 

DOC Dissolved Organic Carbon 

DT50 Degradation half-life or period required for 50 percent dissipation / degradation 

DT90 Period required for 50 percent dissipation / degradation 

DTH Delayed Time Hypersensibility 

E Explosive (Symbols and indications of danger for dangerous substances and preparations 

according to Annex III of Directive 67/548/EEC) 

EASE Estimation and Assessment of Substance Exposure Physico-chemical properties [Model] 

EbC50 Effect Concentration measured as 50 % reduction in biomass growth in algae tests 

EC European Communities 

EC10 Effect Concentration measured as 10 % effect 

EC50 median Effect Concentration 

ECB European Chemicals Bureau 

ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals 

ECVAM European Centre for the Validation of Alternative Methods 

EDC Endocrine Disrupting Chemical 

EEC European Economic Communities 

EG Ethylene Glycol 

EGBE 2-Butoxyethanol 

EGBEA 2-Butoxyethanol acetate 

EINECS European Inventory of Existing Commercial Chemical Substances 

ELINCS European List of New Chemical Substances 

EN European Norm 

EPA Environmental Protection Agency (USA) 

ErC50 Effect Concentration measured as 50 % reduction in growth rate in algae tests 

ESD Emission Scenario Document 

EtOH Ethanol 

EU European Union 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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EUSES European Union System for the Evaluation of Substances [software tool in support of 

the Technical Guidance Document on risk assessment] 

F(+) (Highly) flammable (Symbols and indications of danger for dangerous substances and 

preparations according to Annex III of Directive 67/548/EEC) 

FAO Food and Agriculture Organisation of the United Nations 

FELS Fish Early Life Stage 

G6P Glucose 6 Phosphate 

G6PD Glucose 6 Phosphate Dehydrogenase 

GE Glycol Ether 

GGT Gamma Glutamyl Transpeptidase 

GI Gastro Intestinal 

GLP Good Laboratory Practice 

GLU Glucose 

GP Guinea Pig 

Hb Haemoglobin 

HCT Haematocrit 

HEDSET EC/OECD Harmonised Electronic Data Set (for data collection of existing substances) 

HELCOM Helsinki Commission -Baltic Marine Environment Protection Commission 

HGB Haemoglobin 

HPLC High Pressure Liquid Chromatography 

HPVC High Production Volume Chemical (> 1000 t/a) 

HSE Health and Safety Executive (UK) 

IARC International Agency for Research on Cancer 

IC Industrial Category 

IC50 median Immobilisation Concentration or median Inhibitory Concentration

IL2 Inter Leukine 2 

ILO International Labour Organisation 

INF Interferon 

INRS Institut National de Recherche et de Sécurité 

IP Intra Peritoneal 

IPA Intra Peritoneal 

IPCS International Programme on Chemical Safety 

ISO International Organisation for Standardisation 

IUCLID International Uniform Chemical Information Database (existing substances) 

IUPAC International Union for Pure and Applied Chemistry 

IV Intra Venous 

JEFCA Joint FAO/WHO Expert Committee on Food Additives 

JMPR Joint FAO/WHO Meeting on Pesticide Residues 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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Koc organic carbon normalised distribution coefficient 

Kow octanol/water partition coefficient 

Kp solids-water partition coefficient 

L(E)C50 median Lethal (Effect) Concentration 

LAEL Lowest Adverse Effect Level 

LAS Alkyl Sulfonate 

LC50 median Lethal Concentration 

LD50 median Lethal Dose 

LDH Lactate DeHydrogenase 

LEV Local Exhaust Ventilation 

LLNA Local Lymph Node Assay 

LN Lymph Node 

LOAEC Low Observable Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOEC Lowest Observed Effect Concentration 

LOED Lowest Observed Effect Dose 

LOEL Lowest Observed Effect Level 

MAC Maximum Allowable Concentration 

MATC Maximum Acceptable Toxic Concentration 

MC Main Category 

MCH Mean Corpuscular Haemoglobin 

MCHC Mean Cell Haemoglobin Concentration 

MDA Malonyl DiAldehyde 

MeOH Methanol 

MITI Ministry of International Trade and Industry, Japan 

MOE Margin of Exposure 

MOS Margin of Safety 

MW Molecular Weight 

N Dangerous for the environment (Symbols and indications of danger for dangerous 

substances and preparations according to Annex III of Directive 67/548/EEC 

NAEL No Adverse Effect Level 

nBuOH n-Butanol 

NICNAS National Industrial Chemicals Notification and Assessment Scheme (Australia) 

NIOSH National Institute for Occupational Safety and Health (USA) 

NK Natural Killer 

NOAEC No Observable Adverse Effect Concentration 

NOAEL No Observed Adverse Effect Level 

NOEL No Observed Effect Level 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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NOEC No Observed Effect Concentration 

nPrOH n-Propanol 

NTP National Toxicology Program (USA) 

NZ Natural Killer 

O Oxidizing (Symbols and indications of danger for dangerous substances and preparations 

according to Annex III of Directive 67/548/EEC) 

OECD Organisation for Economic Cooperation and Development 

OEL Occupational Exposure Limit 

OJ Official Journal 

OSPAR Oslo and Paris Convention for the protection of the marine environment of the Northeast 

Atlantic 

P Persistent 

PBT Persistent, Bioaccumulative and Toxic 

PBPK Physiologically Based PharmacoKinetic modelling 

PBTK Physiologically Based ToxicoKinetic modelling 

PCV Packed cell volume 

PEC Predicted Environmental Concentration 

PEG PolyEthylene Glycol 

PGME Propylene Glycol Methyl Ether 

pH logarithm (to the base 10) (of the hydrogen ion concentration {H+

} 

pKa logarithm (to the base 10) of the acid dissociation constant 

pKb logarithm (to the base 10) of the base dissociation constant 

PNEC Predicted No Effect Concentration 

PO Per os 

POP Persistent Organic Pollutant 

PPE Personal Protective Equipment 

QSAR (Quantitative) Structure-Activity Relationship 

R phrases Risk phrases according to Annex III of Directive 67/548/EEC 

RAR Risk Assessment Report 

RBC Red Blood Cell 

RC Risk Characterisation 

RfC Reference Concentration 

RfD Reference Dose 

RNA RiboNucleic Acid 

RPE Respiratory Protective Equipment 

RWC Reasonable Worst Case 

S phrases Safety phrases according to Annex III of Directive 67/548/EEC 

SAR Structure-Activity Relationships 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

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SBR Standardised birth ratio 

SCE Sister Chromatic Exchange 

SDS Safety Data Sheet 

SEM Scanning electron microscopy 

SETAC Society of Environmental Toxicology And Chemistry 

SGOT Serum Glutamic Oxaloacetic Transaminase 

SGPT Serum Glutamic Pyruvic Transaminase 

SNIF Summary Notification Interchange Format (new substances) 

SSD Species Sensitivity Distribution 

STEL Short-Term Exposure Limit 

STP Sewage Treatment Plant 

T(+) (Very) Toxic (Symbols and indications of danger for dangerous substances and 

preparations according to Annex III of Directive 67/548/EEC) 

TDI Tolerable Daily Intake 

TG Test Guideline 

TGD Technical Guidance Document

TLV Threshold Limit Value 

TNsG Technical Notes for Guidance (for Biocides) 

TNO The Netherlands Organisation for Applied Scientific Research 

TWA Time-Weighted Average 

UC Use Category 

UDS Unscheduled DNA Synthesis 

UN United Nations 

UNEP United Nations Environment Programme 

US EPA Environmental Protection Agency, USA 

UV Ultraviolet Region of Spectrum 

UVCB Unknown or Variable composition, Complex reaction products of Biological material 

vB very Bioaccumulative 

Vd Volume of Distribution 

vP very Persistent 

vPvB very Persistent and very Bioaccumulative 

v/v volume per volume ratio 

w/w weight per weight ratio 

WBC White Blood Cell 

WEC Whole Embryo Culture 

WHO World Health Organization 

WWTP Waste Water Treatment Plant 

Xn Harmful (Symbols and indications of danger for dangerous substances and preparations 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2ABBREVIATIONS

RAPPORTEUR FRANCE 305

according to Annex III of Directive 67/548/EEC) 

Xi Irritant (Symbols and indications of danger for dangerous substances and preparations 

according to Annex III of Directive 67/548/EEC) 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2APPENDIX A

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Appendix A 

Methods of calculation of consumer exposures 

Scenario 1 : Household surface cleaners

inhalation

Vr x Cair x te Eei = 

Wb x hd

Eei = external exposure by inhalation 

Vr = respiratory volume in one day 

Cair = concentration in air 

te = duration of exposure

Wb = mean bodyweight for a consumer (60 kg) 

hd = hours in one day 

Eii = Eei x absi

Eii = internal exposure by inhalation 

absi = percentage of absorption by inhalation 

Dermal

Cp x Qp x Qh

Eed = 

Wb

Eed = external dermal exposure 

Cp = concentration of EGBE in the cleaning product 

Qp = Quantity of product used 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2APPENDIX A

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Qh = Quantity of EGBE transferred to the hands 

Wb = mean bodyweight for a consumer (60 kg) 

Eid = Eei x absi

Eid = internal dermal exposure 

absi = percentage of absorption by skin

Scenario 2 : indoor air

inhalation

Vr x Cair x te Eei = 

Wb x hd

Eei = external exposure by inhalation 

Vr = respiratory volume in one day 

Cair = concentration in air 

te = duration of exposure

Wb = mean bodyweight for a consumer (60 kg) 

hd = hours in one day 

Eii = Eei x absi

Eii = internal exposure by inhalation 

absi = percentage of absorption by inhalation

Scenario 3 : painting

Inhalation

Vr x Cair x te Eei = 

Wb x hd
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2APPENDIX A

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Eei = external exposure by inhalation 

Vr = respiratory volume in one day 

Cair = concentration in air 

te = duration of exposure

Wb = mean bodyweight for a consumer (60 kg) 

hd = hours in one day 

Eii = Eei x absi

Eii = internal exposure by inhalation 

absi = percentage of absorption by inhalation

CONSEXPO model parameters:

Contact scenario: evaporation due to Painting 

Duration of contact per event: 133 min 

Frequency of contact: 10/year 

Person uses product (room volume, ventilation and release area personal). 

Personal volume=5.000000 m3. 

Mean event concentration (average case): 3.772e+02 mg/m3 

Year average (average case): 9.539e-01 mg/m3 

Mean event concentration (cumulative worst case): 3.772e+02 mg/m3 

Year average (cumulative worst case): 9.539e-01 mg/m3 

Exposure estimates based on the following parameters: 

Release area: 28.000 m2 

Room volume: 25.000 m3 

Room Ventilation rate: 12.5 m3/hr 

Product amount: 5.000 kg 

product density: 1.300 mg/cm3 

Weight fraction: 3% 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2APPENDIX A

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Fraction to upper layer: 1.000 fraction 

Layer exchange rate: 1.000 1/min 

Temperature: 293.000 Kelvin 

Molecular weight matrix: 400.000 g/mol 

Uptake 

Model: fraction model 

Cumulative worst case estimate: 7616 mg/year : 0.2979 mg/(kg.day) 

Uptake estimates based on the following parameters: 

Absorbed fraction: 60 % 

Inhalation rate: 25300 cm3/min 

Respirable fraction: 100% 

WALLPAINT EXPOSURE MODEL (WPEM)

Resident DIY model 

Room volume 25m3 

Painted area 28m2

Air changes 0.5 per hour 

Paint quantity:5kg, density 1.3, type: flat 

Model type: empirical 

Body mass: 60kg 

Events per year: 10 

Active/total lifetime: 40/70 years 

No sinks 

EGBE content: 3% 

Painting time: 133 minutes 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2APPENDIX A

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Dermal 

C prod x TH der x AREA der x n

Eed =

D x BW

where : 

Eed = external exposure 

C prod = concentration of EGBE in paint (kg/m3) 

TH der = thickness of product layer on skin 

AREA der = area of contact between paint and skin 

n = number of events (n = 1) 

D = dilution. There is no dilution so D = 1 

BW = mean bodyweight for a consumer (60 kg) 

Eid = Eei x absi

Eid = internal dermal exposure 

absi = percentage of absorption by skin 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2 APPENDIX B 

RAPPORTEUR FRANCE 311

Appendix B 

Risk characterisation without using PbPk extrapolation 

In the kinetic studies, if the pbpk is not taken into account, some data can be used to derive 

MOS for interspecies differences. It is clearly demonstrated that rodent blood cells are much 

more sensitive than human ones (at least 100 times), for this effect, a conservative factor of 

0.1 will be used. For dermal penetration is was also demonstrated than rat skin was 2-3 more 

permeable than human one, for this end-point a correcting factor of 0.5 will be taken into 

account. 

The risk characterisation for repeat dose then becomes as follows: 

Table: Assessment factors applied for the calculation of minimal MOS for occupational exposure 

Interspecies differences 0.1 for greater sensitivity of rodents blood cells 

0.5 for dermal absorption 

Dermal: Additional factor of 4 to account for allometric 

scaling of rats to humans 

Intraspecies differences 5 (default for workers) 

Duration of study No factor required 

Uncertainties concerning dermal 

exposure 

0.2 for dermal and for scenarios using data derived 

from measurements with a less volatile solvent.* 

Type of effect 1 

Extrapolation LOAEL to NOAEL 3 for oral and inhalation The effects were mild at this 

dose and there is evidence to show that the LOAEL is 

near the threshold level for effects of concern (eg 

NOAECs from other studies) 

Factor of 1 for dermal exposure 

Confidence of the database 1 

Minimal MOS 1.5 for oral and inhalation 

1 for dermal exposure 

Note that the critical LOAEC used for the inhalation risk characterisation is derived from a 

whole body inhalation exposure study (as are all similar studies). These animals will have 

been subjected to exposure by both the dermal and inhalation routes. 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2 APPENDIX B 

RAPPORTEUR FRANCE 312

Exposure will be compared with the LOAEC of 31 ppm (152 mg/m3) derived from a 14-week 

study in rats. 

Risk characterisation 

Oral exposure 

No oral exposure has been identified for workers, therefore there are no concerns for this 

route of exposure. 

Inhalation 

Risk assessment for inhalation 

exposure

Risk assessment for dermal 

exposure to liquid EGBE

Scenario

8-hour 

TWA 

inhalation 

(mg/m3

)

MOS1 Conclusion Estimated 

Skin 

exposure 

mg/day 

(mg/kg 

bw/d

MOS2 Conclusion

1 - Manufacture 12 12.7 ii 42 

(0.6) 

250 ii 

2 - Formulation 15.7 9.7 ii 2000 

(28.57) 

5.3 ii 

3.1 Coating/Painting

3.11 –Industrial

-spraying 58.1 2.6 ii 2000 

(28.57) 

5.3 ii 

-other works 30.4 5 ii 430 

(6.14) 

24 ii 

3.12 - decorative 30.4 5 ii 70 

(1) 

150 ii 

3.2 Printing

3.21 Silk 

screening

20 7.6 ii 23 

(0.33) 

454 ii 

3 - Use of 

end 

products

3.22 General 

printing 

5 30.4 ii 168 

(2.4) 

63 ii 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2 APPENDIX B 

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3.3 Cleaning - 

spraying 

49 3.1 ii 250 

(3.57) 

42 

3.3 Cleaning - 

wiping 

49 3.1 ii 1040 

(14.86) 

10 ii 

For exposure via the inhalation route, conclusion (ii) is drawn for all scenarios. 

Dermal exposure: 

Exposure values are compared with the NOAEL of 150 mg/kg. 

See previous table 

For exposure via the dermal route, conclusion (ii) is drawn for all scenarios. 

Combined exposure: 

For the combined exposures the estimated internal doses are calculated from the biological 

exposure data. The inhalation LOAEC is chosen for comparison as this is lower than the 

equivalent for the dermal route. The LOAEC of 152 mg/m3 would lead to an internal doses of 

152 mg/m3

*10m3/day*0.6/70kg = 13 mg/kg/day This internal dose should be compared with 

internal dose calculated . This LOAEC should be compared with internal doses calculated 

from exposures in each scenario (inhalation + dermal). The internal doses are calculated as 

follow : 

Inhalation exposure will give internal dose of: 

X (value of the 8-hour TWA inhalation (mg/m3

)) x 10 m3 (inhaled air during a workday) x 0.6 

(percentage of absorption by inhalation) / 70 (mean bw of a worker) = Y (inhalation internal dose). 

This value does not take into account the possible dermal absorption of vapour during the 8hr TWA. It 

has been demonstrated that dermal absorption of vapour EGBE could count for 39 % of the internal 

dose of EGBE. To take into account this value (which is not negligible) the value of internal dose due 

to dermal exposure to vapours (Z) should by added to the former value (Y). Z represent 39 % of the 

total internal dose and can be calculated as follow : 

Z = 0.39/0.61 x Y = 0.64 Y 

The total internal dose due to inhalation exposure (inhalation output + dermal vapour penetration 

output) is Y + Z = 1.64 Y 

For dermal exposure internal dose is calculated for a 70 kg bw worker with a percentage of absorption 

of 30 % (liquid EGBE, worst case) 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2 APPENDIX B 

RAPPORTEUR FRANCE 314

The values are summarized in the following table: 

Scenario 

Internal dose 

after exposure 

to 8-hour 

TWA (mg/kg 

bw) 

Y+Z 

Internal dose after 

Dermal exposure 

to liquid EGBE 

(mg/kg bw) 

worst case (based 

on maximal dose)

Total internal 

dose 

(inhalation + 

dermal 

exposure) 

MOS Ccl 

1 - Manufacture 1.69 0.18 1.87 7.0 ii 

2 - Formulation 2.2 8.58 10.78 1.2 iii 

3 - Use of products 

3.1 

Coating/Painting 

- industrial 

- Spraying 

- Other 

works 

- decorative 

3.2 Printing 

- silk screening 

- general 

printing 

3.3 Cleaning 

spraying 

3.3 Cleaning 

wiping 

8.17 

4.28 

4.28 

2.81 

0.70 

6.89 

6.89 

8.58 

1.84 

0.30 

0.10 

0.72 

1.07 

4.46 

16.75 

6.12 

4.58 

2.91 

1.42 

7.96 

11.35 

0.77 

2.1 

2.8 

4.5 

9.2 

1.63 

1.14 

iii 

ii 

ii 

ii 

ii 

ii 

iii 

1) internal dose for exposure to EGBE vapour is due to inhalation uptake (10 m3 a workday, a 

worker of 70 kg bw and 60 % of absorption.

The minimal MOS required for the inhalation route is 1.5 whilst for the dermal route it is 1. 

According to the results obtained a conclusion iii is reached for scenario 2, scenario 3.1: 

industrial spraying and scenario 3.3 cleaning wiping. 

There are no concerns for repeat dose exposure for the other scenarios. (conclusion ii). 
EU RISK ASSESSMENT – 2-BUTOXYETHANOL CAS 111-76-2 

RAPPORTEUR FRANCE 315

European Commission 

EUR [ECB: click here to insert EUR No.] - European Union Risk Assessment Report

[ECB: click here to insert SUBSTANCE NAME, and volume no.] 

Editors: (keep this updated) 

Luxembourg: Office for Official Publications of the European Communities 

[ECB: insert year] – VIII pp., [ECB: insert number of pages] pp. – 17.0 x 24.0 cm 

Environment and quality of life series 

ISBN [ECB: insert ISBN No.] 

Price (excluding VAT) in Luxembourg: EUR [ECB:insert price] 

The report provides the comprehensive risk assessment of the substance 2-butoxyethanol. It 

has been prepared by France in the frame of Council Regulation (EEC) No. 793/93 on the 

evaluation and control of the risks of existing substances, following the principles for 

assessment of the risks to man and the environment, laid down in Commission Regulation 

(EC) No. 1488/94. 

This part of the evaluation considers the emissions and the resulting exposure to human 

populations in all life cycle steps. The scenarios for occupational exposure, consumer 

exposure and humans exposed via the environment have been examined and the possible 

risks have been identified. 

The human health risk assessment concludes that there is no concern for workers, 

consumers, for humans exposed via the environment and for human health (physicochemical properties).