Appendix A - acetonitrile, 2,2’2’’‐nitrilotris‐ (CAS# 7327‐60‐8) 

Robust summaries ‐ IUCLID5 

4 Physical and chemical properties 

Endpoint summary: Physical and chemical properties 

UUID IUC5-c85658bc-64a8-440c-93d9-68ee4a4a0e2a 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-12 11:11:49 CET 

Remarks 

 

Administrative Data 

 

Discussion 

This substance is not produced nor transported as an aerosol nor as a gas under pressure. NTAN has no 

physical/chemical properties that need classification and labelling. 

4.1 Appearance/physical state/colour 

Endpoint summary: Appearance/physical state/colour 

UUID IUC5-c9dce4cf-526a-4e2f-81d6-4a5e7bbef844 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:09:18 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The substance is an off-white, odourless, organic powder (wet cake). 

Key value for chemical safety assessment 

Physical state at 20oC and 1013 hPa 

solid 

Endpoint study record: 7327-60-8, Appearance/physical state/colour, Huybens, 2010, CoA, RS 

UUID IUC5-04964d1f-cfff-48fb-8b5e-e212375bf042 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:09:41 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period May 2006 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Signed certificate of analysis. 

Data source 

1
Reference 

Referenc

e type 

Author Year Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Huyben

s ICM 

201

0 

Certificat

e of 

analysis 

NTAN 

 Chelates 

(FC-C), 

Deventer, 

The 

Netherland

s 

Batch 

numbe

r CFC 

7538 

AkzoNobe

l 

Functional 

Chemicals 

bv 

 2010-

06-10 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

equivalent or similar to EPA OPPTS OPPTS 830.6303 (Physical State) 

equivalent or similar to EPA OPPTS 830.6304 (Odor) 

equivalent or similar to EPA OPPTS 830.6302 (Color) yes (visual) 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number CFC7538Purity 94,1 %Water 3.2 %Expiration date April 2010

Results and discussion 

Physical state at 20oC and 1013 hPa 

solid 

Form 

other: Powder (wet cake) 

Colour 

off-white 

Odour 

odourless 

Substance type 

organic 

Overall remarks, attachments 

Overall remarks 

see:7327-60-8, Density, Knuif, 2010, rs for odour.

2
Attached background material 

Attached document Remarks 

7327-60-8, appearance,Huybens,2010, CoA, REF.doc / 187.5 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANBased on results in this report: The substance is a off-white, odourless, organic powder 

(wet cake). 

4.2 Melting point/freezing point 

Endpoint summary: Melting range 

UUID IUC5-1b91ae06-cf61-4282-a672-ed0285aad53d 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:10:13 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The melting range of NTAN (dried) is 125.8– 127.7°C. 

Discussion 

Key study: 7327-60-8, Melting point, Boerdijk, 2006, RS 

Supportedby: 

7327-60-8, physchem, melting point, Witteveen, 2003, RS

The melting point of the substance is 128°C. 

7327-60-8, Melting point/freezing point, EPISuite, SS 

EPISuite calculation: 92.78°C. 

Endpoint study record: 7327-60-8, Melting point, Boerdijk, 2006, RS 

UUID IUC5-5f9adc7b-d7b9-41f6-8ccc-3e21baa72bd4 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:07:57 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period May 2006 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies According to OECD guidline. 

Data source 

3
Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laborator

y 

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repo

rt 

date 

study 

report 

Boerdij

k GB 

200

6 

Determination 

of the melting 

point of 

nitrilotriacetonitr

ile 

 Akzo 

Nobel 

Technolog

y & 

Engineerin

g bv 

Deventer 

The 

Netherlan

ds 

2006059

8-DSC 

AkzoNob

el 

Function

al 

Chemical

s bv 

 2006-

05-19 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 102 (Melting point / Melting Range) no data 

Type of method 

thermal analysis 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number 2005L07241-01 (CFC-7538)Purity 94 w/w%Storage requirements Avoid contact with: 

aluminium, zinc, nickel, copper and copper alloys. Store in: PVC, PE, Stainless steel, bituminized 

tanks.Stability Stable under storage and handling conditions.Expiration date October 2008Sample 

preparationThe consigned sample was a wet cake. The water was removed by drying it in a desiccator 

filled with silica gel. The desiccator was set at vacuum at 20-25°C. After constant weight, the material is 

tested in the DSC. 

Any other information on materials and methods incl. tables 

 

Apparatus Mettler Toledo STAR system 

DSC pan type Standard aluminum, 40 μl; closed 

Scanning rate 10.0 °C/min 

Weight 5 mg 

4
Temperature scanning range 25°C – 150°C 

Following recommendations in method: ASTM E 537-02 

Results and discussions 

Melting / freezing point 

Melt./Freez. pt. Atm. 

pressure 

Decomposition Decomp. 

temp. 

Sublimation Subl. 

temp. 

Remarks 

> 125.8 — < 

127.7 °C 

 

Any other information on results incl. tables 

Experiment Scanning rate

K/min 

Tonset °C Tend-set°C Trange°C 

Screening DSC 

 

Measurement 1 

Measurement 2 

Average value

10.0 

 

2.0 

2.0 

125.8 

 

125.8 

125.9 

125.8

132.3 

 

127.6 

127.8 

127.7

6.5 

 

1.8 

1.9 

The melting range of NTAN (dried) is 125.8 ± 0.5°C – 127.7 ± 0.5°C. 

Overall remarks, attachments 

Overall remarks 

Since the accuracy of the DSC sample temperature in between 25°C and at least 420°C*2is ± 0.5°C, a 

melting point would be given, if Trange< 1.0. 

The highest temperature at which the DSC is accurately calibrated. This is the melting point of 

Zinc(419.5°C) 

Above this temperature value, the accuracy decreases slowly. 

Attached background material 

Attached document Remarks 

CoA NTAN CFC7538.docx / 471.46 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document)  

7327-60-8, melting point, Boerdijk, 2006, REF.doc / 186 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTAN 

Based on results in this report: The melting range of NTAN (dried) is 125.8– 127.7°C. 

Endpoint study record: 7327-60-8, Melting point, Witteveen, 2003, RS 

UUID IUC5-bee3f550-c25b-481d-a18f-363b261e544e 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:08:05 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study; robust study summary 

Study result type experimental result Study period april 2003 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies Similar to According to OECD guidline. 

5
Data source 

Reference 

Referenc

e type 

Author Year Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Wittevee

n AJ 

200

3 

Thermal 

Analysi

s of 

Nitrils 

 Chemicals 

Research 

Arnhem 

The 

Netherland

s 

CPPM0300

9 

AkzoNobe

l 

Functional 

Chemicals 

bv 

 2003-

05-07 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, physchem, boiling point,Witteveen,Thermal analysis of nitrils,rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

equivalent or similar to OECD Guideline 102 (Melting point / Melting Range) 

Type of method 

thermal analysis 

Principles of method if other than guideline 

1. TGAEquipment: TA Instruments TGA 2950Temperature program: 25°C -> 700°C at 2°C/min. Sample 

holders: PlatinumSample size: 6 ± 1 mg.Atmosphere: Air, 100 ml./min.2. DSCEquipment: TA Instruments 

DSC 2920Temperature program: -100°C ->decomposition temperature at 10°/min.Sample holders: 

Aluminum, standard pansSample size: 6 ± 1 mg.Atmosphere: Helium, 25 ml./min. 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Sample code AppearanceCFC 5858 WhiteCFC 5861 WhiteNTAN batches / 5857 / 5858 are production 

samples, Lima, 2001/2002Since CFC samples 5143 / 5144 / 5861 contain impurities the data are not 

used in this data base.According to certificate of analysis of CFC-5858, 5859, 6058:Specification + typical 

information of NTANAnalysis of Result Method referenceAppearance Off-white wet crystals Visual, 

specificationAssay (NTAN) Min 90 % Calculated from water contentSpecificationWater Max 10.0 % Loss 

on drying (160°C) specificationChemical purity NTAN > 99% typical 

Results and discussions 

6
Melting / freezing point 

Melt./Freez. pt. Atm. pressure Decomposition Decomp. temp. Sublimation Subl. temp. Remarks 

128 °C 

Any other information on results incl. tables 

 

 TGA DSC

 RT -> 700°C, 2°C/min., air RT®decomposition, 10°C/min.,helium

 % H2O temp. of 

0.5% wt. 

temp. of 

1.0% wt. 

main 

 based on loss 

relative to

loss 

relative to

Melting peak 

 water-free 

product 

water-free 

product 

water-free 

product 

Onset temperature 

sample Code (°C) (°C) 

NTAN CFC 

5858 

0.0 126 125 127 

NTAN CFC 

5861 

0.0 127 134 127 

 

The two samples (5858 and 5861)in this data base reported show a sharp melting peak at 128°C. 

The other samples mentioned in the report do notshow a sharp melting peak,because of impurities. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, melting point,Witteveen,2003, REF.doc / 187 KB (application/msword) 

7327-60-8, Huybens, 2010, CoA NTAN CFC5858-5859-6058,ref.docx / 37.76 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANBased on results in this report: The melting point of the substance is 128°C. 

Endpoint study record: 7327-60-8, Melting point/freezing point, EPISuite, SS 

UUID IUC5-8e7308d9-227c-4ac3-9415-bf02a4666bd6 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 00:01:39 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

EPISuite 

 

7
Applicant's summary and conclusion 

Conclusions 

MP: 92.78°C 

4.3 Boiling point 

Endpoint summary: Boiling point 

UUID IUC5-7090fa05-25a8-426f-925e-083a2b7e70aa 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:10:45 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Melting starts at 127°C. At the same temperature the evaporation of the substance starts too. So during 

melting the substance evaporates, starting both at 127°C. 

Key value for chemical safety assessment 

Boiling

point 

at 101 

325 Pa 

127 °C 

Discussion 

Key study: 7327 -60 -8, physchem, boiling point, Witteveen, 2003, RS

Supported by 7327-60-8, Boiling point, EPISuite, SS 

321.24°C 

Endpoint study record: 7327-60-8, Boiling point, Witteveen, 2003, RS 

UUID IUC5-c2dcc7db-5441-4cd5-80fb-b72fdb73bfd1 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:08:18 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period April 2003 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies According to OECD guidline. 

Data source 

Reference 

Referenc

e type 

Author Year Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Wittevee

n AJ 

200

3 

Thermal 

Analysi

s of 

Nitrils 

 Chemicals 

Research 

Arnhem 

The 

Netherland

s 

CPPM0300

9 

AkzoNobe

l 

Functional 

Chemicals 

bv 

 2003-

05-07 

8
Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, physchem, melting point,Witteveen,Thermal analysis of nitrils,rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

equivalent or similar to OECD Guideline 103 (Boiling point/boiling range) 

Type of method 

differential scanning calorimetry (and Thermogravimetric analysis) 

Principles of method if other than guideline 

Experimental conditions1. TGAEquipment: TA Instruments TGA 2950Temperature program: 25°C -

>700°C at 2°C/min. Sample holders: PlatinumSample size: 6 ± 1 mg.Atmosphere: Air, 100 ml./min.2. 

DSCEquipment: TA Instruments DSC 2920Temperature program: -100°C ->decomposition temperature 

at 10°/min.Sample holders: Aluminum, standard pansSample size: 6 ± 1 mg.Atmosphere: Helium, 25 

ml./min. 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Sample code AppearanceCFC 5858 WhiteCFC 5861 WhiteNTAN batches / 5857 / 5858 are production 

samples, Lima, 2001/2002Since CFC samples 5143 / 5144 / 5861 contain impurities the data are not 

used in this data base.According to certificate of analysis of CFC-5858, 5859, 6058:Specification + typical 

information of NTANAnalysis of Result Method referenceAppearance Off-white wet crystals Visual, 

specificationAssay (NTAN) Min 90 % Calculated from water contentSpecificationWater Max 10.0 % Loss 

on drying (160°C) specificationChemical purity NTAN > 99% typical 

Results and discussions 

Boiling point 

Boiling 

pt. 

Atm. 

pressure 

Decomposition Decomp. 

temp. 

Remarks 

> 120 °C The substance evaporates during melting and 

before boiling. 

9
Any other information on results incl. tables 

 

 TGA DSC

 RT -> 700°C, 2°C/min., air RT®decomposition, 10°C/min.,helium

 % H2O temp. of 

0.5% wt. 

temp. of 

1.0% wt. 

main 

 based on loss 

relative to

loss 

relative to

Melting peak 

 water-free 

product 

water-free 

product 

water-free 

product 

Onset temperature 

sample Code (°C) (°C) 

NTAN CFC 

5858 

0.0 126 125 127 

NTAN CFC 

5861 

0.0 127 134 127 

 Weight loss starts right after melting and the TGA curves suggest volatilization rather than 

decomposition. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8,boiling point,Witteveen,2003, REF.doc / 191.5 KB (application/msword) 

7327-60-8, Huybens, 2010, CoA NTAN CFC5858-5859-6058,ref.docx / 37.81 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANBased on results in this report: melting starts at 127°C. At the same temperature the 

evaporation of the substance starts too.So during melting the substance evaporates, starting both at 

127°C. 

Endpoint study record: 7327-60-8, Boiling point, EPISuite, SS 

UUID IUC5-a0fab734-9cb2-4b85-bf47-f4b3899728be 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:01:23 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

EPISuite 

 

Applicant's summary and conclusion 

Conclusions 

10
BP: 321.24°C 

4.4 Density 

Endpoint summary: Density 

UUID IUC5-ce70ae9a-7d59-45ee-b860-647b5f0ce85d 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:06:53 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

7327-60-8, NTANThe relative density is 1.298 with a standard deviation of 0.001. 

Key value for chemical safety assessment 

Relative density at 20°C 

1.298 

Discussion 

Key study: 7327-60-8, Density, Knuif, 2010, RS

Endpoint study record: 7327-60-8, Density, Knuif, 2010, RS 

UUID IUC5-2a0bae99-d5c6-4e8c-8f7b-7e412773be12 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:08:37 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period April 2010 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies According to OECD 109. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laborator

y 

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Knuif 

MJM 

201

0 

Determination 

of the relative 

density of 

nitrilotriacetonitri

le (NTAN) 

 Akzo 

Nobel 

Technolog

y and 

Engineerin

g B.V. 

Departme

nt Powder 

Technolog

y Deventer 

The 

Netherlan

2.416.98

4 - 

AkzoNob

el 

Function

al 

Chemical

s B.V. 

 2010-

04-13 

11
ds 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 109 (Density of Liquids and Solids) (July 1995) no 

according 

to 

other guideline: DIN 51913, Testing of carbon materials – Determination of 

density by gas pycnometer (volumetric) using helium as the measuring gas – 

Solid materials, May 2001 

no 

Type of method 

pycnometer method 

GLP compliance 

no (Internal Management System) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number CFC-7538Description Off-white odourless powderPurity (NTAN) 94.1 m/m % , certificate of 

analysis included in the report.Water 3.2 m/m %Storage requirements Store in a cool, well ventilated 

area. The thermal stability depends on temperature and time of exposure. Keep container tightly 

closed.Expiration date April 2011 

Any other information on materials and methods incl. tables 

Test principle 

 

The volume of a sample of known mass is determined by measuring the changes of gas volume within 

the pycnometer upon introducing the sample. The volume change is obtained indirectly by measuring the 

changes of the pressure within the pycnometer and calculating the volume using the pressure-volume 

relationship for ideal gases. The volume obtained by this procedure is related to the solid alone without its 

gas accessible pores. 

The pycnometer is placed inside of a climate chamber and acclimatized at a temperature of 20.0+0.5oC. 

Measurement is carried out at this temperature. 

 

Sample preparation 

 

None, relative density of the sample was determined “as is”. 

Sample was not dried because of possible evaporation of crystal water during heating. 

 

Test conditions 

 Test equipment

 

12
Apparatus Type Supplier Apparatus 

number Calibration 

Analytical 

balance AG 204 Mettler 2106-2A002 29.09.2009 

Climate 

box SLH 60/40 Weiss Enet 3122-3S001 09.06.2009 

Helium 

pycnometer 

AccuPyc 1340 

Gas Pycnometer Micromeritics 2109-5S001 25.03.2010 

Gas

Gas Type Supplier 

Helium 5.0 Air Liquide 

Results and discussion 

Density 

Type Density Temp. Remarks 

relative density 1.2979 20 °C St. Dev. 0.0007 RSD 0.05% 

Any other information on results incl. tables 

The true density of the NTAN was determined in triplicate. 

True density of NTAN

 

Sample 

Sample 

weight 

(g) 

Volume 

(cm3

) 

True density 

(g/cm3) 

Temperature 

(oC) 

CFC-7538.1 3.5491 2.7352 1.2976 20.0 

CFC-7538.2 3.3541 2.5853 1.2974 20.0 

CFC-7538.3 3.3547 2.5833 1.2986 20.0 

 

The relative density (D4

20), is theratioof the truedensityof a material at a temperature of 20oC and the 

density of water at a temperature of 4oC (density = 0.999975g/cm3

). 

The relative density has no dimension. 

Relative density of NTAN:

 

Sample 

/ 

Relative Density 

NTAN 

Batch No.: CFC-7538 

Average 1.2979 

St. Dev. 0.0007 

RSD (%) 0.05 

Overall remarks, attachments 

Overall remarks 

Control pycnometer

Control sample 

Standard 

volume 

(cm3

) 

Measured 

volume 

(cm3

) 

Deviation 

(%) 

Temperature 

(oC) 

St.0 (empty vial) 0.0000 0.0011 - 20.1 

Standard 135 6.3717 6.3719 +0.003% 20.1 

Remark: The measured volume of0 should be within 0.05% of full scale (10 cm3). 

The measured volume of the Standard 135 should be within 0.01%. 

13
Attached background material 

Attached document Remarks 

7327-60-8, relative density, Knuif, 2010, REF.doc / 192 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTAN 

Based on results in this report: The relative density is 1.298 with a standard deviation of 0.001. 

4.5 Particle size distribution (Granulometry) 

Endpoint summary: Particle size distribution (Granulometry) 

UUID IUC5-c077fbb7-0815-490e-8594-c4c0a8a0cfbf 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:09:44 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

10% of the particles is smaller than 90-120 μm, 50% of the particles is smaller than 185-250 μm and 90% 

of the particles is smaller than 305-370 μm. In addition, no NTAN particles smaller than 10 μm were 

found. Therefore, the respirable fraction is considered to be negligible. 

Discussion 

Key study: 7327-60-8, Particle size distribution (Granulometry), Reichwein, 2010, RS 

Endpoint study record: 7327-60-8, Particle size distribution (Granulometry), Reichwein, 2010, rs 

UUID IUC5-53337715-0d38-43f5-8ab2-589a986d6073 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:08:46 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period January 2005 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies According to NEN-ISO 13320-1. 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Reichwei

n AM 

201

0 

Particle 

size 

distributio

n of 

 AkzoNobel 

Chelates 

(FC-C), 

Deventer, 

FC-C 

M1002

8 

Akzo 

Nobel 

Functiona

l 

 2010-

03-10 

14
NTAN The 

Netherland

s 

Chemical

s 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Type of distribution 

volumetric distribution 

Test guideline 

Qualifier Guideline Deviations 

according to other guideline: NEN-ISO 13320-1 no data 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

CFC 5858 (lot no. 801L023873-05)CFC 5859 (lot no. 801L023874-05) CFC 6058 (lot no. 801L024271-

05). These commercial samples produced by AkzoNobel in Lima (U.S.) were tested.The actual product is 

produced commercially as a wet cake with a very low dustiness 

Any other information on materials and methods incl. tables 

Laser diffraction 

Equipment: Sympatec HELOS with RODOS and GRADIS 

RODOS: dry dispersion with aerosol jet 

GRADIS: dry dispersion by gravity 

In order to be able to measure the particle size distribution by laser diffraction, NTAN was measured as 

such in a dry air stream (RODOS). 

Results and discussions 

Particle size 

Percentile Mean St. dev. Remarks 

D10 > 92 — < 120 μm Different samples 

D50 > 185 — < 246 μm Different samples 

D90 > 304 — < 371 μm Different samples 

Particle size distribution at different passages 

No. Size Distribution Remarks 

 < 10 μm 0 % 

Any other information on results incl. tables 

The first graph for each sample shows a cumulative value for the particle size distribution and the second 

graph shows a frequency value for the particle size distribution. 

The key data that characterize the particle size distributions for these three NTAN samples are given in 

the table below. 

15
Characteristics

CFC 5858

lot no.

801L023873-05

CFC 5859

lot no.

801L023874-05

CFC 6058

lot no.

801L024271-05

Q3 ( 10.00 μm ) [%] 0.0 0.0 0.0 

X3 ( Q=10.00 % ) [μm] 92 100 120 

X3 ( Q=50.00 % ) [μm] 185 200 246 

X3 ( Q=90.00 % ) [μm] 304 332 371 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, Particle size distribution, Reichwein, 2010, REF.doc / 190 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8,NTAN:10% of the particles is smaller than 90-120 μm, 50% of the particles is smaller than 185-

250 μm and 90% of the particles is smaller than 305-370 μm. In addition, no NTAN particles smaller than 

10 μm were found. Therefore, the respirable fraction is considered to be negligible. 

4.6 Vapour pressure 

Endpoint summary: Vapour pressure 

UUID IUC5-8a164af6-6c5e-4432-9265-701a9ac4b48c 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:36:34 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

NTAN was determined to have a vapour pressure of 0.000104 Pa at 25°C. 

Discussion 

Key study: 7327-60-8, Vapour pressure, Elliott 2009, RS 

Supported by: 

7327-60-8, Vapour pressure, Grybat, 2006, SS 

The vapour pressure is < 0.001 mbar at 80°C. The substance starts to decompose above 80°C. 

7327-60-8 Vapour pressure, EPISuite, SS 

EPISuite calculation: 6.17x10-5mmHg 

Endpoint study record: 7327-60-8, Vapour pressure, Elliott, 2009, RS 

UUID IUC5-d1e3904a-7f00-4e61-b32e-301e34403b7a 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 10:26:14 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period September-October 2009 

Reliability 1 (reliable without restriction) 

16
Rationale for reliability incl. deficiencies Well conducted and reported study under GLP 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Elliott 

T 

201

0 

Nitrilotriacetonitri

le (CAS# 7327-

60-8): Vapor 

pressure 

determination by 

the Gas 

Saturation 

Method 

 ABC 

Laboratorie

s Inc., 

Columbia, 

Missouri, 

USA 

64965 Akzo 

Nobel 

Function

al 

Chemical

s LLCChelates 

 2010-

02-05 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 104 (Vapour Pressure Curve) 

according to EPA OPPTS 830.7950 (Vapor Pressure) 

Type of method 

gas saturation method 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

See CoA included in the report.Batch no: CFC 9161Expiration date: June 2, 2012Purity: 99.6 +/- 

1.5%Water: 0.4 +/- 0.2% 

Results and discussions 

Vapour pressure 

0.000104 Pa 

25 °C 

Remarks SD = 4.22E-6 Pa 

Transition / decomposition 

Transition / 

decomposition 

no 

Overall remarks, attachments 

Overall remarks 

17
Given this vapour pressure, NTAN was calculated to have a Henry's Law Constant of 1.85E-9 in 

deionised water at 25 degrees C. 

Attached background material 

Attached document Remarks 

7327-60-8, Vapour pressure, Elliott, 2010, ref.docx / 37.32 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

NTAN was determined to have a vapour pressure of 0.000104 Pa at 25 degrees C. 

Endpoint study record: 7327-60-8, Vapour pressure, Grybat, 2006, SS 

UUID IUC5-9b4ebe37-f7dc-4d6b-b60e-2cc869f3f087 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2010-12-21 10:41:07 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period 2006 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Fair report, according to EU Method A.4, signed certificate of analysis 

seperately uploaded. 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laboratory 

Report 

no. 

Owner 

compan

y 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Grybat 

A, 

Wollma

nn R, 

Fischer 

K 

200

6 

Vapour 

pressure 

determination 

of 

nitrilotriacetonit

rile 

 Laboratory 

for 

Thermophysi

cal 

Properties 

(LTP GmbH), 

Oldenburg, 

Germany 

2006059

8-VP 

AkzoNob

el 

Function

al 

Chemica

ls bv 

 2006-

08-03 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.4 (Vapour Pressure) no data 

Type of method 

18
gas saturation method 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number 2005L07241-01 (CFC-7538)Product code D2765Description off-white odourless powder 

(wet cake)Purity 94.1 w/w%Storage requirements Avoid contact with: aluminium, zinc, nickel, copper and 

copper alloys. Store in: PVC, PE, Stainless steel, bituminized tanks.Stability Stable under storage and 

handling conditions.Expiration date October 2008 

Any other information on materials and methods incl. tables 

To make sure that no decomposition product was found in the cryo trap, the original and the frozen 

sample were measured by HPLC with UV/Vis detection. 

For the identification of the substances at the top of the observed peak the flow was stopped and a 

spectrum scan was performed. 

The test substance is measured as such. 

Results and discussions 

Vapour pressure 

< 0.001 mBar 

80 °C 

Remarks No substance could be found in the cryo trap after several days even at the highest 

temperature. 

Transition / decomposition 

Transition / 

decomposition 

yes 

at > 80 °C 

Any other information on results incl. tables 

Several vapour pressure measurements with the gas saturation method were performed at temperatures 

between 60 and 120 °C for NTAN. No substance could be found in the cryo trap after several days even 

at the highest temperature. 

The substance starts to decompose above 80°C. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

CoA NTAN CFC7538.docx / 471.46 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document)  

7327-60-8, Vapor pressure, Grybat, 2006, ref.docx / 37.43 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANBased on results in this report: The determined vapour pressure was < 0.001 mbar at 

80°C. The substance starts to decompose above 80°C. 

19
Endpoint study record: 7327-60-8 Vapour pressure, EPISuite, SS 

UUID IUC5-5721ae7d-cbd3-43f2-b9a6-3a4a1eb98eb6 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 00:06:28 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

EPISuite 

 

Applicant's summary and conclusion 

Conclusions 

VP: 6.17x10-5 mmHg 

Endpoint study record: 7327-60-8, Vapour pressure, Daubert & Danner, 1989, S 

UUID IUC5-81f93014-e2b8-465a-ad21-936d4c1ecd03 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:17:41 CEST 

Remarks 

 

Administrative Data 

Purpose flag disregarded study 

Study result type experimental result 

Reliability 4 (not assignable) 

Rationale for reliability incl. deficiencies Original report was not available. 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

publication Daubert 

& 

Danner 

1989 

Applicant's summary and conclusion 

Conclusions 

VP: 3.17 x 10-7 mmHg 

4.7 Partition coefficient 

Endpoint summary: Partition coefficient 

UUID IUC5-acbbe867-6765-4f36-84b8-87cb0d2b95e2 

Dossier UUID 0 

20
Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:19:37 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Calculation withKOWWIN: Log Kow =-1.3903, indicating that NTAN is not expected to be susceptible to 

bioaccumulation. 

Key value for chemical safety assessment 

Log

Kow 

(Log

Pow)

-1.3903 

Discussion 

Key study: 7327-60-8, Partition coefficient, Zvinavashe, 2010, RS 

Software used: KOWWIN v1.67 (Sept. 2008) for log Kow 

A computional method has been used because the substance does not sufficiently dissolve in octanol 

due to its polarity and therefore high solubility in water. The calculated log Kow is less than the 

bioconcentration threshold (log Kow =3) indicating that NTAN is not expected to be susceptible to 

bioaccumulation 

Endpoint study record: 7327-60-8, Partition coefficient, EPISuite, Zvinavashe, 2010, RS 

UUID IUC5-8cda2be8-bfa7-4e71-b84a-9bd4b65ba2f9 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:32:37 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type estimated by calculation Study period June 2010 

Reliability 1 (reliable without restriction) 

Rationale for reliability 

incl. deficiencies 

A computional method has been used because the substance does not 

sufficiently dissolve in octanol due to its polarity and therefore high solubility in 

water. 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

other: 

Calculatio

n 

Zvinavash

e E 

201

0 

Calculate

d octanolwater 

partition 

coefficient 

(log Kow) 

and 

organic 

carbon 

 Akzo Nobel 

T&E, 

Arnhem, 

the 

Netherland

s 

341.17

0 

Akzo 

Nobel 

Functiona

l 

Chemical

s BV 

 2010-

06-04 

21
partition 

coefficient 

(log Koc) 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Partition coefficient type 

octanol-water 

Test guideline 

Qualifier Guideline Deviations 

no guideline required 

Type of method 

other: calculation 

Principles of method if other than guideline 

Software used: KOWWIN v1.67 (Sept. 2008) for log Kow 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

NTANSMILES : C(#N)CN(CC(#N))CC(#N) 

Analytical method 

other: calculation 

Any other information on materials and methods incl. tables 

The calculated log Kow value represents the neutral or unionized form. It should be noted that in the 

ionized form, the log Kow value is probably lower. The fragment constants used for estimating the log 

Kow value are derived from models with compounds in the log Kow range of 0 – 5. Estimations for log 

Kow values outside this range are generally less accurate. 

Results and discussions 

Partition coefficient 

Type Partition coefficient Temp. pH Remarks 

log Pow -1.3903 Calculation, so no temperature or pH mentioned. 

Any other information on results incl. tables 

Log Kow calculation

 

SMILES : C(#N)CN(CC(#N))CC(#N) 

CHEM : Acetonitrile, 2,2',2''-nitrilotrisMOL FOR: C6 H6 N4 

MOL WT : 134.14

-------+-----+--------------------------------------------+---------+-------- 

 TYPE | NUM | LOGKOW FRAGMENT DESCRIPTION | COEFF | VALUE 

22
-------+-----+--------------------------------------------+---------+-------- 

 Frag | 3 | -CH2- [aliphatic carbon] | 0.4911 | 1.4733 

 Frag | 1 | -N< [aliphatic attach] |-1.8323 | -1.8323 

 Frag | 3 | -C#N [cyano, aliphatic attach] |-0.9218 | -2.7654 

 Factor| 3 | -N-CH-C#N [aminomethylcyano] correction | 0.5017 | 1.5051 

 Const | | Equation Constant | | 0.2290 

-------+-----+--------------------------------------------+---------+-------- 

 Log Kow = -1.3903

Overall remarks, attachments 

Overall remarks 

Despite this inaccuracy, the calculated log Kow value is in accordance with the high water solubility (3 

g/L) for this compound. The calculated log Kow is less than the bioconcentration threshold (log Kow =3) 

indicating that NTAN is not expected to be susceptible to bioaccumulation. 

Attached background material 

Attached document Remarks 

7327-60-8, partition coefficient log Kow. calculation, Zvinavashe, 2010, REF.doc / 198 KB 

(application/msword) 

 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANCalculation withKOWWIN: Log Kow =-1.3903, indicating that NTAN is not expected to 

be susceptible to bioaccumulation. 

Executive summary 

The calculated log Kow is less than the bioconcentration threshold (log Kow =3) indicating that NTAN is 

not expected to be susceptible to bioaccumulation. 

4.8 Water solubility 

Endpoint summary: Water solubility 

UUID IUC5-504e482e-fd01-4768-801f-29c72af72602 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 21:37:14 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The water solubility is 3.06 g/L at 20°C. 

Key value for chemical safety assessment 

Water 

solubility

3.06 g/L at the 

temperature 

of 

20 °C 

Discussion 

Key study: 7327-60-8, Water solubility, Elliott, 2009, RS

Supported by: 

7327-60-8, Water solubility, EPISuite, SS 

EPISuite calculation: 1x106 mg/L 

Endpoint study record: 7327-60-8, Water solubility, Elliott, 2009, RS 

UUID IUC5-2cac6b88-3633-44ee-a5db-af7e61908cfe 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

23
Date 2011-07-14 21:24:31 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental 

result 

Study 

period 

18 August, 2009 till 30 November 

2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

GLP, according to OECD Guideline 105. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Elliott 

T 

200

9 

Nifrilotriacetonitri

le (CAS# 7327-

60-8): 

Determination of 

Water Solubility 

 ABC 

Laboratorie

s, Inc. 7200 

E. ABC 

Lane 

Columbia, 

Missouri 

65202 

ABC 

Study 

No. 

64964 

Akzo 

Nobel 

Function

al 

Chemical

s LLCChelates 

 2009-

11-30 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but not willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 105 (Water Solubility) no 

according 

to 

other guideline: U.S. Environmental Protection Agency Product Properties Test 

Guidelines OPPTS 830.7840 

no 

Type of method 

flask method 

GLP compliance 

yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

24
Product Name: Nitrilotriacetonitrile (NTAN)Lot Number: CFC 9161Purity: 99.6% a.i.Expiration Date: June 

02, 2012 

Any other information on materials and methods incl. tables 

Preliminary Test

Approximately 0.1 g of test substance was weighed into a graduated mixing cylinder 

(10 mL volume). Water was added in incremental volumes (to equal 0.1, 0.4, 1,2, 

and 10 nt total volume) with 10 minutes of agitation between additions. Since the sample did 

not dissolve after the addition of 10 mL of reagent water, the solution was quantitatively 

transferred to 100 -ml cylinders and brought to a volume of 100 mL with water. The test 

substance still did not show visual solubility, so the solution was quantitatively transferred to a 

1000 -ml graduated cylinder and tested at volumes of 250, 500, and 1,000 mL. 

The test substance did not show visual solubility at a volume of 1,000 mL, therefore the 

solubility of all solutions was estimated to be less than 0.0001 g/mL. 

Method Validation

The analytical method was validated for recovery of the test substance in triplicate at nominal 

concentration levels of 41.4 and 414μg/mL. Samples were prepared by transferring 10 mL of 

the 2.O7-mg/mI~ nitrilotriacetonitrile solution to a 25-mL volumetric flask and 

bringing to volume with reagent water for a concentration of 828μg/mL. The low level (41.4- 

[tg/mL) method validation samples were prepared by transferring 0.5 mL of the 828 -μg/niL 

solution to a 10-mL volumetric flask and bringing to volume with acetonitrile. The high level 

(414 -μglmL) method validation samples were prepared by transferring 5-nt of the 828μg/mL 

solution to a 10-nt volumetric flask and bringing to volume with acetonitrile. 

The analytical method was validated for linearity of response by injecting six standards ranging 

from 20.5 to 656μg/ml(Section 2.1.2). 

The analytical method was validated for system precision and suitability by injecting a mid 

range standard (82.0 -μg/mL), as prepared above, ten consecutive times. Suitability was also 

shown by injecting a blank sample to check for interference. 

The samples were analyzed using the GC equipment and conditions described in the report. 

Definitive Test

Definitive test samples were prepared at three proposed sample points in reagent water by 

transferring approximately I g of the test substance into 7 -ml Teflon® centrifuge tubes. To each 

tube was added 5 mL of reagent water. These amounts allowed for an excess of test substance in 

all samples. A quality control sample at each of the 3 time points was also prepared by adding 

approximately 10 mg of the test substance to 2-oz amber bottles. Fifty milliliters of reagent 

water was added to each bottle. The test and quality control samples were capped and shaken by 

hand to mix. They were placed on a shaker in an incubator at 30°C. 

After approximately 24 hours, the test and quality control samples for the first time point were 

removed from the shaker in the 30°C incubator and were placed on a shaker in the 20°C 

environmental chamber. After approximately 48 hours, the test and quality control samples for 

the second time point were removed from the shaker in the 30°C incubator and were placed on a 

shaker in the 20°C environmental chamber. After approximately 72 hours, the test and quality 

control samples for the third time point were removed from the shaker in the 30°C incubator and 

were placed on a shaker in the 20°C environmental chamber. 

After allowing each of the samples to remain on the shaker in the 20°C chamber for 

approximately 24 hours, the samples were centrifuged for 30 minutes at approximately 45,000 x 

g and 20°C. The supematant of each sample was transferred to a 14 -ml amber vial with a 

disposable pipet. The pH of each supematant was measured after transferred to the amber vial. 

The samples were diluted by transferring 1 mL to 25 -ml volumetric flasks, and bringing the 

flasks to volume with acetonitrile. The diluted samples were further diluted by transferring 1 mL 

to I O-mL volumetric flasks and bringing the flasks to volume with acetonitrile. The samples 

were analyzed by GC using the conditions described in the report. 

Results and discussions 

Water solubility 

> 3.02 — < 3.1 g/L 

25
Temp. 20 °C 

pH 6.9 

Any other information on results incl. tables 

Solubility

The solubility of nitrilotriacetonitrile was determined to be 3,060 ± 41.1μg/ml(3.06 g/L) at 

20+/-0.5°C, with less than 1% variability from the mean in each of the three replicates. 

A representative chromatogram of a definitive test sample is presented in the report. The pH of 

the supernatants were 6.90, 6.88, and 6.87. 

Method Validation

Acceptable recoveries of nitrilotriacetonitrile were obtained at levels similar to those seen in the water 

solubility samples. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, water solubility, Elliot, 2009, REF.doc / 191 KB (application/msword) 

Applicant's summary and conclusion 

Interpretation of results 

soluble (1000-10000 mg/L) 

Conclusions 

7327-60-8, NTAN,According to this report: The water solubility is 3.06 g/l at 20°C. 

Endpoint study record: 7327-60-8, Water solubility, EPISuite, SS 

UUID IUC5-a682fbf0-8d23-4d96-a6b5-1eb7c531b31b 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:24:12 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

EPISuite 

 

Applicant's summary and conclusion 

Interpretation of results 

very soluble (> 10000 mg/L) 

Conclusions 

WS: 1x10E6 mg/l 

4.10 Surface tension 

Endpoint summary: Surface tension 

UUID IUC5-9a1f8559-89fe-4d6b-bd9d-452f93424887 

Dossier UUID 0 

26
Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:38:57 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Based on the molecular structure the endpoint could be waived. No hydrofobic tail is present. However, 

measurement of the surface tension of an aqueous test substance solution at a concentration of 1.06 g/l 

and at a temperature of 22.0°C resulted in a value of 72.6 mN/m.This is the same surface tension as 

water has. 

Key value for chemical safety assessment 

Surface 

tension 

72.6 in mN/m at 

20°C and 

concentration 

in mg/L 

1060 

Discussion 

Key study: 7327 -60 -8, Surface tension, Van der Linden, 2006, RS

Endpoint study record: 7327-60-8, Surface tension, Van der Linden, 2006, RS 

UUID IUC5-df8bf7d3-78db-41e3-99b0-18ece4399afe 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 21:39:24 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period Januari 2006 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies According to EU A.5 and OECD 115 guidelines. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laborator

y 

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Linde

n van 

der 

EGM 

200

6 

Determination 

of the surface 

tension of an 

aqueous 

solution of 

nitrilotriacetonitr

ile 

 Research 

and 

Technolo

gy 

Chemical

s 

2006059

8-ST 

AkzoNob

el 

Function

al 

Chemical

s bv 

 2006-

01-17 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

27
Type of method 

OECD harmonised ring method 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.5 (Surface Tension) yes (Cleaning procedure) 

according to OECD Guideline 115 (Surface Tension of Aqueous Solutions) yes (Cleaning procedure)

Principles of method if other than guideline 

Deviations from the standard procedureAll glass vessels used for the test were new. All glassware was 

thoroughly washed with tap-water and rinsed with Direct Q Ultrapure water. Before each measurement 

the measuring body was rinsed with Direct Q Ultrapure water and shortly heated above a flame. 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number :2005L07241-01 (CFC-7538)Product code :D2765Description: Off white odourless powder 

(wet cake)Purity: 94.1 w/w%Storage requirements: Avoid contact with: aluminium, zinc, nickel, copper 

and copper alloys. Store in: PVC, PE, Stainless steel, bituminized tanks.Stability : Stable under storage 

and handling conditions.Expiration date: October 2008 

Details on methods 

EquipmentApparatus: Krüss Processor Tensiometer K12 (version 3.1). The measuring body is a 

platinum-iridium ring with a wire radius of 0.185 mm and a ring radius of 9.55 mm (ring no. 5).Water: 

Millipore Direct Q Ultrapure Water system (CAP-CWA lab).Rationale: The test system is recognized by 

international guidelines as recommended apparatus.Validation: The test system is validated before 

measurement using 96% ethanol ([7.3] for reference value) or n-hexane, n-heptane or n-octane ([7.4] for 

reference values). 

Any other information on materials and methods incl. tables 

Preparation of the test solution 

 

An amount of 106.0 mg test substance was dissolved in 100 ml Direct Q Ultrapure water, resulting in a 

clear test solution. The concentration of the test solution was 1.06 g/l. 

 

Sample measurement

 

A clean measurement vessel is filled with the test solution. The time from transferring the test solution to 

the measurement vessel is recorded. The measurement vessel is placed on the mobile sample table. The 

ambient temperature is read from a thermometer in the laboratory. The table is raised until the 

measurement body is just above the surface of the solution. The table automatically moves upward until 

the ring is immersed below the surface of the solution. Subsequently, the table is automatically lowered 

gradually and evenly to detach the ring from the surface until the maximum force is reached. The force is 

read on the tensiometer. The liquid layer attached to the ring must not separate from the ring. This 

measurement is repeated until a constant surface tension value is reached. 

 

DATA interpretation 

28
 

The surface tension is read from the tensiometer in mN/m (SI-sub-unit). 

 

In order to calculate the correct surface tension, the value read on the tensiometer is multiplied by the 

calibration factorФb. This yields a value which is only approximate and requires a correction. The 

additional correction is based on the Harkins-Jordan tables [7.5], see Table 1. The table provides 

corrected values for measurements taken after calibration. Interpolation shall be used for readings 

ranging between the table values. 

 

Correction of the water calibrated surface tension according to Harkins and Jordan

 

Density of the test solutions:ρ ≈ 1 g / cm3

. 

Average ring radius: R = 9.55 mm.. 

Average ring wire diameter: r = 0.185 mm. 

 

Experimental 

value after water 

calibration (mN/m) 

Corrected value 

(mN/m) 

20 18.1 

22 20.1 

24 22.1 

26 24.1 

28 26.1 

30 28.1 

32 30.1 

34 32.1 

36 34.1 

38 36.1 

40 38.2 

42 40.3 

44 42.3 

46 44.4 

48 46.5 

 

 

Experimental 

value after water 

calibration (mN/m) 

Corrected value 

(mN/m) 

50 48.6 

52 50.7 

54 52.8 

56 54.9 

58 57.0 

60 59.1 

62 61.3 

64 63.4 

66 65.5 

68 67.7 

70 69.9 

72 72.0 

74 74 

76 76 

78 78 

 

 

 

Results and discussions 

Surface tension 

72.6 mN/m 

Temp. 22 °C 

Concentration 1.06 g/L 

Any other information on results incl. tables 

Surface tension of Direct Q Ultrapure water at 22.0°C. 

 

Measurement no. Surface tension (mN/m) 

measured meanσg

1 71.23 

2 71.19 71.20 

3 71.19 

 

29
The calibration factorФb= 72.44 / 71.20 = 1.017. 

Surface tension measurement of test solution 

 

The test solution was transferred to the measurement vessel within 1 hour after preparation. Table 3 

shows the individual measurements and the corrected surface tension (calibration correction and Harkins 

& Jordan correction) for the test solution in water at a concentration of 1.06 g/l and at a temperature of 

22.0°C. 

Surface tension of an aqueous test substance solution at 22.0°C

 

 

 

measurement 

 

 

time 

expired1

(min) 

Surface tension (mN/m) 

 

measured 

 

mean2

not 

corrected 

 

mean2

after 

calibration 

correction 

mean after 

Harkins & 

Jordan 

correction 

1 3 71.37 

2 9 71.42 71.38 72.59 - 

3 13 71.35 

1 Time passed since the test solution was transferred to the measurement vessel. 2

 Mean was calculated from measurements 1 - 3. 

 

The surface tension was found to be stable after 3 minutes. 

 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

CoA NTAN CFC7538.docx / 471.46 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document)  

7327-60-8, surface tension, Van der Linden, 2006, REF.doc / 194 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8 NTAN, 

Based on results in this report:The surface tension of an aqueous test substance solution at a 

concentration of 1.06 g/l and at a temperature of 22.0°C is 72.6 mN/m. 

Based on the criteria as outlined in the EEC guideline, the substance is classified as “not surface active”. 

4.11 Flash point 

Endpoint summary: Flash point 

UUID IUC5-0a2fc5c8-5c5b-4d49-bd0a-e724cc0037d3 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:39:49 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The endpoint is waived because it is only a relevant property for liquids, thus this test does not need to be 

performed for substances that are solids or gases at room temperature. 

Endpoint study record: 7327-60-8, Flash point, waiver 

UUID IUC5-50d8504f-77f3-4883-87b5-d433c6207222 

30
Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:36:30 CET 

Remarks 

 

Administrative Data 

Data waiving other justification 

Justification for data 

waiving 

Only a relevant property for liquids, thus it does not need to be done for 

substances that are solids or gases at room temperature. 

4.12 Auto flammability 

Endpoint summary: Auto flammability 

UUID IUC5-821c296a-3d46-48c4-8341-7e6f0a988ea7 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 21:40:09 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The substance did not show self-heating up to its melting point which means that it does not have a selfignition temperature. 

Discussion 

Key study: 7327-60-8, Auto flammability, Nagelhout, 2010, RS

Endpoint study record: 7327-60-8, Auto flammability, Nagelhout, 2010, RS 

UUID IUC5-5ecaaf7d-a7cc-44a8-9074-d6ac44cafb1d 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:40:24 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period March 25 – 26, 2010 

Reliability 1 (reliable without restriction)

Rationale for reliability incl. 

deficiencies 

Performed under Internal management system, according to EU 

Method A.16. 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliograph

ic source 

Testing 

laboratory 

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Nagelho

ut G 

201

0 

Determinati

on of the 

self-ignition 

temperature 

of NTAN 

 Akzo Nobel 

Technology 

and 

Engineering 

B.V. 

2.419.35

7 

Akzo 

Nobel 

Function

al 

Chemical

 2010-

06-15 

31
Department 

HSE 

Safety,Testi

ng & 

Modeling, 

Deventer, 

The 

Netherlands 

s BV 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.16 (Relative Self-Ignition Temperature for Solids) no 

GLP compliance 

no (Internal quality management system) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

Batch number CFC-7538Description Off-white odourless powderCertificate of analysis included in the 

report:Purity 94.1%Water 3.2%Storage requirements Store in a cool, well ventilated area. The thermal 

stability depends on temperature and time of exposure. Keep container tightly closed.Expiration date April 

2011 

Any other information on materials and methods incl. tables 

Test principle 

A certain amount of substance is introduced into a wire mesh cube and placed in an air circulation oven 

at room temperature. The temperature in the centre of the sample is recorded while the temperature of 

the oven is increased to 400°C, or to the melting point if lower, at a rate of 0.5°C/min. When the 

substance ignites the sample temperature will show a very sharp rise above the oven temperature. The 

temperature of the oven at which the sample temperature reaches 400°C by self-heating is called the 

self-ignition temperature. 

Test conditions 

Equipment Temperature programmed air circulation oven 

Thermocouples Rössel Chromel-Alumel, type K; diameter 0.5 mm 

for sample and 1.0 mm for oven 

Recorder Atal MS3 Datalogger 

Sample cube 20 mm sides, wire mesh 0.045 mm 

Scan rate 0.5°C/min 

Temperature range 25°C – 400°C 

Intake 4.66 g 

The test cube was filled with substance, and by tapping it gently, more of the substance was added until 

32
the cube was completely full. The cube was then placed in the centre of the oven at room temperature. 

One thermocouple was placed in the centre of the cube to measure the sample temperature, and the 

other one between the cube and the oven wall to measure the oven temperature. The oven was closed 

and turned on. The temperature of the sample and oven were continuously recorded while the 

temperature of the oven was increased at a rate of 0.5°C/min. The test was stopped after the sample had 

molten completely. 

The thermocouples and recorder were calibrated (and adjusted when necessary) maximum one year 

before this study according to the internal calibration method KALTDR-001 

Results and discussions 

Autoflammability / Self-ignition temperature 

 

Remarks The substance did not show self-heating up to its melting point which means that it does not 

have a self-ignition temperature. 

Any other information on results incl. tables 

Self-ignition temperature test substance 

The temperature/time course of the test is shown in the graph in the report. The temperature of the 

sample remained constant at 126°C due to melting of the sample. No exothermal self-heating occurred 

before melting. The molten sample dripped out of the cube, resulting in an empty cube after complete 

melting of the sample. The recorded value of the thermocouple in the test cube had no relevance 

anymore thereafter, since it measured the air temperature. The test was stopped at that point. 

 

This result means that the test substance did not show self-heating up to its melting point, and 

consequently it does not have a self-ignition temperature. 

Overall remarks, attachments 

Overall remarks 

Calibration 

Calibration date: July 21, 2009 

After correction the deviation of the thermocouples was within the estimated accuracy of the test method, 

which is ±1.5°C. 

Attached background material 

Attached document Remarks 

7327-60-8, self-ignition_temperature, Nagelhout, 2010, REF.doc / 196 KB (application/msword) 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTAN, 

The substance did not show self-heating up to its melting point which means that it does not have a selfignition temperature. 

4.13 Flammability 

Endpoint summary: Flammability 

UUID IUC5-235f78cd-74ba-4179-a941-d9236ff2699f 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 21:41:36 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The substance is not flammable and has no pyrophoric properties. 

Key value for chemical safety assessment 

33
Flammability non flammable 

Discussion 

Key study: 7327-60-8, Flammability, Ruiter, 2002, RS 

Key study: 7327-60-8, Pyrophoric properties, Ruiter, 2002, RS

Justification for classification or non-classification 

The substance is not flammable and has no pyrophoric properties. Therefore it is not classified. 

Endpoint study record: 7327-60-8, Flammability, Ruiter, 2002, RS 

UUID IUC5-b1c119dc-0896-432b-9b45-4ee9f4cb522e 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands 

Date 2010-11-08 15:39:39 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2002 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. 

deficiencies 

Fair report, no detailed information, according to EU Method A.10 , 

purity mentioned. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Ruiter 

AJ 

200

2 

Physicochemical 

propertie

s of 

NTAN 

 AkzoNobel 

Polymer 

Chemicals 

Deventer 

The 

Netherland

s 

PCS 

20020013

0 

AkzoNobe

l 

Functional 

Chemicals 

B.V. 

 2002-

03-11 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, Explosiveness, Ruiter, 2002, rs7327-60-8, Pyrophoric properties, Ruiter, 2002, rs7327-60-

8,Oxidising properties,Ruiter, 2002, rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.10 (Flammability (Solids)) no data 

Principles of method if other than guideline 

No data 

GLP compliance 

no 

34
Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch number : 01036N348704, manufactured by A.N. Lima, Ohio - USASample : CFC 

5422Appearance : white powderPurty > 99% on dry base. 

Results and discussions 

Solid/liquid: Ignition on contact with air 

no 

Solid: Burning time (s) 

 

Remarks No ignition of substance and consequently no propagation of burning 

Aerosol: Flame extension (cm) 

 

Remarks The substance is not produced nor transported as an aerosol. 

Aerosol: Flame projection (cm) 

 

Remarks The substance is not produced nor transported as an aerosol. 

Gas: Lower explosion limit (%) 

 

Remarks The substance is a solid. 

Gas: Upper explosion limit (%) 

 

Remarks The substance is a solid. 

Any other information on results incl. tables 

No ignition of substance and consequently no propagation of burning.

The substance is not flammable. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, Safety, Ruiter, 2002a, REF.doc / 183.5 KB (application/msword) 

Applicant's summary and conclusion 

Interpretation of results 

non flammable 

Conclusions 

7327-60-8,NTAN,The substance is not flammable. 

Endpoint study record: 7327-60-8, Pyrophoric properties, Ruiter, 2002, RS 

UUID IUC5-e2938470-99d6-44f9-a5bc-963ddd90abb7 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands 

Date 2010-11-08 15:39:09 CET 

Remarks 

35
 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2002 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. 

deficiencies 

Fair report, no detailed information, according to EU Method A.10 , 

purity mentioned. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Ruiter 

AJ 

200

2 

Physicochemical 

propertie

s of 

NTAN 

 AkzoNobel 

Polymer 

Chemicals 

Deventer 

The 

Netherland

s 

PCS 

20020013

0 

AkzoNobe

l 

Functional 

Chemicals 

B.V. 

 2002-

03-11 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, Explosiveness, Ruiter, 2002, rs7327-60-8, Flammability, Ruiter, 2002, rs 7327-60-8,Oxidising 

properties,Ruiter, 2002, rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.13 (Pyrophoric Properties of Solids and Liquids) no data 

Principles of method if other than guideline 

No data 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch number : 01036N348704, manufactured by A.N. Lima, Ohio - USASample : CFC 

5422Appearance : white powderPurty > 99% on dry base. 

Results and discussions 

Solid/liquid: Ignition on contact with air 

36
no 

Remarks see: 7327-60-8, Flammability, Ruiter, 2002, rs 

Solid: Burning time (s) 

 

Remarks No ignition of substance and consequently no propagation of burning 

Aerosol: Flame extension (cm) 

 

Remarks The substance is not produced nor transported as an aerosol. 

Aerosol: Flame projection (cm) 

 

Remarks The substance is not produced nor transported as an aerosol. 

Gas: Lower explosion limit (%) 

 

Remarks The substance is a solid. 

Gas: Upper explosion limit (%) 

 

Remarks The substance is a solid. 

Any other information on results incl. tables 

No ignition within five minutes after contact with diatomacéous earth

The substance has no pyrophoric properties. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, Safety, Ruiter, 2002b, REF.doc / 184 KB (application/msword) 

Applicant's summary and conclusion 

Interpretation of results 

other: No pyrophoric properties. 

Conclusions 

7327-60-8,NTAN,The substance has no pyrophoric properties. 

4.14 Explosiveness 

Endpoint summary: Explosiveness 

UUID IUC5-f41bfe02-fd1d-436f-b476-d6f96c756741 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:42:27 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The substance has no explosive properties. 

Key value for chemical safety assessment 

Explosiveness non explosive 

Discussion 

Key study: 7327-60-8, Explosiveness, Ruiter, 2002, RS

37
Justification for classification or non-classification 

The substance has no explosive properties. Therefore the substance is not classified. 

Endpoint study record: 7327-60-8, Explosiveness, Ruiter, 2002, RS 

UUID IUC5-38e4c978-bd66-4204-97c7-0d1677eaa969 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2011-07-14 21:42:46 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2001-2002 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies According to EU Method A.14. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Ruiter 

AJ 

200

2 

Physicochemical 

propertie

s of 

NTAN 

 AkzoNobel 

Polymer 

Chemicals 

Deventer 

The 

Netherland

s 

PCS 

20020013

0 

AkzoNobe

l 

Functional 

Chemicals 

B.V 

 2002-

03-11 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, Flammability, Ruiter, 2002, rs 7327-60-8, Pyrophoric properties, Ruiter, 2002, rs7327-60-

8,Oxidising properties,Ruiter, 2002, rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.14 (Explosive properties) no data 

Principles of method if other than guideline 

No data 

GLP compliance 

no 

Test materials 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

38
Batch number : 01036N348704, manufactured by A.N. Lima, Ohio - USASample : CFC 

5422Appearance : white powderPurty > 99% on dry base. 

Any other information on materials and methods incl. tables 

Method :EC test A.14, part thermal sensitivity, Koenen test 

Sample mass : 26 ml filling in steel tube 

Method : EC test A.14, part mechanical sensitivity (shock), BAM fallhammer apparatus 

Fall height : 0.4 m 

Drop weight : 10 kg 

Method : EC test A.14, part mechanical sensitivity, BAM friction apparatus 

Load : 360 N 

Results and discussions 

Explosive (not specified) 

no 

Any other information on results incl. tables 

Thermal sensitivity, Koenen test

Orifice 

diameter(mm) Result 

 2 no explosion (3 times) 

 6 no explosion (3 times) 

Result : No explosion in the tests with an orifice of 2 and 6 mm diameter. 

Conclusion : The substance is not thermally sensitive (limiting diameter < 2.0 mm). 

Mechanical sensitivity(shock): 

Result: 6 times negative, shock sensitivity is > 40 J. 

Conclusion : The substance is not sensitive to mechanical shock 

Mechanical sensitivity, BAM friction apparatus

Result : No external changes or decomposition effects, friction sensitivity > 360 N 

Conclusion : The substance is not sensitive to mechanical friction. 

Overall remarks, attachments 

Overall remarks 

The substance has no explosive properties.

Attached background material 

Attached document Remarks 

7327-60-8, Safety, Ruiter, 2002c, REF.doc / 187.5 KB (application/msword) 

Applicant's summary and conclusion 

Interpretation of results 

non explosive 

Conclusions 

7327-60-8, NTAN,The substance has no explosive properties. 

4.15 Oxidising properties 

Endpoint summary: Oxidising properties 

UUID IUC5-fb83e887-50d8-4f19-814d-8111257475cc 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:44:07 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

39
The substance has no oxidising properties.

Key value for chemical safety assessment 

Oxidising no 

Discussion 

Key study: 7327-60-8, Oxidising properties, Ruiter, 2002, RS

Justification for classification or non-classification 

The substance has no oxidising properties. Therefore it is not classified.

Endpoint study record: 7327-60-8,Oxidising properties,Ruiter, 2002, RS 

UUID IUC5-c9181310-5a4f-4209-92b8-7efa3522070c 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:43:22 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2002 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies According to EU Method A.17. 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Ruiter 

AJ 

200

2 

Physicochemical 

propertie

s of 

NTAN 

 AkzoNobel 

Polymer 

Chemicals 

Deventer 

The 

Netherland

s 

PCS 

20020013

0 

AkzoNobe

l 

Functional 

Chemicals 

B.V. 

 2002-

03-11 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, Explosiveness, Ruiter, 2002, rs7327-60-8, Flammability, Ruiter, 2002, rs 7327-60-8, 

Pyrophoric properties, Ruiter, 2002, rs 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to EU Method A.17 (Oxidising Properties (Solids)) no data 

Principles of method if other than guideline 

No data 

40
GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch number : 01036N348704, manufactured by A.N. Lima, Ohio - USASample : CFC 

5422Appearance : white powderPurty > 99% on dry base. 

Contact with 

powdered cellulose 

Duration of test (contact time) 

> 147 — < 607 s 

Details on methods 

Reference material: BariumnitrateThe test was repeated with NTAN mixed with Kieselguhr (inert 

material). 

Results and discussions 

Test result 

other: The substance has no oxidising properties. 

Any other information on results incl. tables 

Test with cellulose and as reference Barium nitrate.

The sample mixed with cellulose in various ratios burned faster than the reference material, 

Bariumnitrate. Because of the chemical nature of NTAN oxidizing properties were not to be expected. 

According to test procedure as prescribed in test A17 to demonstrate whether a “false” positive test result 

was observed, only due to burning properties of NTAN on carrier, the test was repeated with NTAN mixed 

with Kieselguhr (inert material). In case propagation of burning is also observed with Kiezelguhr, the 

propagation is only due to burning properties instead of oxidizing properties. 

Test with Kiezelguhr and as reference Barium nitrate.

As expected, Bariumnitrate did not propagate burning. NTAN did show propagation with inert Kiezelguhr. 

Therefore, the propagation as determined in the tests with NTAN and cellulose mixture is due to burning 

properties of the substance on carrier rather than due to oxidizing properties. Even under nitrogen 

atmosphere, NTAN mixed with cellulose showed no propagation or smouldering effect after ignition, 

which demonstrates that air is needed and thus ordinary burning occurs during the original test. 

Overall remarks, attachments 

Overall remarks 

Conclusion : The substance has no oxidising properties.

Attached background material 

Attached document Remarks 

7327-60-8, Safety, Ruiter, 2002d, REF.doc / 189 KB (application/msword) 

Applicant's summary and conclusion 

Interpretation of results 

no oxidising properties 

Conclusions 

7327-60-8, NTANThe substance has no oxidising properties.

4.17 Stability in organic solvents and identity of relevant degradation products 

41
Endpoint summary: Stability in organic solvents and identity of relevant degradation products 

UUID IUC5-6524c674-276f-4cac-9195-4a7b66242201 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:43:36 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Endpoint is waived. Based on the partition coefficient it can be concluded that the substance is extremely 

low soluble in organic solvents and consequently the stability in organic solvent is considered not critical. 

Endpoint study record: 7327-60-8, Stability in organic solvents and identity of relevant 

degradation products, waiver 

UUID IUC5-352bf287-eab1-4951-8395-6e2009756ef9 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands 

Date 2010-11-08 15:42:32 CET 

Remarks 

 

Administrative Data 

Data waiving other justification 

Justification for 

data waiving 

Based on the partition coefficient it can be concluded that the substance is 

extremely low soluble in organic solvents and consequently the stability in organic 

solvent is considered not critical. 

4.21 Dissociation constant 

Endpoint summary: Dissociation constant 

UUID IUC5-326150aa-c2ae-48cb-ac7b-9fe1e23ac71b 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:44:57 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Endpoint is waived, study technically not feasible, no dissociation possible. 

Discussion 

Based on structure: Substance cannot dissociate due to a lack of ionisable groups. 

Endpoint study record: 7327-60-8, Dissociation constant, waiver 

UUID IUC5-c3ac1cab-4e0b-4f63-a54c-c0e69ea81d96 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:43:13 CET 

Remarks 

 

Administrative Data 

Data waiving study technically not feasible 

42
Justification for data waiving Substance cannot dissociate due to a lack of ionisable groups. 

4.22 Viscosity 

Endpoint summary: Viscosity 

UUID IUC5-fd28d626-f2fc-4d7d-992f-2093c9c2fffe 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:45:12 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The endpoint is waived , study technically not feasible, study is not relevant for solids. 

Endpoint study record: 7327-60-8, Viscosity, waiver 

UUID IUC5-177b78b3-2db1-47fa-b04a-b0ebdc3762bb 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:43:41 CET 

Remarks 

 

Administrative Data 

Data waiving study technically not feasible 

Justification for data waiving Property is only relevant to liquids. Substance is a solid. 

4.23 Additional physico-chemical information 

Endpoint summary: Self-reactivity and self-heating 

UUID IUC5-155f9b15-3371-4e90-bea8-889507651718 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:45:31 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

The following endpoints are waived: Selfreactivity and selfheating. 

Discussion 

Self reactivity: testing by DSC; no classification when decomposition energy <300 J/g or when SADT > 

75°C 

Self heating melting point <160°C (mp = 125.8 -127.7°C) See:7327-60-8, Melting range. 

Endpoint study record: 7327-60-8, Self-reactivity, waiver 

UUID IUC5-b3bfc011-bd88-4ba7-980a-4655b581287c 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:44:24 CET 

Remarks 

 

Administrative Data 

43
Data waiving other justification 

Justification for data 

waiving 

testing by DSC; no classification when decomposition energy < 300 J/g or 

when SADT > 75°C 

Endpoint study record: 7327-60-8, Self-heating, waiver 

UUID IUC5-6c79e6a0-91a5-40b7-b274-c8bb7faf7f79 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:44:54 CET 

Remarks 

 

Administrative Data 

Data waiving other justification 

Justification for data 

waiving 

melting point < 160°C (mp = 125.8 -127.7°C)See: 7327-60-8, Melting 

range 

5 Environmental fate and pathways 

Endpoint summary: Environmental fate and pathways 

UUID IUC5-b59a89e2-1885-4491-9217-cb0ee536b1c3 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-11 16:26:28 CET 

Remarks 

 

Administrative Data 

Discussion 

The substance is registered as TII and therefore an environmental fate assessment is not required. 

However, with regard to degradation the following can be concluded: 

Nitrilotriacetonitrile is not biodegraded in the prolonged Closed Bottle test. Nitrilotriacetonitrile should 

therefore not be classified as readily 

biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that nitrilotriacetonitrile 

is persistent in nature because the 

stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. 

With regard to mobility the following can be conlcuded: 

The estimated log Koc values are less than the threshold value of 3, indicating no adsorbing potential for 

this compound. 

5.2 Biodegradation 

Endpoint summary: Biodegradation 

UUID IUC5-f44e5659-6de6-4912-aba2-dde275df3421 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:51:30 CET 

Remarks 

 

Administrative Data 

Discussion 

Nitrilotriacetonitrile is not biodegraded in the prolonged Closed Bottle test. Nitrilotriacetonitrile should 

therefore not be classified as readily 

biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that nitrilotriacetonitrile 

44
is persistent in nature because the 

stringency of the test procedures could account for the recalcitrance in the Closed Bottle test. 

5.2.1 Biodegradation in water: screening tests 

Endpoint study record: 7327-60-8, Biodegradation in water: screening tests, Ginkel van, 2010, RS 

UUID IUC5-0bea0e5d-fb20-41f7-a9a2-548b99139980 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:48:00 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 08-04-2010 - 08-06-2010 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Test performed under GLP according guidelines with (minor) acceptable 

deviations, meeting all validity criteria 

Data source 

Reference 

Referen

ce type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laboratory 

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repo

rt 

date 

study 

report 

Ginkel 

van 

CG 

201

0 

Biodegradability 

of 

Nitrilotriacetonitr

ile (CAS 7327-

60-8) in the 

closed bottle 

test 

 Technology 

& 

Engineering 

Environment

al Chemistry 

2.420.08

1 

AkzoNob

el 

Function

al 

Chemical

s BV 

T10009 

C 

2010-

07-01 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

ready biodegradability 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle 

Test) 

yes (acceptable 

deviations) 

Principles of method if other than guideline 

One minor deviation from the guidelines of the Closed Bottle test was introduced; a) ammonium chloride 

was omitted from the medium to prevent oxygen consumption due to nitrification (omission doesnot result 

in nitrogen limitation as shown by the biodegradation of the reference compound). 

GLP compliance 

yes (incl. certificate) 

Test materials 

45
Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

EC number 230-804-9 

Details on test material 

A sample of nitrilotriacetonitrile was received from AkzoNobel Functional Chemicals on 08-03-2010. The 

following test substance data were submitted by the sponsor, who accepted full responsibility for the 

validity thereof.- chemical name (see annex 2) nitrilotriacetonitrile- CAS reg. No. 7327-60-8- purity (see 

annex 2) 99.6 +/- 1.5- Batch/lot no. CFC 9161- appearance white crystals- stability not relevant in test; 

stable under storage conditions- solubility in water soluble- storage at room temperature in the darkThe 

concentrations cited in this report refer to the as-received sample of nitrilotriacetonitrile. 

Study design 

Oxygen conditions 

aerobic 

Inoculum or test system 

activated sludge, domestic, non-adapted 

Details on inoculum 

Secondary activated sludge (02-04-2010) was obtained from the wastewater treatment plant Nieuwgraaf 

in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic 

wastewater. The activated sludge was preconditioned to reduce the endogenous respirationrates. To this 

end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in 

the BOD bottles (van Ginkel and Stroo, 1992). 

Duration of test (contact time) 

60 d 

Initial test substance concentration 

Initial conc. Based on 

4 mg/L test mat. 

Parameter followed for biodegradation estimation 

O2 consumption 

Details on analytical methods 

The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode 

(WTW Trioxmatic EO 200) and meter(WTW OXI 530) (Retsch, Ochten, The Netherlands). The pH was 

measured using a Knick 765 calimatic pH meter (Elektronische Messgerate GmbH, Berlin, Germany). 

The temperature was measured and recorded with a sensor connected to a data logger. 

Details on study design 

Test bottlesThe test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass 

stoppers.Nutrients and stock solutionsDeionized water used in the Closed Bottle test contained per liter of 

water with 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 

mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent 

nitrification. Sodium acetate and the test substance were added to the bottles using stock solutions of 1.0 

g/L. Test proceduresThe Closed Bottle test was performed according to the study plan. The study plan 

was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 

10 bottles containing inoculum and 16.0 mg/L humic acid, 10 bottles containing inoculum, test substance 

andhumic acid (16.0 mg/L), and 6 bottles containing sodium acetate and inoculum. The concentrations of 

the test substance and sodium acetate in the bottles were 1.0 and 6.7 mg/L, respectively. Each of the 

prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were 

completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved 

46
oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. The 

bottles with the test substance and the bottles with humic acid were placed onmagnetic stirrer plates (600 

rpm). The bottles contained magnetic bars in the bottles. Two duplicate bottles of all series were 

withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28. One extension 

from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by 

measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel 

fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to 

measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. 

After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed 

by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992)Calculation of the 

resultsCalculation of endogenous respiration The endogenous respiration (oxygen depletion in the 

control) was calculated as follows;Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc 

(day 28) Mc is the mean oxygen level in the control bottle inoculated with activated sludge.Calculation of 

the theoretical oxygen demand (ThOD)The ThODs of nitrilotriacetonitrile,and sodium acetate were 

calculated from their molecular formulae and molecular weights The calculated theoretical oxygen 

demand of nitrilotriacetonitrile, is 1.1 mg/mg. The theoretical oxygen demand of sodium acetate is 0.8 

mg/mg. Calculation of the biochemical oxygen demand (BOD)Provided that the oxygen concentrations in 

all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference 

compound bottles were calculated as follows:Oxygen consumption (mg/L) by test substance = Mch - Mt 

Oxygen consumption (mg/L) by reference compound = Mc - Ma Mc or ch = the mean oxygen level in the 

control bottles inoculated with activated sludge n days after the start of the test (C). The control of the test 

substance contained humic acid (CH).Mt or a = the mean oxygen concentration in the bottles containing 

the test compound (t) or the reference compound, sodium acetate (a), and inoculated with activated 

sludge n-days after the start of the test. The biological oxygen demand (BOD) mg/mg of the test 

compound and sodium acetate was calculated by dividing the oxygen consumption by theconcentration of 

the test substance and sodium acetate in the closed bottle, respectively.Calculation of the biodegradation 

percentagesThe biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to 

the theoretical oxygen demand (ThOD). 

Reference substance 

acetic acid, sodium salt 

Any other information on materials and methods incl. tables 

Sodium acetate anhydrous was used as a reference substance in the Closed Bottle test. This compound was 

purchased from Acros organics. 

The substance data were submitted by the supplier. 

· Reference compound acetic acid, sodium salt 

· CAS reg. No. 127-09-3 

· purity >99% 

· batch/lot No. A0206783001 

· appearance white crystals 

· solubility in water soluble in water 

· storage at ambient temperature in the dark 

Concentration sodium acetate in test = 6,7 mg/l 

Results and discussions 

% Degradation of test substance 

%Degr. St. dev. Parameter Sampling time Remarks 

0 O2 consumption 28 d 

0 O2 consumption 60 d 

Details on results 

ToxicityInhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test 

compound in the Closed Bottle test was not determined because possible toxicity of nitrilotriacetonitrile to 

microorganisms degrading acetate is not relevant. A slight inhibition of the endogenousrespiration of the 

inoculum by the test substance was detected. The test substance may therefore be slightly inhibitory to 

47
the inoculum.Test conditionsThe pH of the media was 7.0 at the start of the test. The pH of the medium at 

day 28 was 7.0 (control and test). Temperatures were within the prescribed temperature range of 22 to 

24°C 

Overall remarks, attachments 

Overall remarks 

Oxygen consumption (mg/L) and the percentages biodegradation of nitrilotriacetonitrile (BOD/ThOD) and 

sodium acetate (BOD/ThOD) in the Closed Bottle test. 

Time (days) Oxygen consumption (mg/L) Biodegradation (%) 

 Test substance Acetate Test substance Acetate 

0 0.0 0.0 0 0 

7 0.0 3.6 0 67 

14 0.0 4.0 0 74 

21 0.0 0 

28 0.0 0 

 42 0.0 0 

 60 0.0 0 

Attached background material 

Attached document Remarks 

7327-60-8, biodegradation in water screening tests, van Ginkel, 2010, REF.doc / 194.5 KB 

(application/msword) 

 

Illustration (picture/graph) 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes (endogenous respiration of <1.5 mg/L; differences between replicates at day 28 < 20%; sodium 

48
acetate was degraded 74% after 14 days.; oxygen concentrations >0.5 mg/L in all bottles during the test 

period.) 

Interpretation of results 

inherently biodegradable 

Conclusions 

Test performed under GLP according guidelines with (minor) acceptable deviations, meeting all validity 

criteriaNitrilotriacetonitrile is not biodegraded in the prolonged Closed Bottle test. Nitrilotriacetonitrile 

should therefore not be classified as readilybiodegradable. The lack of biodegradation in the Closed 

Bottle test does not mean that nitrilotriacetonitrile is persistent in nature because thestringency of the test 

procedures could account for the recalcitrance in the Closed Bottle test. 

Executive summary 

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the 

biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was 

determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test 

Guidelines, and in compliance with the OECD principles of Good Laboratory Practice. Nitrilotriacetonitrile 

did cause a slight reduction in the endogenous respiration. The test substance may therefore be slightly 

inhibitory to the inoculum. Nitrilotriacetonitrile was not biodegraded in the Closed Bottle test after 28 and 

60 days (prolonged) and should therefore not be classified as readily biodegradable. The lack of 

biodegradation in the Closed Bottle test does not mean that nitrilotriacetonitrile is persistent in nature 

because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle 

test. The test is valid as shown by an endogenous respiration of 0.9 mg/L and by the total mineralization 

of the reference compound, sodium acetate. Sodium acetate was degraded 74% of its theoretical oxygen 

demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L 

in all bottles during the test period. 

5.4 Transport and distribution 

5.4.1 Adsorption / desorption 

Endpoint summary: 7327-60-8, Adsorption / desorption 

UUID IUC5-a8f3ffa8-6244-43c7-bd29-19fd5e7a43ad 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:47:55 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Calculation with KOWWINv2.00 : Log Koc=1.15 (Estimate from MC) or 0.87 L/kg (Estimate from Log 

Kow.). The estimated log Koc values are less than the threshold value of 3, indicating no adsorbing 

potential for this compound. 

Key value for chemical safety assessment 

Koc 

at 

20 °C 

1.15 

Discussion 

Key study: 7327-60-8, Adsorption / desorption, Zvinavashe,2010, RS 

Software used: KOCWIN v2.00(Jan. 2009) for log Koc 

A computional method has been used because the substance does not sufficiently adsorb onto organic 

material due to its polarity and therefore high solubility in water. 

Endpoint study record: 7327-60-8, Adsorption / desorption, Zvinavashe, 2010, RS 

UUID IUC5-b4eac447-5a2e-4830-b937-03af0b1b5c68 

Dossier UUID 0 

49
Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 15:48:44 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type estimated by calculation Study period June 2010 

Reliability 1 (reliable without restriction) 

Rationale for reliability 

incl. deficiencies 

A computional method has been used because the substance does not 

sufficiently adsorb onto organic material due to its polarity and therefore high 

solubility in water. 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

other: 

calculatio

n 

Zvinavash

e E 

 Calculate

d octanolwater 

partition 

coefficient 

(log Kow) 

and 

organic 

carbon 

partition 

coefficient 

(log Koc) 

 Akzo Nobel 

T&E, 

Arnhem, 

the 

Netherland

s 

341.17

0 

Akzo 

Nobel 

Functiona

l 

Chemical

s BV 

 2010-

06-04 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

7327-60-8, Partition coefficient, Zvinavashe, 2010, rs 

Materials and methods 

Study type 

adsorption/desorption 

Media 

other: not relevant 

Type of method 

other: calculation 

Test guideline 

Qualifier Guideline Deviations 

no guideline required 

Principles of method if other than guideline 

Software used: KOCWIN v2.00(Jan. 2009) for log Koc 

50
GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Compound name: NTANSMILES : C(#N)CN(CC(#N))CC(#N)CHEM : Acetonitrile, 2,2',2''-nitrilotrisDetails on properties of test surrogate or analogue material 

Not relevant 

Study design 

Test temperature 

Not relevant 

Batch equilibrium or other method 

Computational methods 

KOCWIN v2.00(Jan. 2009) for log Koc 

Results and discussions 

Other adsorption coefficients 

Type Value Remarks 

other: log Koc 1.15 L/kg Estimate from MCI 

other: Log Koc 0.87 L/kg Estimate from Log Kow 

Results: Batch equilibrium or other method 

Any other information on results incl. tables 

Log Koc calculation

SMILES : C(#N)CN(CC(#N))CC(#N) 

CHEM : Acetonitrile, 2,2',2''-nitrilotrisMOL FOR: C6 H6 N4 

MOL WT : 134.14 

--------------------------- KOCWIN v2.00 Results --------------------------- 

 

Koc Estimate from MCI:

 --------------------- 

 First Order Molecular Connectivity Index ........... : 4.846 

 Non-Corrected Log Koc (0.5213 MCI + 0.60) .......... : 3.1261 

 Fragment Correction(s): 

 3 Nitrogen to Carbon (aliphatic) (-N-C).. : -0.6382 

 2 Nitrile/Cyanide (-C#N) ............... : -1.3354 

 Corrected Log Koc .................................. : 1.1525 

 

 Estimated Koc: 14.21 L/kg <===========

 Estimated log Koc: 1.15 L/kg <===========

 

Koc Estimate from Log Kow:

 ------------------------- 

 Log Kow (Kowwin estimate) ......................... : -1.39 

 Non-Corrected Log Koc (0.55313 logKow + 0.9251) .... : 0.1562

 Fragment Correction(s): 

51
 3 Nitrogen to Carbon (aliphatic) (-N-C).. : -0.0654 

 2 Nitrile/Cyanide (-C#N) ............... : 0.7843 

 Corrected Log Koc .................................. : 0.8752 

 

 Estimated Koc: 7.503 L/kg <===========

 Estimated log Koc: 0.87 L/kg <===========

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, adsorption-desorption,log Koc. calculation, Zvinavashe, 2010, REF.doc / 195 KB 

(application/msword) 

 

Applicant's summary and conclusion 

Conclusions 

7327-60-8, NTANCalculation with KOWWINv2.00 : Log Koc=1.15 (Estimate from MC) or 0.87 L/kg 

(Estimate from Log Kow.) 

Executive summary 

The estimated log Koc values are less than the threshold value of 3, indicating no adsorbing potential for 

this compound. 

6 Ecotoxicological Information 

Endpoint summary: Ecotoxicological Information 

UUID IUC5-c38bd173-7dc3-4e3a-b9de-c79faaa28691 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-11 16:27:08 CET 

Remarks 

 

Administrative Data 

PNEC oral 

Discussion 

As no Chemical Safety Assessment/Report is required for the transported isolated intermediate NTAN, no 

PNEC values have been calculated. 

Conclusion on classification 

Environmental classification justification 

Acute toxicity tests for algae and Daphnia magna are available for NTAN. Both tests show EC50 values 

far above the threshold for classification of 100 mg/l. Therefore classification as hazardous for the 

environment (according to Directive 67/548/EEC and Regulation 1272/2008) is not required for NTAN 

based on the available data. 

6.1 Aquatic toxicity 

Endpoint summary: Aquatic toxicity 

UUID IUC5-8f1dd59f-e7b1-490f-8602-09085ed8b73b 

Dossier UUID 0 

Author KolckM / Akzo Nobel Industrial Chemicals GmbH / Ibbenbüren / Germany 

Date 2010-10-27 14:20:32 CEST 

Remarks 

 

Administrative Data 

Discussion 

52
The available studies for the aquatic toxicity of NTAN are as follows: 

 

Guideline Test species Endpoint Result Rema

OECD 202 Daphnia magna 48h-EC50 

48h-NOEC 

>1000 mg/l 

≥1000 mg/l 

Key s

GLP, 

OECD 201 Pseudokirchneriella subcapitata 72h-EC50 

72h-NOEC 

>1000 mg/l 

171 mg/l 

Key s

GLP, 

6.1.1 Short-term toxicity to fish 

Endpoint summary: Short-term toxicity to fish 

UUID IUC5-7f9c956b-d9d5-40b0-917d-0dcc84b386af 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:50:48 CEST 

Remarks 

 

Administrative Data 

 

Short description of key information 

EC50 5460 mg/L calculated with ECOSAR. 

Endpoint study record: 7327-60-8, Short-term toxicity to fish, ECOSAR, SS 

UUID IUC5-54c6dd26-e6a4-41d6-a5a8-c827ec293ab2 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:50:16 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

ECOSAR 

 

Applicant's summary and conclusion 

Conclusions 

EC50: 5460 mg/L 

6.1.3 Short-term toxicity to aquatic invertebrates 

Endpoint summary: Short-term toxicity to aquatic invertebrates 

UUID IUC5-ede95e58-b9ee-4c6a-b014-6e261061ec52 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:56:00 CEST 

Remarks 

53
 

Administrative Data 

Short description of key information 

48h-EC50 >1000 mg/l, 48h-NOEC >= 1000 mg/l for Daphnia magna (OECD TG 202) 

Discussion 

The acute toxicity of NTAN toDaphnia magna(water flea) was tested under static conditions for 48 hours 

in accordance with OECD TG 202 under GLP. This test, with a reliability of 1, showed no effects up to the 

highest concentration of 1000 mg/l. The 48h-EC50 is therefore >1000 mg/l, the 48h-NOEC is >= 1000 

mg/l. 

A value was also calculated with ECOSAR. The calculated EC50 value is 260 mg/L. This value turns out 

to be too conservative. 

Endpoint study record: 7327-60-8, Short-term toxicity to aquatic invertebrates, Gyimesi, 2010, RS 

UUID IUC5-9b617e45-2e35-4b2d-a901-bb55189001e4 

Dossier UUID 0 

Author KolckM / Akzo Nobel Industrial Chemicals GmbH / Ibbenbüren / Germany

Date 2010-10-27 14:20:33 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2010-04-20 until 2010-05-28 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Study was well conducted according to standard guideline, with 

chemical analysis and GLP. 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Gyimes

i M 

201

0 

ACUTE 

TOXICIT

Y OF 

NTAN TO 

WATER 

FLEAS, 

DAPHNIA 

MAGNA 

 Akzo Nobel 

T&E, dept. 

RALS, 

Arnhem, 

the 

Netherland

s 

2.419.89

1 

Akzo 

Nobel 

Functiona

l 

Chemical

s BV 

 2010-

06-07 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) no 

GLP compliance 

54
yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Name: NTANChemical name: NitrilotriacetonitrilePurity / active ingredient: 99.6 ± 1.5 %CAS reg. no.: 

7327-60-8Appearance: White crystalline powderWater solubility: 0.268 wt%Lot number: CFC 

9161Stability: Stable under storage and handling conditionsStorage until required: At room 

temperatureSupplier: Akzo Nobel Functional Chemicals - ChelatesExpiry date: June 2, 2012 

Analytical monitoring 

yes 

Details on sampling 

At the start of the test, samples of 10 mL were taken from all test concentrations and the control solution. 

These samples were taken from the additional replicate made for chemical analysis. For the samples at 

the end of the test, 2.5 mL of each test vessel with the same test concentration was pipetted into a 

sample bottle. All samples taken at the start of the test were stored in the refrigerator prior to chemical 

analysis. 

Details on analytical methods 

Gas Chromatography with FIDThe following conditions were found to be suitable for the determination of 

the test substance in the concentration range of 0- 1.2 g/L.Column: DB 17HT, 30m x 0.32mm; 0.15μm 

dFFlame gas: 70 kPa air, 100 kPa H2Carrier gas: 70 kPa HeOven: Starting temperature: 100°CWait time: 

2 min.Rate temperature increase: 10°C / min.Ending temperature: 300°CWait time: 2 min.Detector: Built 

in Flame Ionization DetectorTemperature detector: 300°CTemperature injector: 275 °CInjection volume: 

0.5 μlSplit flow: 15 ml/ min.Integration software: Chromeleon version 6.80 RS7 Build 2528For the 

standards a stock solution was made in test medium. From this solution dilutions were made in the 

concentration range 0 – 1.2 g/L for the calibration standards. (n >= 5). Control standards from the middle 

range of the calibration series were analyzed at a minimum rate of one per ten samples or at least after 

each test series, to check the stability of the analytical system.Samples taken at the start of the test were 

stored at ± 4 oC in the dark until analysis. Three separate stability samples were stored under the same 

conditions as the test samples to check the storage stability, by comparing the concentration of the stored 

stability samples to the fresh stability samples prepared on the day of analysis. Stability samples should 

be within 10% of the expected values. The control standards analyzed in the middle and at the end of the 

sampling series were within 10% of the value calculated with the calibration 

curve.RepeatabilityRepeatability samples for chemical analysis were not measured. The control 

standards measured several times during the analyses show that repeatability was good. All control 

standards did not differ more than 10%. Therefore, this omission did not have an impact on the outcome 

of the test.LOD/LOQThe Limit of Detection (LOD) was calculated using the signal to noise ratio (S/N). The 

LOD is calculated as 3 * N whereas the Limit of Quantification (LOQ) is calculated as 10 * N. S/N was 

determined with the software (i.e. Chromeleon) based on peak height. (noise region; retention time 9.0 – 

9.4 min.) The calibration standard 75 mg/L had a signal to noise ratio of 143.2 resulting in a LOD of 1.57 

mg/L and a LOQ of 5.24 mg/L.System stabilityThe stability samples that were stored under the same 

conditions as the test samples differed less than 10% from the freshly prepared samples, therefore 

storage conditions were considered to be appropriate and no corrections were made for loss.LinearityThe 

analytical method was found to be linear over the range of 75 - 1200 mg/L of standard solutions of test 

substance using the conditions described above The GC calibration analyses gave a linear regression 

with a squared regression coefficient r2 >= 0.998. 

Vehicle 

no 

55
Details on test solutions 

The test medium, so called Dutch Standard Water (DSW), was used, having a pH of approximately 8.2, 

and a conductivity between 550 and 650 μs/cm, containing per liter of deionized water: 200 mg of 

CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3.The test medium was 

aerated before being used in the test. The air was water-saturated and purified by an activated charcoal 

and cotton filter.A stock solution of 1.0000 g/L of NTAN was prepared. An accurately measured amount of 

0.5000 g of test material, weighed out on an analytical balance, was loaded in approximately 480 mL test 

medium. Because NTAN did not dissolve easily the solution was sonicated three times 15 minutes. After 

this the solution was clear and it was decanted in a volumetric flask of 500 mL and filled up to the mark. 

The pH of the stock solution was checked and found to be 8.0, therefore no adjustment was necessary. A 

clear solution was obtained.The stock solution was diluted to obtain the test solutions. The stock solution 

was diluted with freshly prepared test medium.A control containing only test medium was included in the 

test. 

Test organisms 

Test organisms (species) 

Daphnia magna 

Details on test organisms 

The test was performed with Daphnia magna Clone 5 (Origin: Dr .U. Noack-Laboratorien Käthe-PaulusStr. 1, D-31157 Sarstedt, Germany) cultured in conformity with the relevant SOP. The animals used in the 

test were less than 24 hours old at the beginning of the test and were obtained from parent animals 

having an age of 2-4 weeks. 

Study design 

Test type 

static 

Water media type 

freshwater 

Limit test 

no 

Total exposure duration 

48 h 

Test conditions 

Hardness 

12.5 °dH61.2 mg/L Ca and 16.9 mg/L Mg. 

Test temperature 

20.95 - 21.25 °C 

pH 

7.7 - 8.0 

Dissolved oxygen 

8.7 - 9.0 mg/l 

Nominal and measured concentrations 

95, 171, 309, 556 and 1000 mg/L.The concentrations measured were within 80 to 120% of the nominals, 

therefore nominal concentrations were used to calculate the effects. 

Details on test conditions 

TEST SYSTEM- Test vessel: 50 mL glass beakers- Type (delete if not applicable): open- Aeration: noNo. of organisms per vessel: 5- No. of vessels per concentration (replicates): 4- No. of vessels per control 

(replicates): 4TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: DSWConductivity: 614 μs/cm- Culture medium different from test medium: Yes, culture medium is Elendt M4 

mediumOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: 16 h of ambient lightEFFECT 

PARAMETERS MEASURED (with observation intervals if applicable) : immobilityTEST 

56
CONCENTRATIONS- Spacing factor for test concentrations: 1.8- Range finding study- Test 

concentrations: control, 1, 10, 100 and 1000 mg/L. - Results used to determine the conditions for the 

definitive study: No effects were found, therefore it was decided that a limit test at 1000 mg/L would be 

sufficient. In the first definitive test, several animals died in the test concentration. Therefore, a second 

range finding test was performed in which again no effects were found up to 1000 mg/L. 

Reference substance (positive control) 

yes (potassium dichromate) 

Results and discussions 

Effect concentrations 

Duration Endpoint Effect 

conc. 

Nominal/Measured Conc. based 

on 

Basis for 

effect 

Remarks (e.g. 95% 

CL) 

48 h EC50 > 1000 

mg/L 

nominal test mat. mobility 

48 h NOEC >= 1000 

mg/L 

nominal test mat. mobility 

Details on results 

At 48 h one animal in 1000 mg/L was lying on the bottom of the test vessel. It started to swim after gentle 

agitation, therefore it was not considered immobile. 

Results with reference substance 

EC50 of the reference substance was found between 4.7 and 12.3 mg/L. 

Reported statistics and error estimates 

No statistics were performed because no immobile animals were seen during the test. 

Any other information on results incl. tables 

Measured concentration and recovery values of the test samples 

Sample Measured conc. 

t=0 (mg/L) 

% recovery Measured conc. 

t=48 (mg/L) 

% recovery 

Control < LOD - < LOD - 

95 mg/L 98.9 104.1 97.8 103.0 

171 mg/L 181.8 106.3 170.3 99.6 

309 mg/L 297.1 96.1 299.7 97.0 

556 mg/L 516.8 93.0 581.3 104.6 

1000 mg/L 944.9 94.5 946.4 94.6 

Overall remarks, attachments 

Attached full study report 

Attached full study report 

7327-60-8, Short-term toxicity to aquatic invertebrates, Gyimesi, 2010, REF.docx / 40.73 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes 

Conclusions 

The test substance did not give any effect up to the highest test concentration of 1000 mg/l and is 

therefore not toxic to Daphnia magna. 

Executive summary 

The acute toxicity of NTAN toDaphnia magna(water flea) was tested under static conditions for 48 hours 

in accordance with OECD, EEC guidelines for testing chemicals. The test was performed in compliance 

with Good Laboratory Practice (GLP). 

NTAN is soluble in water. During the preliminary test, performed as a non-GLP test, 20 animals per 

57
concentration, divided over 4 replicates, were exposed to control, 1, 10, 100 and 1000 mg/L. No effects 

were found, therefore it was decided that a limit test at 1000 mg/L would be sufficient. In the first definitive 

test, several animals died in the test concentration. Therefore, a second range finding test was performed 

in which again no effects were found up to 1000 mg/L. Still it was decided to perform a full definitive test. 

A definitive test with nominal test concentrations in the range of 95, 171, 309, 566 and 1000 mg/L 

including a control was performed. The results show that the test substance had no effect up to the 

highest test concentration. Based on the test results the EC50 is greater than 1000 mg/L. 

The results of the chemical analyses show that the observed test substance concentrations during the 

test differed less than 20% from the nominal test concentrations. As all concentrations measured are 

close to the nominals the effect data are based on the nominal test concentrations. 

Endpoint study record: 7327-60-8, Short-term toxicity to aquatic invertebrates, ECOSAR, SS 

UUID IUC5-6688ce49-2187-4da6-8020-c946c1e8a6a2 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:52:08 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

ECOSAR 

 

Applicant's summary and conclusion 

Conclusions 

EC50 260 mg/L 

6.1.5 Toxicity to aquatic algae and cyanobacteria 

Endpoint summary: Toxicity to aquatic algae and cyanobacteria 

UUID IUC5-f68f3b6c-0402-4292-9d01-b360de223f1b 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:55:42 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

72h-ErC50 >1000 mg/l, 72h-NOEC 171 mg/l for P. subcapitata (OECD TG 201) 

Key value for chemical safety assessment 

 

EC10/LC10 

or NOEC 

for 

freshwater 

171 mg/L 

58
algae 

Discussion 

The toxicity of NTAN to freshwater algae (P. subcapitata) was assessed in an algal growth inhibition test 

in accordance with OECD TG 201 under GLP. Slight modifications to the guideline were applied to 

ensure good growth and pH control of the cultures. 

No effect was found on growth rate up to 171 mg/L, therefore the 72h-NOEC is 171 mg/L. The 72h-ErC10 

is 652 mg/L. 50% effect was not reached, therefore the 72h-ErC50 is > 1000 mg/L. 

A value was also calculated with ECOSAR. The calculated EC50 value is 152 mg/L and the ChV is 15 

mg/L. 

Endpoint study record: 7327-60-8, Toxicity to aquatic algae and cyanobacteria, Gyimesi, 2010, RS 

UUID IUC5-74de4fcb-f4ba-4e85-8021-31b9b18b2449 

Dossier UUID 0 

Author KolckM / Akzo Nobel Industrial Chemicals GmbH / Ibbenbüren / Germany 

Date 2010-10-27 14:20:34 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 2010-04-19 until 2010-05-28 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Study was well conducted according to standard guideline, with 

chemical analysis and GLP. 

Data source 

Reference 

Referen

ce type 

Autho

r 

Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Report 

no. 

Owner 

compan

y 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Gyime

si, M. 

201

0 

EFFECTS OF NTAN 

ON THE GROWTH 

OF THE 

FRESHWATER 

GREEN ALGA 

PSEUDOKIRCHNERI

ELLA SUBCAPITATA 

 Akzo 

Nobel 

T&E, 

dept. 

RALS, 

Arnhem, 

the 

Netherlan

ds 

2.419.8

92 

Akzo 

Nobel 

Chemic

als BV. 

 2010-

06-07 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 201 

(Alga, Growth 

Inhibition Test) 

yes (The NaHCO3 concentration of the test medium was 150 mg/L 

instead of 50 mg/L, as recommended by the OECD/EEC Guidelines, 

in order to maintain a more constant pH during the test.) 

GLP compliance 

59
yes (incl. certificate) 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Details on test material 

Name: NTANChemical name: NitrilotriacetonitrilePurity / active ingredient: 99.6 ± 1.5 %CAS reg. no.: 

7327-60-8Appearance: White crystalline powderWater solubility: 0.268 wt%Lot number: CFC 

9161Stability: Stable under storage and handling conditionsStorage until required: At room 

temperatureSupplier: Akzo Nobel Functional Chemicals - ChelatesExpiry date: June 2, 2012 

Analytical monitoring 

yes 

Details on sampling 

At the start of the test, samples of 10 mL were taken from all test concentrations and the control solution. 

These samples were taken from the additional replicate made for chemical analysis. For the samples at 

the end of the test, 3.33 mL of each test vessel with the same test concentration was pipetted into a 

sample bottle. For the control the same was done, but only 1.66 mL was taken from each test vessel. The 

samples were filtered using an Acrodisk GHP 0.45 μm filter. All samples taken at the start of the test were 

stored in the refrigerator prior to chemical analysis. 

Details on analytical methods 

Gas Chromatography with FIDThe following conditions were found to be suitable for the determination of 

the test substance in the concentration range of 0- 1.2 g/L.Column: DB 17HT, 30m x 0.32mm; 0.15μm 

dFFlame gas: 70 kPa air, 100 kPa H2Carrier gas: 70 kPa HeOven: Starting temperature: 100°CWait time: 

2 min.Rate temperature increase: 10°C / min.Ending temperature: 300°CWait time: 2 min.Detector: Built 

in Flame Ionization DetectorTemperature detector: 300°CTemperature injector: 275 °CInjection volume: 

0.5 μlSplit flow: 15 ml/ min.Integration software: Chromeleon version 6.80 RS7 Build 2528For the 

standards a stock solution was made in test medium. From this solution dilutions were made in the 

concentration range 0 – 1.2 g/L for the calibration standards. (n >= 5).Control standards from the middle 

range of the calibration series were analyzed at a minimum rate of one per ten samples or at least after 

each test series, to check the stability of the analytical system.Samples taken at the start of the test were 

stored at ± 4 oC in the dark until analysis. Three separate stability samples were stored under the same 

conditions as the test samples to check the storage stability, by comparing the concentration of the stored 

stability samples to the fresh stability samples prepared on the day of analysis. Stability samples should 

be within 10% of the expected values. The control standards analyzed in the middle and at the end of the 

sampling series were within 10% of the value calculated with the calibration 

curve.RepeatabilityRepeatability samples for chemical analysis were not measured. The control 

standards measured several times during the analyses show that repeatability was good. All control 

standards did not differ more than 10%. Therefore, this omission did not have an impact on the outcome 

of the test.LOD/LOQThe Limit of Detection (LOD) was calculated using the signal to noise ratio (S/N). The 

LOD is calculated as 3 * N whereas the Limit of Quantification (LOQ) is calculated as 10 * N. S/N was 

determined with the software (i.e. Chromeleon) based on peak height. (noise region; retention time 9.0 – 

9.4 min.) The calibration standard 75 mg/L had a signal to noise ratio of 259.5 resulting in a LOD of 0.87 

mg/L and a LOQ of 2.89 mg/LSystem stabilityThe stability samples that were stored under the same 

conditions as the test samples differed less than 10% from the freshly prepared samples, therefore 

storage conditions were considered to be appropriate and no corrections were made for loss.LinearityThe 

analytical method was found to be linear over the range of 75 - 1200 mg/L of standard solutions of test 

substance using the conditions described above The GC calibration analyses gave a linear regression 

with a squared regression coefficient r2 >= 0.9994. 

Vehicle 

no 

Details on test solutions 

A stock solution of 1.0002 g/L of NTAN was prepared. An accurately measured amount of 0.5001 g of 

test material, weighed out on an analytical balance, was loaded in approximately 480 mL test medium. 

60
Because NTAN did not dissolve easily the solution was sonicated three times 20 minutes. After this the 

solution was clear and it was transferred to a volumetric flask of 500 mL and filled up to the mark. The pH 

of the stock solution waschecked and found to be 8.3, therefore no adjustment was necessary. A clear 

solution was obtained.The test solutions were prepared by addition of the adequate amounts of stock 

solution to the test vessels, then dilution water was added up to the required final volume. A fourth 

replicate was made for chemical analysis. 

Test organisms 

Test organisms (species) 

Pseudokirchnerella subcapitata 

Details on test organisms 

Freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae 

and Protozoa, Institute of Freshwater Ecology,The Windermere Laboratory, Cumbria, Ambleside, United 

Kingdom. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were 

stored at 4°C until required.The initial stock culture was inoculated with P. subcapitata from a sloped agar 

tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was 

regularly transferred to fresh medium to act as inoculum for testing.The absorbance of an exponentially 

growing stock culture was measured. The cell density was determined using the calibration curve 

described below. From this algal culture a dilution was prepared to obtain an initial cell density of 

approximately 1*10E4 cells/mL in the test medium. 

Study design 

Test type 

static 

Water media type 

freshwater 

Limit test 

no 

Total exposure duration 

72 h 

Test conditions 

Test temperature 

22.1 - 22.6 °C 

pH 

8.2 - 9.8The pH increased with 1.6 pH units. This was a little more than recommended in the guideline, 

but it is not considered to have an impact on the outcome of the test. 

Nominal and measured concentrations 

95, 171, 309, 556 and 1000 mg/LThe concentrations measured were within 80 to 120% of the nominals. 

Only the lowest test concentration at t = 72h differed 21.5%. As the measured concentration at the start of 

the test was close to nominal and also all other measured test concentrations, this is not taken into 

account. Nominal concentrations were therefore used to calculate the effects. 

Details on test conditions 

TEST SYSTEM- Test vessel:- Initial cells density: 1* 10E4 cells/ml- Control end cells density: 1.44 * 10E6 

cells/ml- No. of vessels per concentration (replicates): 3- No. of vessels per control (replicates): 

6GROWTH MEDIUM- Standard medium used: yes, OECD mediumTEST MEDIUM / WATER 

PARAMETERS- Culture medium different from test medium: noOTHER TEST CONDITIONS- Sterile test 

conditions: yes- Adjustment of pH: no- Photoperiod: continuous light- Light intensity and quality: 98.6 - 

99.6 μmol/m2/sEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- 

Determination of cell concentrations: spectrophotometerTEST CONCENTRATIONS- Spacing factor for 

test concentrations: 1.8- Range finding studyFrom a range finding test it was decided that a limit test at 

1000 mg/L would be sufficient. In the first definitive test, no growth was found in the test concentration.A 

second range finding test was performed in which no effects were found up to 100 mg/L. 1000 mg/L gave 

61
a significant effect. 

Reference substance (positive control) 

yes (potassium dichromate) 

Results and discussions 

Effect concentrations 

Duration Endpoint Effect 

conc. 

Nominal/Measured Conc. based 

on 

Basis for 

effect 

Remarks (e.g. 95% 

CL) 

72 h NOEC 171 mg/L nominal test mat. growth rate 

72 h LOEC 309 mg/L nominal test mat. growth rate 

72 h EC10 652 mg/L nominal test mat. growth rate 

72 h EC50 > 1000 

mg/L 

nominal test mat. growth rate 

Results with reference substance (positive control) 

The EC50 values of the reference compound, potassium dichromate, were in the range of 0.25-2.0 mg/L. 

Reported statistics and error estimates 

Where possible, the EC10,20,50,80 values were computed from the best fitted line (leastsquares method) 

through the points given by the probit of the percentage of inhibition and the logarithm of the 

concentration of the test substance. The Lowest Observed Effect Concentration (LOEC)was determined 

by comparison of the growth at each concentration and the control using threshold values from the 

William’s test (9.5) The No Observed Effect Concentration (NOEC) was derived from the results as the 

first concentration below the LOEC value, where growth shows no significant inhibition relative to the 

control values. Confidence limits were computed on the basis of Fieller’s theorem if possible (9.6). All 

computations were performed using the TOXCALC 5.0 program. 

Any other information on results incl. tables 

Extinctions measured in the algal growth inhibition test with NTAN 

Conc. Time (hours) Area Inhib. Specific Inhib. 

(mg/L) 0 24 48 72 % gr. rate % 

0 0.007 0.027 0.193 0.795 14.400 0.067 

0 0.006 0.028 0.198 0.810 14.784 0.069 

0 0.006 0.028 0.208 0.851 15.516 0.070 

0 0.006 0.028 0.199 0.854 15.336 0.070 

0 0.006 0.025 0.187 0.822 14.592 0.070 

0 0.006 0.029 0.203 0.828 15.144 0.070 

Mean 0.006 0.028 0.198 0.827 14.962 0.069 0.0 

 

95 0.006 0.028 0.199 0.803 14.724 0.070 

95 0.007 0.028 0.186 0.810 14.436 0.068 

95 0.006 0.026 0.175 0.763 13.620 0.069 

Mean 0.006 0.027 0.187 0.792 14.260 4.7 0.069 1.1 

 

171 0.006 0.028 0.177 0.811 14.292 0.069 

171 0.006 0.026 0.181 0.818 14.424 0.069 

171 0.006 0.023 0.163 0.730 12.864 0.068 

Mean 0.006 0.026 0.174 0.786 13.860 7.4 0.069 1.3 

 

309 0.007 0.026 0.144 0.697 12.024 0.065 

309 0.006 0.028 0.173 0.760 13.584 0.068 

62
309 0.006 0.026 0.173 0.755 13.476 0.069 

Mean 0.006 0.027 0.163 0.737 13.028 12.9 0.067 3.0 

 

556 0.007 0.022 0.118 0.599 10.128 0.064 

556 0.007 0.024 0.129 0.637 10.896 0.065 

556 0.007 0.024 0.128 0.597 10.392 0.064 

Mean 0.007 0.023 0.125 0.611 10.472 30.0 0.065 7.1 

 

1000 0.007 0.017 0.069 0.325 5.544 0.056 

1000 0.007 0.020 0.075 0.331 5.832 0.055 

1000 0.007 0.019 0.085 0.422 7.140 0.059 

Mean 0.007 0.019 0.076 0.359 6.172 58.7 0.057 18.2 

Average specific growth rate and coefficient of variation of average specific growth rates during the whole 

test period and themean coefficient of variation for section-by-section specific growth rates in the control 

cultures 

Time (hours) mean SD ± CV% Mean 

0-24 24-48 48-72 

72 hours 

test CV% 

0.056 0.082 0.059 0.0657 0.01412 21.48 

18.47 

0.064 0.082 0.059 0.0681 0.01190 17.47 

0.064 0.084 0.059 0.0688 0.01306 18.97 

0.064 0.082 0.061 0.0689 0.01126 16.36 

0.059 0.084 0.062 0.0683 0.01348 19.72 

0.066 0.081 0.059 0.0684 0.01151 16.82 

 mean 0.0681 

 stdev 0.0012 

 CV% 1.7226 

Overall remarks, attachments 

Attached full study report 

Attached full study report 

7327-60-8, Toxicity to aquatic algae and cyanobacteria, Gyimesi, 2010, REF.docx / 40.69 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

Applicant's summary and conclusion 

Validity criteria fulfilled 

yes 

Conclusions 

The NOEC and ErC10 are greater than 100 mg/l and the ErC50 is greater than 1000 mg/l, therefore 

NTAN can be considered as not toxic to freshwater green algae. 

Executive summary 

In order to predict effects of chemicals in an aquatic environment, the toxicity to freshwater algae was 

assessed. The algal toxicity was determined in the Algal Growth Inhibition Test in accordance with OECD, 

EEC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. Slight 

modifications to the guideline were applied to ensure good growth and pH control of the cultures. 

The toxicity of NTAN to an exponentially growing culture ofP. subcapitatawas determined over an 

exposure period of 72 hours. 

No effect was found on growth rate up to 171 mg/L, therefore NOEC is 171 mg/L and LOEC is 309 mg/L. 

The ErC10 is 652 mg/L. 50% effect was not reached, this gives an ErC50 > 1000 mg/L. 

The test was conducted in a mineral salts medium in acclimatized illuminated orbital incubator. The 

63
maximum variation in pH was 1.6 units. 

The test is valid as shown by: 

- the increase of the absorbance of the control over 72 h by a factor of 144 

- the mean coefficient of variation for section-by-section specific growth rates in the control cultures is 

18.5%. 

- the coefficient of variation of average specific growth rates during the whole test period in the replicate 

control cultures is 1.7%. 

The results of the chemical analyses show that the concentrations in the test solutions at the start and the 

end of the test are close to the initial nominal concentration. The measured concentration stayed within 

80 to 120% of the nominal. Only the lowest test concentration at t = 72h differed 21.5%. As the measured 

concentration at the start of the test was close to nominal and also all other measured test 

concentrations, this is not taken into account. Nominal concentrations are used to calculate the effects. 

Endpoint study record: 7327-60-8, Toxicity to aquatic algae and cyanobacteria, ECOSAR, SS 

UUID IUC5-bc6c214b-0d43-481c-9842-3d3d3a4e0c39 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 21:54:42 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type estimated by calculation 

Reliability 2 (reliable with restrictions) 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

other: 

ECOSAR 

 

Applicant's summary and conclusion 

Conclusions 

Algae EC50: 152 mg/LAlgae ChV: 15 mg/L

7 Toxicological information 

Endpoint summary: Toxicological information 

UUID IUC5-68e89f10-3569-4c31-9aaf-0bebf98c0a9b 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-12 11:04:05 CET 

Remarks 

 

Administrative Data 

Workers 

Long-term exposure - local effects 

Discussion 

As no Chemical Safety Assessment Report is required for the transported isolated intermediate NTAN, no 

DNEL values have been calculated for the worker. 

Because of the oral LD50 value of 50 -200 mg/kg bw; the test substance NTAN needs classification in 

GHS cat. III and R25 according to DSD-DPD. Based on the results described, NTAN would need 

64
classification in GHS cat. IV and R21according to DSD-DPD for acute dermal toxicity. 

Due to the minimal degree of skin and eye irritation observed, NTAN does not meet the criteria for 

classification as a skin and eye irritant. 

Based on the chemical structure of NTAN it is very unlikely that NTAN has skin sensitising properties. 

Based on the severe toxicity observed following repeated oral treatment at 45 mg/kg bw/day (death and 

severe clinical signs of toxicity), NTAN should be classified with R48/22 and STOT repeated exposure 

Cat. 2. 

Because both the Ames test and the chromosome aberration test showed that NTAN was not mutagenic, 

no classification for genotoxicity is needed. 

In a combined subacute/reproduction/developmental toxicity study, NTAN did not show 

reproduction/developmental toxicity at a level up to 45 mg/kg bw, although this level resulted in severe 

parental toxicity, including death. As such no classification for reproduction toxicity is warranted. 

General population 

Long-term exposure - local effects 

Discussion 

As no Chemical Safety Assessment/Report is required for the transported isolated intermediate NTAN, no 

DNEL values have been calculated for the general population. 

7.2 Acute Toxicity 

Endpoint summary: Acute Toxicity 

UUID IUC5-21685578-d503-47e5-a5b8-df7aa0bcf4d3 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-15 15:23:58 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

Several acute oral toxicity studies were available, and several types of NTAN have been tested, viz. a 

standard batch, a recrystallized bath and a lab washed batch. In addition, a batch was tested with 

unknown purity. One dermal acute toxicity was available. 

Discussion 

Although there were slight differences among six acute oral toxicity studies, viz. mortality varied between 

40 to 80% at 200 mg/kg bw, it was concluded that the LD50 value was ca. 200 mg/kg bw. The batch with 

unknown purity showed an LD50 value between ca. 50 and ca. 200 mg/kg bw. Therefore it was 

concluded that the LD50 was between 50 and 200 mg/kg bw, probably close to 200 mg/kg bw. The acute 

dermal LD50 value was > 200 mg/kg bw (highest dose tested). Three TSCA 8(e) notifications showed 

that the acute dermal LD50 value was (1) > 794 and < 1260 mg/kg bw, (2) > 1000 mg/kg bw, and (3) > 

2000 mg/kg bw. These results indicate that NTAN is much more toxic via the oral route than via the 

dermal route. 

Justification for classification or non-classification 

Because of the oral LD50 value of 50 -200 mg/kg bw; the test substance NTAN needs classification in 

GHS cat. III and R25 according to DSD-DPD. Based on the results described above, NTAN would need 

classification in GHS cat. IV and R21according to DSD-DPD for acute dermal toxicity. 

7.2.1 Acute toxicity: oral 

Endpoint study record: 7327-60-8, Acute toxicity: oral, batch 3554B, Glaza 2001a, RS 

UUID IUC5-1fe1cc41-41e6-4e98-ae8a-57b344fc5360 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 10:59:36 CET 

Remarks 

65
 

Administrative Data 

 

Purpose flag key study 

Study result type experimental result Study period April-May 2001 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laboratory

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Glaza 

SM 

200

1 

Acute oral 

toxicity study of 

Nitrilotriacetonitr

ile (Batch 

3554B) in rats 

 Covance 

Laboratori

es Inc., 

Madison, 

WI, USA 

1040060

0 

Akzo 

Nobel 

Chemical

s Inc., 

Madison, 

WI, USA 

 2001-

06-27 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no: 3554BDate of receipt: 3 April 2001Purity: 95 wt%Moisture: 5 wt% 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

66
Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on oral exposure 

The test material was mixed with corn oil; 10 mg/mL for the low dose, 40 mg/mL for the high dose. Single 

gavage application at 5 mL/kg bw. 

Doses 

50 and 200 mg/kg bw 

No. of animals per sex per dose 

5 

Control animals 

no 

Details on study design 

Duration of observation period following administration: 14 daysFrequency of observations and weighing: 

several times on the day of treatment and once daily thereafterNecropsy of survivors performed: yesOther 

examinations performed: body weight (weekly) 

Statistics 

Not applicable 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 > 50 — < 200 mg/kg bw test mat. 

Mortality 

None at 50 mg/kg bwFour males and two females (60%) at 200 mg/kg bw. 

Clinical signs 

At 50 mg/kg bw: staggered gait, wet/dark-stained urogenital area on the day of treatmentAt 200 mg/kg 

bw: hypoactivity, staggered gait, clonic convulsions, wet area around the mouth, red-stained face, 

lacrimation, wet urogenital area. In surviving animals these changes had disappeared by Day 4 (day of 

treatment = day 1). 

Body weight 

Surviving animals showed body weight gain throughout the study period. 

Gross pathology 

Only in animals that died (200 mg/kg bw); changes consisted of moist, red oral discharge. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, acute toxicity, oral, batch 3554B, Glaza, 2001a, ref.docx / 38.15 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

67
OECD GHS 

Conclusions 

The oral LD50 was between 50 and 200 mg/kg bw (close to 200 mg/kg bw). 

Executive summary 

An acute oral toxicity study was carried out in Sprague Dawley rats. Two groups of 5 male and 5 female 

rats each were dosed by single gavage at 50 or 200 mg/kg bw and kept for 14 days observation. None of 

the rats dosed with 50 mg/kg died; 60% mortality was observed at 200 mg/kg bw. Therefore the LD50 

was between 50 and 200 mg/kg bw, close to 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, recrystallized, Glaza 2001b, RS 

UUID IUC5-a14c3607-92e9-44d3-899c-27763bdc9b65 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2010-12-21 11:05:07 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study 

Study result type experimental result Study period April-May 2001 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laboratory

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Glaza 

SM 

200

1 

Acute oral 

toxicity study of 

Nitrilotriacetonitr

ile 

(Recrystallized) 

in rats 

 Covance 

Laboratori

es Inc., 

Madison, 

WI, USA 

1030695

3 

Akzo 

Nobel 

Chemical

s Inc., 

Madison, 

WI, USA 

 2001-

06-18 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) 

GLP compliance 

yes 

Test materials 

68
Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no: RecrystallizedDate of receipt: 3 April 2001Purity: 95 wt%Moisture: 5 wt% 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on oral exposure 

The test material was mixed with corn oil; 40 mg/mL Single gavage application at 5 mL/kg bw. 

Doses 

200 mg/kg bw 

No. of animals per sex per dose 

5 

Control animals 

no 

Details on study design 

Duration of observation period following administration: 14 daysFrequency of observations and weighing: 

several times on the day of treatment and once daily thereafterNecropsy of survivors performed: yesOther 

examinations performed: body weight (weekly) 

Statistics 

Not applicable 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 ca. 200 mg/kg bw test mat. 

Mortality 

Two males and three females (50%) died within one day 

Clinical signs 

Hypoactivity, staggered gait, prostation, tremors, clonic convulsions, wet area around the mouth, 

flaccidity, red-stained face, dyspnoea, and wet urogenital area. In surviving animals these changes had 

disappeared by Day 5 (day of treatment = day 1). 

Body weight 

Surviving animals showed body weight gain throughout the study period. 

Gross pathology 

69
Only in animals that died; changes consisted of moist, red oral discharge. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7326-60-8, acute toxicity, oral, recrystallized, Glaza, 2001b, ref.docx / 38.14 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

The oral LD50 was ca. 200 mg/kg bw 

Executive summary 

An acute oral toxicity study with NTAN Recrystallized was carried out in Sprague Dawley rats. One group 

of 5 male and 5 female rats was dosed by single gavage at 200 mg/kg bw and kept for 14 days 

observation. 50% mortality was observed at 200 mg/kg bw. Therefore the LD50 was ca. 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, lab washed, Glaza 2001c, RS 

UUID IUC5-a81c8fd4-4fe3-44a9-8551-02c8f436801b 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-15 14:58:09 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study 

Study result type experimental result Study period April-May 2001 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliograph

ic source 

Testing 

laboratory

Report 

no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Glaza 

SM 

200

1 

Acute oral 

toxicity study of 

Nitrilotriacetonitr

ile (Lab washed) 

in rats 

 Covance 

Laboratori

es Inc., 

Madison, 

WI, USA 

1040059

9 

Akzo 

Nobel 

Chemical

s Inc., 

Madison, 

WI, USA 

 2001-

06-18 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

70
Test type 

acute toxic class method 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no: Lab washedDate of receipt: 3 April 2001Purity: 95 wt%Moisture: 5 wt% 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on oral exposure 

The test material was mixed with corn oil; 40 mg/mL Single gavage application at 5 mL/kg bw. 

Doses 

200 mg/kg bw 

No. of animals per sex per dose 

5 

Control animals 

no 

Details on study design 

Duration of observation period following administration: 14 daysFrequency of observations and weighing: 

several times on the day of treatment and once daily thereafterNecropsy of survivors performed: yesOther 

examinations performed: body weight (weekly) 

Statistics 

Not applicable 

Results and discussions 

Effect levels 

71
Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 > 200 mg/kg bw test mat. 

Mortality 

Two males and two females (40%) were found dead on the day of treatment. 

Clinical signs 

Hypoactivity, staggered gait, tremors, clonic convulsions, wet area around the mouth, red-stained face, 

dyspnoea, non-formed faeces, dark-stained urogenital area. In surviving animals these changes had 

disappeared by Day 3 (day of treatment = day 1). 

Body weight 

Surviving animals showed body weight gain throughout the study period. 

Gross pathology 

Only in animals that died; changes consisted of moist, red oral discharge. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, acute toxicity, oral, lab washed, Glaza, 2001c, ref.docx / 38.2 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because 40% of the animals died, the oral LD50 was slightly higher than 200 mg/kg bw . 

Executive summary 

An acute oral toxicity study was carried out with NTAN Lab washed in Sprague Dawley rats. One group of 

5 male and 5 female rats was dosed by single gavage at 200 mg/kg bw and kept for 14 days observation. 

40% mortality was observed at 200 mg/kg bw. Therefore the LD50 was slightly higher than 200 mg/kg 

bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, batch 01013N34603, Glaza 2001d, RS 

UUID IUC5-6d9c369b-0223-4ebf-9847-f11e3f686311 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-15 15:15:27 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag key study 

Study result type experimental result Study period April-May 2001 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study 

Data source 

Reference 

Referenc Autho Yea Title Bibliograph Testing Report Owner Compan Repor

72
e type r r ic source laboratory no. compan

y 

y study 

no. 

t date 

study 

report 

Glaza 

SM 

200

1 

Acute oral 

toxicity study of 

Nitrilotriacetonitr

ile (batch 

01013N34603) 

in rats 

 Covance 

Laboratori

es Inc., 

Madison, 

WI, USA 

1010633

0 

Akzo 

Nobel 

Chemical

s Inc., 

Madison, 

WI, USA 

 2001-

05-04 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no: 01013N346403Purity: 94.43 wt%Moisture: 5.57 wt%White powderNTAB 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on oral exposure 

The test material was mixed with distilled water to a specific concentration for each doselevel (10 mg/mL 

for the 50 mg/kg dose level and 40 mg/mL for the 200 mg/kg doselevel). Single gavage application at 5 

mL/kg bw. 

73
Doses 

50 and 200 mg/kg bw 

No. of animals per sex per dose 

5 

Control animals 

no 

Details on study design 

Duration of observation period following administration: 14 daysFrequency of observations and weighing: 

several times on the day of treatment and once daily thereafterNecropsy of survivors performed: yesOther 

examinations performed: body weight (weekly) 

Statistics 

Not applicable 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 > 50 — < 200 mg/kg bw test mat. 

Mortality 

Five male and 3 female animals treated at 200 mg/kg were found dead on the day of test material 

administration. No other mortality was observed. The estimated oral LDso values in male and female rats 

were determined to be between 50 and 200 mg/kg of body weight. 

Clinical signs 

Clinical signs of toxicity were observed in all ten animals treated at 200 mg/kg and included hypoactivity, 

staggered gait, prostration, tremors, clonic convulsions, redstained face, dyspnea, wet area/foam around 

the mouth, few feces, and pale appearance. The two surviving females returned to a normal appearance 

by Day 3 after treatment. 

Body weight 

All animals surviving to the end of the observation period exhibited body weight gain during the study. 

Gross pathology 

Test material-related findings observed at the macroscopic necropsy examinations were limited to those 

animals treated at 200 mg/kg and dying on the day of test material administration. These findings 

included dry or moist red oral discharge. One female that also died on the day of test material 

administration had no test material-related findings. There were no other test material-related findings. 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

None of the animals died at 50 mg/kg bw and 8/10 died at 200 mg/kg bw. The oral LD50 is therefore 

between 50-200 mg/kg bw. 

Executive summary 

The test material, NTAN, was evaluated for its acute oral toxicity potential in male and female rats when 

administered as a single gavage dose at levels of 50 and 200 mg/kg of body weight. 

All animals treated at 50 mg/kg appeared normal and exhibited body weight gain throughout the study. 

All mortality in the animals treated at 200 mg/kg (five males and three females) occurred on the day of 

test material administration. Clinical signs of toxicity were observed in the ten animals treated at 200 

mg/kg and included hypoactivity, staggered gait, prostration, tremors, clonic convulsions, red-stained 

face, dyspnea, wet area/foam around the mouth, few feces, and pale appearance. The two surviving 

74
females returned to a normal 

appearance by Day 3 after treatment and exhibited body weight gain during the study. Test materialrelated findings observed at the macroscopic necropsy examinations were limited to those animals 

treated at 200 mg/kg and dying on the day of test material administration. These findings included dry or 

moist red oral discharge. There were no other test material-related findings. Based on the mortality 

observed in the study, the 

estimated oral LD50 values for male and female rats were detelmined to be between 50 and 200 mg/kg. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, batch 3554B, Kukulinski, 2001a, SS 

UUID IUC5-2ca5f8a3-9b0e-47fe-bb12-89f34943157a 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 13:42:26 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period April-May 2001 

Reliability 2 (reliable with restrictions) 

Rationale for reliability 

incl. deficiencies 

Limited reported study, no CoA included in the report (but most probaly 

similar to that in the acute oral study of Glaza (2001a). 

Data source 

Reference 

Referenc

e type 

Author Year Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Kukulinsk

i M 

200

1 

Acute oral 

toxicity 

study 

OPPTS 

870.1100, 

D.O.T. 

Design 49 

CFR 

173.132; 

Compound

: NTAN Lot 

3554B 

 Tox 

Monitor 

Lab. Inc., 

Oak Park, 

IL 60302, 

USA 

01-034 Akzo 

Nobel 

Functiona

l 

Chemical

s LLC, 

Dobbs 

Ferry NY 

10522-

3401, 

USA 

 2001-

05-17 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

yes 

Test guideline 

75
Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) no 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Lot no: 3554B (most probably similar to that used in acute oral study by Glaza (2001a). 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Doses 

200 mg/kg 

No. of animals per sex per dose 

5 

Control animals 

no 

Any other information on materials and methods incl. tables 

10% suspension in corn oil was made. This corresponds to about 100 mg/mL. Thus an animal received 

about 2 mL/kg bw (this was not indicated in the report). 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 < 200 mg/kg bw test mat. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, acute toxicity, oral, batch 3554B, Kukulinski 2001, ref.docx / 38.36 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

76
Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because 60% died at 200 mg/kg bw, the LD50 value was slightly lower than 200 mg/kg, and as such 

toxicity category III OECD GHS would be required. 

Executive summary 

An acute oral toxicity study with NTAN was carried out in Sprague Dawley rats. One group of 5 male and 

5 female rats was dosed by single gavage at 200 mg/kg bw and kept for 14 days observation. 60% 

mortality was observed at 200 mg/kg bw. Therefore the LD50 was < 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, recrystallized, Kukulinski, 2001b, SS 

UUID IUC5-29354066-6691-429d-a22b-dc94ea624b81 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 13:47:42 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period April-May 2001 

Reliability 2 (reliable with restrictions) 

Rationale for reliability 

incl. deficiencies 

Limited reported study, no CoA included in the report (but most probaly 

similar to that in the acute oral study of Glaza (2001b). 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Kukulinsk

i M 

200

1 

Acute oral 

toxicity study 

OPPTS 

870.1100, 

D.O.T. 

Design 49 

CFR 

173.132; 

Compound: 

NTAN 

Recrystallize

d 

 Tox 

Monitor 

Lab. Inc., 

Oak Park, 

IL 60302, 

USA 

01-

037 

Akzo 

Nobel 

Functiona

l 

Chemical

s LLC, 

Dobbs 

Ferry NY 

10522-

3401, 

USA 

 2001-

05-17 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

77
yes 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) no 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Lot no: Recrystallized (most probably similar to that used in acute oral study by Glaza (2001b)). 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Doses 

200 mg/kg 

No. of animals per sex per dose 

5 

Control animals 

no 

Any other information on materials and methods incl. tables 

10% suspension in corn oil was made. This corresponds to about 100 mg/mL. Thus an animal received 

about 2 mL/kg bw (this was not indicated in the report). 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 > 200 mg/kg bw test mat. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, acute toxicity, oral, recrystallized, Kukulinski 2001b, ref.docx / 38.42 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

78
Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because 40% died at 200 mg/kg bw, the LD50 value was slightly higher than 200 mg/kg, and as such 

toxicity category III OECD GHS would be required. 

Executive summary 

An acute oral toxicity study with NTAN Recrystallized was carried out in Sprague Dawley rats. One group 

of 5 male and 5 female rats was dosed by single gavage at 200 mg/kg bw and kept for 14 days 

observation. 40% mortality was observed at 200 mg/kg bw. Therefore the LD50 was > 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, lab washed, Kukulinski, 2001c, SS 

UUID IUC5-8660db17-7669-410a-8dcf-5151357a28c5 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands 

Date 2010-12-21 13:51:40 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period April-May 2001 

Reliability 2 (reliable with restrictions) 

Rationale for reliability 

incl. deficiencies 

Limited reported study, no CoA included in the report (but most probaly 

similar to that in the acute oral study of Glaza (2001c). 

Data source 

Reference 

Referenc

e type 

Author Year Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

company 

Compan

y study 

no. 

Repor

t date 

study 

report 

Kukulinsk

i M 

200

1 

Acute oral 

toxicity 

study 

OPPTS 

870.1100, 

D.O.T. 

Design 49 

CFR 

173.132; 

Compound

: NTAN 

Lab 

washed 

 Tox 

Monitor 

Lab. Inc., 

Oak Park, 

IL 60302, 

USA 

01-036 Akzo 

Nobel 

Functiona

l 

Chemical

s LLC, 

Dobbs 

Ferry NY 

10522-

3401, 

USA 

 2001-

05-17 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

79
Test type 

acute toxic class method 

Limit test 

yes 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) no 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Lot no: Lab washed (most probably similar to that used in acute oral study by Glaza (2001c). 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Doses 

200 mg/kg 

No. of animals per sex per dose 

5 

Control animals 

no 

Any other information on materials and methods incl. tables 

10% suspension in corn oil was made. This corresponds to about 100 mg/mL. Thus an animal received 

about 2 mL/kg bw (this was not indicated in the report). 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 ca. 200 mg/kg bw test mat. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

80
7327-60-8, acute toxicity, oral, lab washed, Kukulinski 2001c, ref.docx / 38.38 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because 50% died at 200 mg/kg bw, the LD50 value was ca. 200 mg/kg, and as such toxicity category III 

OECD GHS would be required. 

Executive summary 

An acute oral toxicity study with NTAN Lab washed was carried out in Sprague Dawley rats. One group of 

5 male and 5 female rats was dosed by single gavage at 200 mg/kg bw and kept for 14 days observation. 

50% mortality was observed at 200 mg/kg bw. Therefore the LD50 was ca. 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, batch 01013N34603, Kukulinski, 2001d, SS 

UUID IUC5-5cec13b4-333b-4462-9b4e-07139aa0f75f 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-15 15:05:36 CEST 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period April 28, 2001 to May 12, 2001 

Reliability 2 (reliable with restrictions) 

Rationale for reliability 

incl. deficiencies 

Limited reported study, no CoA included in the report (but most probaly 

similar to that in the acute oral study of Glaza (2001c). 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Kukulinsk

i M 

200

1 

Acute oral 

toxicity study 

OPPTS 

870.1100, 

D.O.T. 

Design 49 

CFR 

173.132; 

Compound: 

batch 

01013N3460

3 

 Tox 

Monitor 

Lab. Inc., 

Oak Park, 

IL 60302, 

USA 

01-

035 

Akzo 

Nobel 

Functiona

l 

Chemical

s LLC, 

Dobbs 

Ferry NY 

10522-

3401, 

USA 

 2001-

05-17 

Data access 

data submitter is data owner 

Data protection claimed 

81
yes, but willing to share 

Materials and methods 

Test type 

acute toxic class method 

Limit test 

yes 

Test guideline 

Qualifier Guideline Deviations 

according to EPA OPPTS 870.1100 (Acute Oral Toxicity) no 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Lot no: 01013N34603white powderNTAN 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Doses 

200 mg/kg 

No. of animals per sex per dose 

5 

Control animals 

no 

Any other information on materials and methods incl. tables 

10% suspension in corn oil was made. This corresponds to about 100 mg/mL. Thus an animal received 

about 2 mL/kg bw (this was not indicated in the report). 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

male/female LD50 ca. 200 mg/kg bw test mat. 

Applicant's summary and conclusion 

82
Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because 40% died at 200 mg/kg bw, the LD50 value was ca. 200 mg/kg, and as such toxicity category III 

OECD GHS would be required. 

Executive summary 

NTAN was tested for acute oral toxicity in accordance with OPPTS Guidelines and D.O.T. Design 49 CFR 

173.132. 

NTAN was administered by gavage at a dose of 200 mg/kg body weight to five male and five female rats. 

There was 40% mortality at the 200 mg/kg dose level during the 14 day observation period. The acute 

oral LD50 was determined to be greater than 200 mg/kg body weight. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, Dow, 1967, SS 

UUID IUC5-5bb1e390-cd09-4242-aa46-8ad68707d563 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 13:57:19 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period 1967 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies Limited reported study, used as supporting evidence 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

The 

Dow 

Chemic

al 

Compan

y 

196

7 

Toxicological 

properties and 

industrial 

handling 

hazards of 

Nitrilotriacetonitri

le 

 Biochemic

al 

Research 

Laboratory

, Dow, 

Midland, 

Michigan, 

USA 

12 

4198 

The Dow 

Chemica

l 

Compan

y 

 1967-

11-28 

Data access 

data submitter has Letter of Access 

Materials and methods 

Test type 

standard acute method 

Limit test 

no 

Test guideline 

83
Qualifier Guideline Deviations 

no guideline followed 

Principles of method if other than guideline 

Study was carried out in 1967; no guideline available 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Test animals 

Species 

rat 

Strain 

no data 

Sex 

female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Doses 

2% solution: 25, 50, 100, 200, 398 mg/kg bw10% solution: 63, 126, 252, 500, 1000, 2000 mg/kg bw

No. of animals per sex per dose 

2 

Control animals 

no 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

female LD50 > 50 — < 250 mg/kg bw test mat. see below 

Any other information on results incl. tables 

In case of the 2% solution: 

0% (0/2) of the animals died at 50 and 100 mg/kg bw 

50% (1/2) of the animals died at 25 mg/kg bw 

100% (2/2) of the animals died at 200 and 398 mg/kg bw. 

This means that the LD50 value of the 2% solution is somewhere between 100 and 200 mg/kg bw.

In case of the 10% solution: 

50% (1/2) of the animals died at 126 and 252 mg/kg bw 

100% (2/2) of the animals died at 63, 126, 500, 1000, and 2000 mg/kg bw. 

This means that the LD50 value of the 10% solution is somewhere between 50 and 250 mg/kg bw.

Overall remarks, attachments 

Attached background material 

84
Attached document Remarks 

7327-60-8, overview, Dow, 1967, ref.docx / 36.73 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document)  

Applicant's summary and conclusion 

Interpretation of results 

Toxicity Category III 

Criteria used for interpretation of results 

OECD GHS 

Conclusions 

Because LD50 value was between ca. 50 and 250 mg/kg bw, classification in GHS Cat. III is needed. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, Younger, 1965, SS 

UUID IUC5-64f0a109-08ee-4348-8420-58dd58525b5b 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 22:50:46 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Repo

rt no. 

Owner 

compa

ny 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Young

er, 

F.M. 

196

5 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

179 

Monsan

to 

 1965-

12-02 

Applicant's summary and conclusion 

Executive summary 

LD50 (Rats, Mixed Sex) The diluted compound, 10% in corn oil, was fed by stomach tube to Sprague 

Dawley strain albino male and female rats. Doses 100, 126, 158, 200 mg/kg. After the approximate 

Minimum Lethal Dose was determined groups of male end finale rate were fed in increasing doses at 

increments of 0.1 fractional leg intervals at four levels designed to blanket the toxicity range thereby 

supplying data for calculation of the LD50 which was done according to a modification of the method of S. 

J. de Beer. Observations were made for toxic symptoms and the viscera of the animals that succumbed 

were examined macroscopically. 

The oral LD50 for male and female rats was placed at 150 per kilogram with lower and upper limits of 150 

to 174 milligrams per kilogram. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, Younger, 1966, SS 

UUID IUC5-c7fde2ad-aa40-4cf6-a2f1-0cc0c5e90bb2 

Dossier UUID 0 

85
Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 23:13:40 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Repo

rt no. 

Owner 

compa

ny 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Young

er, 

F.M. 

196

6 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

216 

Monsan

to 

 1966-

01-28 

Applicant's summary and conclusion 

Executive summary 

LD50 (Rats, Mixed Sex) The diluted compound, 10% in corn oil, was fed by stomach tube to Sprague 

Dawley strain albino male and female rats. Doses 126, 158, 200, 251 mg/kg. After the approximate 

Minimum Lethal Dose was determined groups of male end finale rate were fed in increasing doses at 

increments of 0.1 fractional leg intervals at four levels designed to blanket the toxicity range thereby 

supplying data for calculation of the LD50 which was done according to a modification of the method of S. 

J. de Beer. Observations were made for toxic symptoms and the viscera of the animals that succumbed 

were examined macroscopically. 

The oral LD50 for male and female rats was placed at 198 per kilogram with lower and upper limits of 170 

to 321 milligrams per kilogram. 

Endpoint study record: 7327-60-8, Acute toxicity: oral, Eastman Kodak, 1971, SS 

UUID IUC5-8508d0c8-762c-434c-a57a-84ec39d26354 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 23:19:33 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Reference Author Year Title Bibliographic Testing Report Owner Company Report 

86
type source laboratory no. company study no. date 

study 

report 

Eastman 

Kodak 

1971 Eastman 

Kodak 

 

Applicant's summary and conclusion 

Conclusions 

LD50 50 mg/kg in rats and 50-100 mg/kg in mice. 

7.2.3 Acute toxicity: dermal 

Endpoint study record: 7327-60-8, Acute toxicity, dermal, Babish, 1978, RS 

UUID IUC5-2aac1a1e-ccc3-4e72-b390-693fc8a27a46 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 16:05:30 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 1978 

Reliability 2 (reliable with restrictions) 

Rationale for reliability 

incl. deficiencies 

Study appears well conducted with limited reporting of methods and results 

and a single dose tested only and not a limit dose. 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company

study no. 

Report 

date 

study 

report 

Babish 

JG 

1978 Acute 

Dermal 

Toxicity 

Study in 

Rabbits 

 Food and 

Drug 

Research 

Laboratories 

K067479-

003 

The Dow 

Chemical 

Company 

 1978-

09-20 

Data access 

data submitter has Letter of Access 

Materials and methods 

Test type 

standard acute method 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

 other guideline: 16 CFR 1500. 40 no 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

87
Identifier Identity 

CAS number 7327-60-8 

Details on test material 

purity> 99%White Crystalline material 

Test animals 

Species 

rabbit 

Strain 

other: albino 

Sex 

no data 

Administration / exposure 

Type of coverage 

occlusive 

Vehicle 

unchanged (no vehicle) (moistened with saline) 

Details on dermal exposure 

The animals were prepared by clipping the skin of the trunk free of hair. Approximately one-half of the 

animals was further prepared by making epidermal abrasions every 2 or 3 centimeters longitudinally over 

the area of exposure. The abrasions were sufficiently deep to penetrate the stratum corneum (horny layer 

of the epidermis) but not to distrub the derma; that is, not to obtain bleeding.The sleeve was slipped onto 

the animal which is then placed in a comfortable but immobilized position in a multiple animal holder. 

Selected doses of liquids and solutions are introduced under the sleeve. If there is slight leakage from the 

sleeve, which may occur during the first few hours of exposure, it is collected and reapplied. Dosage 

levels are adjusted in subsequent exposures (if necessary) to enable a calculation of a dose that would 

be fatal to 50 percent of the animals. This can be determined from mortality ratios obtained at various 

doses employed. At the end of 24 hours the sleeves or screens are removed, the volume of unabsorbed 

material (if any) is measured, and the skin reactions are noted. The subjects are cleaned by thorough 

wiping, observed for gross symptoms of poisoning, and then observed for 2 weeks.In this study only one 

dose level was chosen. 

Duration of exposure 

24 hours 

Doses 

200 mg/kg bw 

No. of animals per sex per dose 

10 

Control animals 

not required 

Results and discussions 

Effect levels 

Sex Endpoint Effect level Based on 95% CL Remarks 

no data LD0 200 mg/kg bw test mat. no mortalities observed 

Mortality 

No mortality observed 

Clinical signs 

Decreased activity and some nasal discharge was observed, but not indicated in how many animals or at 

what stage after dosing these effects started and ceased. 

88
Body weight 

Not assessed 

Gross pathology 

No pathology reported 

Applicant's summary and conclusion 

Conclusions 

A dose of 200 mg/kg was not lethal to adult albino rabbits following occlusive dermal exposure (intact and 

abraded skin). The LD0, therefore is 200 mg/kg bw. 

Endpoint study record: 7327-60-8, Acute toxicity: dermal, Younger, 1965, SS 

UUID IUC5-7f12b6e5-683e-48f1-9fbb-ca936df622a8 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 22:50:58 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Repo

rt no. 

Owner 

compa

ny 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Young

er, 

F.M. 

196

5 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

179 

Monsan

to 

 1965-

12-02 

Applicant's summary and conclusion 

Executive summary 

The diluted compound was applied in increasing doses at inernen of 0.2 fractional leg intervals to the 

closely clipped, intact skin of New Zealand 

white male and female rabbits. 

The treated area were covered with plastic strips and the animals placed in wooden stocks for periods up 

to twenty four hours, after which time they were assigned to individual cages. Observations were made 

for toxic symptoms and the viscera of the animals that subcomed were examined macroscopically. 

Tle Minimum Lethal Dose by Skin Absorption in male and female rabbits was found to be greater than 

794 milligrams per kilogram and less than 1260 milligrams per kilogram. 

It was classed as moderately toxic by skin absorption in male and female rabbits. 

Endpoint study record: 7327-60-8, Acute toxicity: dermal, Younger, 1966, SS 

UUID IUC5-c6077f91-3665-447e-a69f-68754b0783b1 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 23:40:18 CEST 

Remarks 

89
 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Repo

rt no. 

Owner 

compa

ny 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Young

er, 

F.M. 

196

6 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

216 

Monsan

to 

 1966-

01-28 

Applicant's summary and conclusion 

Executive summary 

The diluted compound was applied in increasing doses at inernen of 0.2 fractional leg intervals to the 

closely clipped, intact skin of New Zealand 

white male and female rabbits. 

The treated area were covered with plastic strips and the animals placed in wooden stocks for periods up 

to twenty four hours, after which time they were assigned to individual cages. Observations were made 

for toxic symptoms and the viscera of the animals that subcomed were examined macroscopically. 

Tle Minimum Lethal Dose by Skin Absorption in male and female rabbits was found to be greater than 

2000 milligrams per kilogram. 

It was classed as non-lethal by skin absorption in male and female rabbits. 

Endpoint study record: 7327-60-8, Acute toxicity: dermal, Eastman Kodak, 1971, SS 

UUID IUC5-b022c6bc-281d-42e5-b48a-381d36993ff8 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 23:21:41 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1971 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

study 

report 

Eastman 

Kodak 

1971 Eastman 

Kodak 

 

90
Applicant's summary and conclusion 

Conclusions 

LD50 (guinea pig) >1000 mg/kg; No clinical signs. 

7.3 Irritation / corrosion 

Endpoint summary: Irritation / corrosion 

UUID IUC5-8ad7b7f3-e018-4683-9d6e-20e4188359e4 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-15 15:26:07 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

One skin and one eye irritation test were available, and two supporting studies. 

Key value for chemical safety assessment 

Skin irritation / corrosion 

slightly irritating 

Eye irritation 

slightly irritating 

Discussion 

In the skin irritation test, 4 out of 5 rabbits showed slight erythema at the end of the 4 -h treatment period. 

Slight erythema was still observed in 2 out of these 4 animals 48 h after treatment. No corrosivity was 

seen. No further observations were made. In the eye irritation test, some conjunctival reddening was seen 

in all animals and slight corneal opacity in one animal. The eye changes were fully reversible within 7 

days. 

The supporting studies showed no skin and eye irritation following application of 10% and the undiluted 

NTAN. 

Justification for classification or non-classification 

Due to the minimal degree of skin and eye irritation observed, this substance does not meet the criteria 

for classification as a skin and eye irritant. 

7.3.1 Skin irritation / corrosion 

Endpoint study record: 7327-60-8, Skin irritation / corrosion, Babish, 1978, RS 

UUID IUC5-a7c19fce-3937-403f-98f9-d192c6f2268f 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 16:12:38 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. 

deficiencies 

Study is reported sufficeintly to understand what was done. Predates 

GLP. Study only designed to assess corrosivity 

Data source 

91
Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Company

study no. 

Report 

date 

study 

report 

Babish 

JG 

1978 Corrosive 

Dermal 

Study 

with 

Rabbits 

 Food and 

Drug 

Research 

Laboratories 

Inc. 

K067479-

005 

The Dow 

Chemical 

Company 

 1978-

09-20 

Data access 

data submitter has Letter of Access 

Materials and methods 

Type of method 

in vivo 

Test guideline 

Qualifier Guideline Deviations 

according to other guideline: see below 

Principles of method if other than guideline 

Guideline for corrosivity assay followed: The method employed is patterned after 21 CFR 191.11 as 

described in 49 CFR 173.240 

GLP compliance 

no 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test materials 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

White Crystalline powderPurity >99% 

Test animals 

Species 

rabbit 

Strain 

other: albino 

Test system 

Type of coverage 

occlusive 

Preparation of test site 

shaved 

Vehicle 

unchanged (no vehicle) (moistened with saline) 

Amount/concentration applied 

0.5 g 

Duration of treatment / exposure 

4 hours 

Observation period 

92
48 hours 

Number of animals 

5 

Control animals 

no 

Details on study design 

5 healthy, acclimated animals were shaved (back). 0.5 g of test material was applied under a 1 inch 

square gauze. An impervious rubber wrap was then applied to hold the material in place. Exposure 

duration was 4 hours and observations were made at the end of the 4 hours and then at 48 hours. No 

further observations were made.Scores were assigned based on the scoring scale below. 

Any other information on materials and methods incl. tables 

Scoring system 

Erythema and Eschar Formation: 

No Erythema 0 

Slight erythema 1

Well defined Erythema 2 

Moderate to Severe Erythema 3 

Severe Erythema 4 

Slight Eschar Formation 5 

Severe Eschar Formation, Ulceration 6 

Results and discussions 

Irritation / corrosion results 

Irritation 

parameter 

Basis Time 

point 

Score Max. 

score 

Reversibility Remarks 

overall 

irritation 

score 

mean 4 h ca. 1 6 other: see below score of 1 was 

seen in 4 out of 5 

animals 

overall 

irritation 

score 

mean 48 h ca. 

0.5 

6 other: at 48 h 2 out of 5 animals still 

showed slight erythema, no further 

observations were made 

 

Irritant/corrosive response data 

In 4 out of 5 animals there was a slight erythema (grade 1 out of a possible 6) at 4 hours. At 48 hours this 

had dissapeared in 2 of those affected. No further observations were made.No corrosivity was observed. 

Applicant's summary and conclusion 

Interpretation of results 

other: not-corrosive 

Criteria used for interpretation of results 

EU 

Conclusions 

The substance was slightly irritating after a 4 hour exposure. No corrosivity was observed. Due to the 

minimal degree of irritation observed it is unlikely that this substance would meet the criteria for 

classification as a skin irritant. 

Endpoint study record: 7327-60-8, Skin irritation / corrosion, Dow, 1967, SS 

UUID IUC5-4b85f3b6-a745-4f77-a554-131b8401d4d1 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 14:09:26 CET 

Remarks 

93
 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period 1967 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies Limited reported study, used as supporting evidence 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

The 

Dow 

Chemic

al 

Compan

y 

196

7 

Toxicological 

properties and 

industrial 

handling 

hazards of 

Nitrilotriacetonitri

le 

 Biochemic

al 

Research 

Laboratory

, Dow, 

Midland, 

Michigan, 

USA 

12 

4198 

The Dow 

Chemica

l 

Compan

y 

 1967-

11-28 

Data access 

data submitter has Letter of Access 

Materials and methods 

Type of method 

in vivo 

Test guideline 

Qualifier Guideline Deviations 

no guideline followed 

Principles of method if other than guideline 

No guideline available at that time 

GLP compliance 

no 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test materials 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Test animals 

Species 

rabbit 

Strain 

no data 

Test system 

Type of coverage 

no data 

94
Preparation of test site 

other: intact and abraded 

Vehicle 

other: Dowanol DPM 

Duration of treatment / exposure 

repeated application, either 3 or 9 times 

Observation period 

not indicated 

Number of animals 

not indicated 

Control animals 

no 

Results and discussions 

Any other information on results incl. tables 

undiluted, intact skin, 9 applications on belly: no irritation observed 

undiluted, abraded skin, 3 applications on belly: no irritation observed 

10% solution, intact skin, 9 applications on ear: slight exfoliation 

10% solution, intact skin, 9 applications on belly: slight exfoliation 

10% solution, abraded skin, 3 applications on belly: slight hyperaemia and exfoliation 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, overview, Dow, 1967, ref.docx / 36.61 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document)  

Applicant's summary and conclusion 

Interpretation of results 

not irritating 

Criteria used for interpretation of results 

expert judgment 

Endpoint study record: 7327-60-8, Skin irritation / corrosion, Younger, 1965, SS 

UUID IUC5-7a6b7fb5-d8a8-4e46-83b0-f0c28d82d5bc 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 22:51:10 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

Repo

rt no. 

Owner 

compa

Compa

ny 

Repo

rt 

95
y ny study 

no. 

date 

study 

report 

Young

er, 

F.M. 

196

5 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

179 

Monsan

to 

 1965-

12-02 

Applicant's summary and conclusion 

Executive summary 

The finely ground powder and a 10.0% solution-suspension in corn oil were applied to the clipped intact 

skin of albino rabbits and removed after twenty four hours. The applications were covered with plastic 

strips to retard evaporation and avoid concentration. Observations vere made over period of several days 

for irritation. The data, scored according to the method of praise, Woodard and Calvery (journal of pharm. 

and Exp. Therapentics, Volume. 82, December, 1944). 

The compound was classed as non-irritating to intact rabbit skin when applied as a finely ground powder. 

The compound was classed as non-irritating to intact rabbit skin when applied as a 10.0% solutionsuspession in corn oil. 

7.3.2 Eye irritation 

Endpoint study record: 7327-60-8, Eye irritation, Babish, 1978, RS 

UUID IUC5-07578f64-161e-4691-aa51-523b11ee726e 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-08 16:48:03 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period 1978 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. 

deficiencies 

Apears to be well conducted and reported sufficiently to make an 

assessment of the endpoint. Prior to GLP. 

Data source 

Reference 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Company

study no. 

Report 

date 

study 

report 

Babish 

JG 

1978 Rabbit 

Eye 

Irritation 

Study with 

fluorescein 

 Food and 

Drug 

Research 

laboratories 

Inc. 

K067479-

004 

The Dow 

Chemical 

Company 

 1978-

09-20 

Data access 

data submitter has Letter of Access 

Materials and methods 

Type of method 

in vivo 

Test guideline 

Qualifier Guideline Deviations 

96
equivalent or similar to OECD Guideline 405 (Acute Eye Irritation / Corrosion) 

Principles of method if other than guideline 

method according to 16 CFR 1500.42. 

GLP compliance 

no 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test materials 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

White Crystalline powderPurity >99% 

Test animals 

Species 

rabbit 

Strain 

New Zealand White 

Details on test animals and environmental conditions 

6 animals between 2 and 4 kg purchased from Dutchland Lab. Inc., Denver, Pennsylvania 

Test system 

Vehicle 

unchanged (no vehicle) 

Any other information on materials and methods incl. tables 

The eyes of each rabbit were examined with sodium fluorescein and an ultraviolet lamp (prior to 

instillation of the test material) and only those rabbits whose eyes were free of irritation and corneal 

lesions were used. 50 mg of the test material was instilled in one eye of each rabbit by gently pulling the 

lower lid away from the eyeball to form a cup into which the test substance was dropped. The lids were 

then held together for one second and the animal released. The untreated eye served as a control. The 

eyes were examined and the grade of ocular reaction recorded at 24, 48 and 72 hours for all animals 

using a scalar score (see attachment). The eyes were again examined with sodium fluorescein and an 

ultraviolet lamp at 72 hours after application of the test material to confirm the presence or absence of 

corneal lesion. 

Results and discussions 

Overall irritation / corrosion results 

Irritation parameter Basis Time point Score Max. score Reversibility Remarks 

cornea score mean average of 24, 48 and 72 0.11 4 fully reversible 

iris score mean 24, 48, 72 hr mean 0 2 other: no effects 

conjunctivae score mean 24, 48, 72 hr mean 1.1 3 fully reversible 

chemosis score mean 24, 48, 72 hours mean 0 4 other: no effects 

Irritant/corrosive response data 

In all animals there was some conjunctival reddening that did not persist to 7 days. One animal had a 

slight corneal opacity that did not persist to 7 days. This substance is not considered irritating to the eyes. 

97
Any other information on results incl. tables 

 Cornea Iris Conjuctiva red 

Animal 24 48 72mean 24 48 72mean 24 48 72mean

1 0 1 1 0.666667 0 0 0 0 2 1 0

2000 0 0 0 0 0210

3000 0 0 0 0 0211

4000 0 0 0 0 0220

5000 0 0 0 0 0210

6000 0 0 0 0 0210

Mean 0.111111 0

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

K-067479-004 scoring system.pdf / 76.66 KB (application/pdf) Scoring system 

Applicant's summary and conclusion 

Interpretation of results 

not irritating 

Criteria used for interpretation of results 

EU 

Conclusions 

The test material was not irritating to the eyes. 

Endpoint study record: 7327-60-8, Eye irritation, Dow, 1967, SS 

UUID IUC5-f5a7e59c-91dd-416d-8069-f9d97edcf56a 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 14:26:24 CET 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1967 

Reliability 2 (reliable with restrictions) 

Rationale for reliability incl. deficiencies Limited reported study, used as supporting evidence 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laboratory

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

The 

Dow 

Chemic

al 

Compan

y 

196

7 

Toxicological 

properties and 

industrial 

handling 

hazards of 

Nitrilotriacetonitri

le 

 Biochemic

al 

Research 

Laboratory

, Dow, 

Midland, 

Michigan, 

USA 

12 

4198 

The Dow 

Chemica

l 

Compan

y 

 1967-

11-28 

98
Data access 

data submitter has Letter of Access 

Materials and methods 

Type of method 

in vivo 

Test guideline 

Qualifier Guideline Deviations 

no guideline followed 

Principles of method if other than guideline 

No guideline available at that time 

GLP compliance 

no 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test materials 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Test animals 

Species 

rabbit 

Strain 

no data 

Test system 

Vehicle 

unchanged (no vehicle) 

Duration of treatment / exposure 

No information 

Observation period 

No information 

Number of animals 

No information 

Control animals 

no 

Results and discussions 

Overall irritation / corrosion results 

Irritation 

parameter 

Basis Time point Score Max. 

score 

Reversibility Remarks 

overall irritation 

score 

other: qualitative 

judgement 

not 

indicated 

 no irritation 

observed 

Any other information on results incl. tables 

The undiluted NTAN applied with washed and unwashed eyes did not show eye irritation. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

99
7327-60-8, overview, Dow, 1967, ref.docx / 36.67 KB (application/vnd.openxmlformatsofficedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

not irritating 

Criteria used for interpretation of results 

expert judgment 

Conclusions 

No irritations was observed following application of the undiluted NTAN. 

Endpoint study record: 7372-60-8, Eye irritation, Younger, 1965, SS 

UUID IUC5-e2574f5d-f79b-4a93-b001-9c9d48658386 

Dossier UUID 0 

Author LondenK / Akzo Nobel T&E / Arnhem / Netherlands

Date 2011-07-14 23:10:06 CEST 

Remarks 

 

Administrative Data 

Purpose flag supporting study 

Study result type experimental result Study period 1965 

Reliability 4 (not assignable) 

Rationale for reliability 

incl. deficiencies 

Very limited report only summarizing the results. The studies were 

submitted to U.S. EPA under TSCA section 8(e). 

Data source 

Reference 

Referen

ce type 

Author Yea

r 

Title Bibliograp

hic source 

Testing 

laborator

y 

Repo

rt no. 

Owner 

compa

ny 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Young

er, 

F.M. 

196

5 

Toxicological 

Investigation Of: 

NITRILOTRIACETONIT

RILE (NTAN) 

 Younger 

Laboratori

es 

Y-65-

179 

Monsan

to 

 1965-

12-02 

Applicant's summary and conclusion 

Executive summary 

Onehundred (100.0) milligrams of finely ground sample were placed in the conjunctival sac of the right 

eye of each of three albino rabbits. observations made over a period of several days for inflamation. The 

eyes were rinsed with warm isotonic aline solution after twenty-four hours. 

The compound was classed as a slight eye irritant. The average maximum score as 8.3 out of a possible 

110 in one hour. 

7.4 Sensitisation 

Endpoint summary: Sensitisation 

UUID IUC5-076a3341-27a1-454e-bc38-d69885f047f0 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-11 16:30:57 CET 

Remarks 

 

100
Administrative Data 

Skin sensitisation 

Short description of key information 

The OECD toolbox was used to establish skin sensitising properties (QSAR). 

Key value for chemical safety assessment 

Skin sensitisation 

not sensitising 

Discussion 

Based on the chemical structure of NTAN it is very unlikely that NTAN has skin sensitising properties.

Respiratory sensitisation 

Short description of key information 

No information available. 

Justification for classification or non-classification 

Based on the QSAR analysis, NTAN does not need classification for skin sensitisation. 

7.4.1 Skin sensitisation 

Endpoint study record: 7327-60-8, Skin sensitisation, Anonymous, 2010, RS 

UUID IUC5-135e1783-2383-4c6c-ad07-6236af9b11d1 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 14:31:55 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type (Q)SAR Study period March 2010 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Generally recogized OECD toolbox was used to establish skin 

sensitising properties 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

other 

company 

data 

Anonymou

s 

201

0 

OECD 

Toolbox 

applicatio

n study 

history - 

CAS # 

7327-60-8 

 Akzo 

Nobel 

Functional 

Chemicals 

BV 

 Akzo 

Nobel 

Functiona

l 

Chemical

s BV 

 2010-

03-26 

Data access 

other: OECD toolbox is publicly available 

Materials and methods 

Type of method 

other: QSAR 

Type of study 

101
other: OECD toolbox 

Test guideline 

Qualifier Guideline Deviations 

no guideline followed 

Principles of method if other than guideline 

QSAR analysis was done using the OECD toolbox. 

GLP compliance 

no 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Test animals 

Species 

other: QSAR 

Strain 

other: QSAR 

Sex 

no data 

Test system 

Traditional sensitisation test 

Route of induction exposure 

other: QSAR 

Route of challenge exposure 

other: QSAR 

Vehicle 

other: QSAR 

Concentration 

Not applicable 

No. of animals per dose 

Not applicable 

Results and discussion 

Traditional sensitisation test 

Results of test (except LLNA) 

Reading Hours after 

challenge 

Group Dose level No. with + 

reactions 

Total no. 

in group 

Clinical observations 

other: 

QSAR 

0 other: 

QSAR 

not 

applicable 

0 0 based on chemical structure, 

skin sensitisation is not very 

likely 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, NTAN-OECDToolbox-SkinSensitisation, ref.docx / 37.61 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

102
Applicant's summary and conclusion 

Interpretation of results 

not sensitising 

Conclusions 

Based on QSAR, it is very unlikely that NTAN has skin sensitising properties. 

7.5 Repeated dose toxicity 

Endpoint summary: Repeated dose toxicity 

UUID IUC5-7c599930-7d66-48e8-a38a-61f02b571006 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands 

Date 2011-03-30 11:57:52 CEST 

Remarks 

 

Administrative Data 

Short description of key information 

A combined 28-day oral repeated dose study and reproductive/development study in rats was available. 

The dose levels for this study were based on a 14-day range-finding study. 

Key value for chemical safety assessment 

Repeated dose toxicity: oral 

Effect 

level 

NOAEL 10 mg/kg bw/day 

Test 

type 

subacute 

Species rat 

Discussion 

Oral administration of NTAN to Sprague-Dawley rats at doses of 1, 10 and 45 mg/kg/day resulted in overt 

signs of parental toxicity at the 45 mg/kg/day dose including death, clinical signs of toxicity (including 

convulsions, labored breathing, etc.), decreased food consumption, body weight, body weight gain, and 

motor activity changes. It was noted that the clinical signs of toxicity occurred in females only (6/15) and 

the change in motor activity was only exhibited in males. It is difficult to discern whether the clinical signs 

of toxicity and change in motor activity are indicative of a neurotoxic effect as they occurred at a dose 

level which produced overt systemic toxicity (death). Interpretation of these types of indicators “as 

neurotoxic effects is dependent on the dose at which such changes occur and the possibility that damage 

to other organ systems may contribute to or cause such changes indirectly”. 

Treatment-related gross necropsy findings were low/singular in incidence, limited to the 45 mg/kg group 

and consisted of red fluid/blood in the cranial cavity/cranium (observed in four animals that died 

spontaneously during the study), pigmentation of the brain, thymus and liver, urinary bladder dilatation 

and an enlarged thymus. No treatment-related histopathological changes were observed in the tissues 

examined from rats in the 45 mg/kg group; all findings were considered spontaneous or incidental 

changes commonly observed in rats of this age and strain. Based on the results of this study, the noobserved-adverse-effect level (NOAEL) for systemic toxicity of NTAN was 10 mg/kg/day. the NOAEL in 

the 14 -day range-finding study was 25 mg/kg bw/day. 

Justification for classification or non-classification 

Based on the severe toxicity observed at 45 mg/kg bw (death and severe clinical signs of toxicity), NTAN 

should be classified with R48/22 and STOT repeated exposure Cat. 2. 

7.5.1 Repeated dose toxicity: oral 

Endpoint study record: 7327-60-8, Repeated dose toxicity: oral, 14-day, Doyle, 2010, SS 

UUID IUC5-f1f5dd5e-85ea-416a-91a8-bfacfde25380 

Dossier UUID 0 

103
Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 14:56:19 CET 

Remarks 

 

Administrative Data 

 

Purpose flag supporting study 

Study result type experimental result Study period July 2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laborator

y 

Repor

t no. 

Owner 

compan

y 

Compan

y study 

no. 

Repor

t date 

study 

report 

Doyle 

A 

201

0 

14-Day rangefinding oral 

(gavage) toxicity 

study of 

Nitrilotriacetonitril

e (NTAN) in rats 

 Experimur

, Chicago, 

IL, USA 

09-

570 

Akzo 

Nobel 

Function

al 

Chemical

s LLCChelates 

 2010-

07-13 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Test type 

subacute 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

no guideline followed 

Principles of method if other than guideline 

range-finding test, primarily according to OECD 407 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no.: CFC 9161Expiry date: 02 June 2012Purity: 99.6 +/- 1.5%Water: 0.4 +/- 0.2% 

104
Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Analytical verification of doses or concentrations 

yes 

Duration of treatment / exposure 

14 days 

Frequency of treatment 

daily 

Doses/concentrations 

1, 5, 25, 50 mg/kg bw 

Basis actual ingested 

No. of animals per sex per dose 

5 

Control animals 

yes, concurrent vehicle 

Examinations 

Any other information on materials and methods incl. tables 

clinical signs 

body weight 

food intake 

gross necropsy 

Results and discussions 

Effect levels 

Endpoint Effect level Based 

on 

Sex Basis for effect level / Remarks 

NOAEL 25 mg/kg bw/day (actual dose 

received) 

test mat. male/female clinical signs and death in one male at 

50 mg/kg bw 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, repeated dose toxicity, 14-day, Doyle, 2010, ref.docx / 37.83 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

Based on the results of this study, the no-observed- adverse-effect level (NOAEL) for oral administration 

of NTAN to Sprague- Dawley rats for 14 consecutive days was 25 mg/kg/day. 

105
Executive summary 

The objective of this study was to evaluate the potential toxicity of Nitrilotriacetonitrile (NTAN) following 

repeated daily oral administration for a period of 14 days and to select dose levels for a longer term study. 

NTAN was administered by oral gavage as a suspension in a vehicle of corn oil to Sprague-Dawley rats 

at dose levels of 0 (vehicle only), 1, 5, 25 and 50 mg/kg/day for 14 consecutive days. Each group 

consisted of 5 rats/sex and were dosed once daily, 7 days/week, for 14 days at a dosing volume of 2 

ml/kg. Standard toxicological evaluations included daily clinical observations, weekly body weights and 

food consumption, and gross evaluations at necropsy. Slight decreases in food consumption, body weight 

and body weight gain were noted during the first week of treatment in animals receiving NTAN; however, 

the alterations were transient in nature and generally resolved by the end of the study. Treatment-related 

clinical signs of toxicity included salivation and red discoloration around the mouth and were noted for one 

male in the 50 mg/kg group that died spontaneously on day 13 of the study. Treatment-related gross 

necropsy findings were also noted for this animal and included red fluid in the cranial cavity. All other 

clinical signs and gross necropsy observations were considered unrelated to treatment with NTAN. Based 

on the results of this study, the no-observed- adverse-effect level (NOAEL) for oral administration of 

NTAN to Sprague- Dawley rats for 14 consecutive days was 25 mg/kg/day. 

Endpoint study record: 7327-60-8, Repeated dose toxicity: oral, 28-day, Doyle, 2010, RS 

UUID IUC5-4f3b4c37-b4e3-4ef6-a845-ccf32fe51e0b 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 15:01:19 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental 

result 

Study 

period 

September-November 

2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Well conducted and reported study 

Data source 

Reference 

Referen

ce type 

Auth

or 

Yea

r 

Title Bibliograp

hic source 

Testing 

laborato

ry 

Repo

rt no. 

Owner 

compan

y 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Doyle 

A 

201

0 

Combined oral 

repeated dose and 

reproductive/developm

ental toxicity screening 

test of 

Nitrilotriacetonitrile 

(NTAN) in rats 

 Experim

ur, 

Chicago, 

IL, USA 

09-

574 

Akzo 

Nobel 

Function

al 

Chemica

ls LLCChelates 

 2010-

10-19 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

106
Also used for developmental/reproduction toxicity 

Materials and methods 

Test type 

subacute 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 

Reproduction / Developmental Toxicity Screening Test) 

 

Principles of method if other than guideline 

range-finding test, primarily according to OECD 407 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no.: CFC 9161Expiry date: 02 June 2012Purity: 99.6 +/- 1.5%Water: 0.4 +/- 0.2% 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Details on test animals and environmental conditions 

See further 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on oral exposure 

Dose formulations of the test material were prepared approximately every 2 weeks as suspensions in a 

vehicle of corn oil at graded concentrations of 0.5, 5.0 and 22.5 mg/ml, which corresponded to target 

doses of 1, 10 and 45 mg/kg/day, when administered at a dosing volume of 2 ml/kg. Dose levels of the 

test material were selected by the Sponsor based on the results of a 14-day range-finding study 

(Experimur Study No.: 09-570). Test material formulations of NTAN were previously shown to be stable 

for a minimum of 4 weeks when stored at room temperature. Test material formulations were stored at 

room temperature (approximately 22 ± 3 degrees C), and thoroughly mixed on a stir plate prior to and 

during dosing. 

107
Analytical verification of doses or concentrations 

yes 

Details on analytical verification of doses or concentrations 

Samples of the test material formulations were collected from the first preparation (i.e., prior to dosing, 

week 0), mid-way through the study (approximately week 3), and towards the end of the study (week 4), 

and analyzed for concentration. Samples for homogeneity (homogeneity is performed on duplicate 

samples collected from the top, middle and bottom of the test material formulation) were collected from 

the low and high dose formulations of the 1st preparation and analyzed concurrently with the first 

concentration analyses. A validated gas chromatography (GC) with flame ionization detection (FID) 

method was used to determine concentration and homogeneity of the NTAN dose formulations. The 

analytically-determined concentrations met the acceptance criterion of ± 15% of the target concentration 

as specified in the study protocol. Samples for homogeneity were collected from the low and high dose 

formulations at study initiation and were found to be within the protocol-specified acceptance criteria of ± 

15%. 

Duration of treatment / exposure 

2 weeks prior to mating, during mating, and until sacrifice (males: 31 doses; females: a minimum of 48 

doses) 

Frequency of treatment 

daily 

Doses/concentrations 

1, 10, 45 mg/kg bw 

Basis actual ingested 

No. of animals per sex per dose 

10 rats/sex main study, 5 additional rats/sex in the control and high dose groups (recovery animals kept 

for 14 days after the last treatment; these animals were not mated) 

Control animals 

yes, concurrent vehicle 

Details on study design 

See below 

Positive control 

Not used 

Examinations 

Observations and examinations performed and frequency 

CAGE SIDE OBSERVATIONS: Yes- Time schedule: dailyDETAILED CLINICAL OBSERVATIONS: YesTime schedule: weeklyBODY WEIGHT: Yes - Time schedule for examinations: weeklyFOOD 

CONSUMPTION: Yes, weekly FOOD EFFICIENCY: NoWATER CONSUMPTION AND COMPOUND 

INTAKE (if drinking water study): NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes - 

Time schedule for collection of blood: prior to necropsy- Anaesthetic used for blood collection: sodium 

pentobarbital- Animals fasted: Yes- How many animals: 5 rats/sex/group (main and recovery)CLINICAL 

CHEMISTRY: Yes - Time schedule for collection of blood: prior to necropsy- Animals fasted: Yes- How 

many animals: 5 rats/sex/group (main and recovery)URINALYSIS: NoNEUROBEHAVIOURAL 

EXAMINATION: Yes - Time schedule for examinations: males after 29 doses, females after post-partum 

Day 4- Dose groups that were examined:- Battery of functions tested: sensory activity / grip strength / 

motor activityOTHER: see at developmental/reproduction toxicity 

Sacrifice and pathology 

GROSS PATHOLOGY: Yes HISTOPATHOLOGY: Yes 

Statistics 

See below 

Any other information on materials and methods incl. tables 

108
Data were manually collected, entered into Excel spreadsheets, and means and standard deviations were 

calculated using the same software. Data were analyzed for homogeneity of variance using Levene's test. 

When the variances were homogeneous (p > 0.001), the data were further analyzed by one-way analysis 

of variance (ANOVA). When a significant F value was observed (p<=0.05) each treatment group was 

compared to the vehicle control group using Dunnett’s two-tailed t-test. Statistical significance was 

declared at p<=0.05 for Dunnett’s test. 

When Levene’s test was significant (p<=0.001), an appropriate transformation was applied to the data 

(e.g., log-transformation), and the analyses were performed on the transformed data. When the variancestabilizing transformation was not achieved, Kruskal-Wallis test was performed followed 

by Mann-Whitney U (MWU) statistic. Minimal statistical significance was declared at p<=0.05). 

Total motor activity data were analyzed using ANOVA, while interval motor activity data were analyzed 

using repeated measures analysis of variance. 

All analyses were performed with Systat 10. Minor differences during recalculation of results, as 

presented in this report, may be noted due to digital 

storage of numbers in computers or calculators as well as software calculating or rounding issues. 

Results and discussions 

Effect levels 

Endpoint Effect level Based 

on 

Sex Basis for effect level / Remarks 

NOAEL 10 mg/kg bw/day 

(actual dose received) 

test 

mat. 

male/female death, clinical signs, decreased food intake and 

BW, and motor activity changes at 45 mg/kg bw 

Results of examinations 

Clinical signs and mortality 

yes 

Body weight and weight gain 

yes 

Food consumption and compound intake (if feeding study) 

yes 

Food efficiency 

not examined 

Water consumption and compound intake (if drinking water study) 

not examined 

Ophthalmoscopic examination 

not examined 

Haematology 

no effects 

Clinical chemistry 

no effects 

Urinalysis 

not examined 

Neurobehaviour 

yes 

Organ weights 

no effects 

Gross pathology 

yes 

Histopathology: non-neoplastic 

no effects 

109
Histopathology: neoplastic 

no effects 

Details on results 

CLINICAL SIGNS AND MORTALITY: one male and three females were found dead. Clinical signs 

consisted of discoloration around the mouth and nose, discolored/wet inguinal fur, convulsions, labored 

breathing, irritability, head weaving, loss of righting reflex, repetitive behavior, cyanosis, salivation, 

hypoactivity, discolored paws and rough hair coat. However, these signs were primarily on a single but 

different day of the study, which is suggestive of the acute nature of this observed toxicity.BODY WEIGHT 

AND WEIGHT GAIN: reduced at 45 mg/kg bwFOOD CONSUMPTION: reduced at 45 mg/kg bwFOOD 

EFFICIENCY: not measuredWATER CONSUMPTION: not measured OPHTHALMOSCOPIC 

EXAMINATION: not measuredHAEMATOLOGY: no treatment-related changesCLINICAL CHEMISTRY: 

no treatment-related changesURINALYSIS: not measuredNEUROBEHAVIOUR: No treatment-related 

differences in grip strength or F.O.B. parameters were noted across treatment groups for core or recovery 

animals. Statistically significant differences in motor activity assessments were observed for males in the 

45 mg/kg group, compared to controls, and included decreased totals for move episodes, stereotypy 1 

moves and episodes, and stereotypy 2 moves and time, suggestive of a change in pattern of behavior for 

these animals.The mean values for these parameters generally did not fall within their respective 

concurrent control ranges and were likely treatment-related. However, this change was only present at 

the dose level that produced overt systemic toxicity (death). Following the recovery period, total move 

episodes remained significantly decreased for males of the 45 mg/kg group, compared to controls, 

indicating that effects on total move episodes were not reversible within the 14-day period following 

cessation of dosing. No significant differences in motor activity parameters were noted for females across 

treatment groups for core or recovery animals.ORGAN WEIGHTS: no treatment-related changesGROSS 

PATHOLOGY: limited to the 45 mg/kg group; consisted of red fluid/blood in the cranial cavity/cranium 

(observed in four animals that died spontaneously during the study), pigmentation of the brain, thymus 

and liver, urinary bladder dilatation and an enlarged thymus. Pigmentation of the thymus was also 

observed in one male of the 45 mg/kg group animal following the recovery period; whereas, one female in 

the 45 mg/kg group was observed with this finding at the end of the treatment 

period.HISTOPATHOLOGY: NON-NEOPLASTIC, no treatment-related changes 

Overall remarks, attachments 

Overall remarks 

It should be noted that the clinical signs of toxicity occurred in females only(6/15) and the change in motor 

activity was only exhibited in males. It is difficult to discern whether the clinical signs of toxicity and 

change in motor activity are indicative of a neurotoxic effect as they occurred at a dose level which 

produced overt systemic toxicity (death). Interpretation of these types of indicators “as neurotoxic effects 

is dependent on the dose at which such changes occur and the possibility that damage to other organ 

systems may contribute to or cause such changes indirectly” (Guidelines for Neurotoxicity Risk 

Assessment, EPA/630/R-95/001F, April 1998, page 20). Treatment-related gross necropsy findings were 

low/singular in incidence, limited to the 45 mg/kg group and consisted of red fluid/blood in the cranial 

cavity/cranium (observed in four animals that died spontaneously during the study), pigmentation of the 

brain, thymus and liver, urinary bladder dilatation and an enlarged thymus. No treatment-related 

histopathological 

changes were observed in the tissues examined from rats in the 45 mg/kg group; all findings were 

considered spontaneous or incidental changes commonly observed in rats of this age and strain. 

Attached background material 

Attached document Remarks 

7327-60-8, repeated dose toxicity, 28-day, Doyle, 2010, ref.docx / 37.81 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Conclusions 

Based on the results of this study, the no-observed- adverse-effect level (NOAEL) for parental systemic 

toxicity following oral administration of NTAN to Sprague-Dawley rats for 31 days (males) or at least 48 

days (females) was 10 mg/kg/day. 

110
Executive summary 

Nitrilotriacetonitrile (NTAN) was administered to male and female Sprague-Dawley rats by oral gavage at 

concentrations of 0.5, 5.0 or 22.5 mg/ml equivalent to doses of 1, 10 or 45 mg/kg/day, when administered 

at a constant dosing volume of 2 ml/kg. The vehicle control group received vehicle (corn oil) only. Ten 

rats/sex/group were selected for cohabitation (i.e., core/parental animals) and 5 additional rats/sex in the 

control and high dose groups were designated as recovery animals in order to evaluate reversibility of 

any NTAN-induced effects. The parental rats were administered NTAN for 2 weeks prior to mating, during 

the mating period, and until sacrifice (males: 31 doses; females: a minimum of 48 doses). 

Recovery animals were treated for the same duration as the parental rats, but were not mated, and were 

held for 14 days after treatment to evaluate recovery from any adverse treatment-related effects. Due to 

the spontaneous deaths of 2 high dose recovery females, only 3 unmated recovery females remained. In 

order to increase the size of the female high dose recovery group, one core parental high dose dam was 

held without treatment for approximately 14 days and was necropsied with the recovery animals. By doing 

this, there was one additional high dose female (n = 4 instead of n = 3) to evaluate for recovery potential 

from treatment-related effects. 

Body weights, body weight gain and food consumption were measured during the study. In addition, 

clinical observations, a functional observational battery with automated motor activity, organ weights, and 

clinical and diagnostic pathology were evaluated. 

Four rats in the 45 mg/kg/day group died spontaneously during the study; male #536 was found dead on 

study day 19 (during treatment/ cohabitation), female #592 was found dead on day 3 (during 

treatment/pre-cohabitation), female #599 (designated as a recovery animal) was 

found dead on day 30 (during the treatment period), and female #586 was found dead on lactation day 3. 

All four deaths were considered to be treatment-related, although microscopically the cause of death for 

these animals was undetermined. Treatment-related clinical signs of toxicity were limited to animals in the 

45 mg/kg group (6/15 females) and were often noted within a few hours following dose administration. 

These clinical signs consisted of discoloration around the mouth and nose, discolored/wet inguinal fur, 

convulsions, labored breathing, irritability, head weaving, loss of righting reflex, repetitive behavior, 

cyanosis, salivation, hypoactivity, discolored paws and rough hair coat. However, these signs were 

primarily on a single but different day of the study, which is suggestive of the acute nature of this 

observed toxicity. Treatment-related decreases in mean food consumption were observed for male and 

female rats in the 45 mg/kg group during the study. These reductions in food consumption began over 

study days 0-4 for both sexes and continued for the remainder of the treatment period for females. 

Corollary reductions in body weight were also observed: mean body weight and/or body weight gain were 

reduced compared to concurrent control values in both sexes in the 45 

mg/kg group beginning over study days 1-4 and these changes generally persisted for males and females 

throughout the treatment phase of the study. No treatment-related changes in clinical chemistry or 

hematology profiles were observed in animals treated with NTAN. 

No treatment-related differences in grip strength or F.O.B. parameters were noted across treatment 

groups for core or recovery animals, or in motor activity parameters for females. Statistically significant 

differences in motor activity assessments were observed for males in the 45 mg/kg group, compared to 

controls, and included decreased totals for move episodes, stereotypy 1 moves and episodes, and 

stereotypy 2 moves and time, suggestive of a change in pattern of behavior for these animals. Following 

the recovery period, total move episodes remained significantly decreased for males of the 45 mg/kg 

group, compared to controls. These changes in motor activity were likely treatment-related but were only 

present at the dose level that produced overt systemic toxicity (death). 

Absolute and relative organ weights were not considered to be affected by treatment with NTAN. 

Treatment-related gross necropsy findings were low/singular in incidence, limited to the 45 mg/kg group 

and consisted of red fluid/blood in the cranial cavity/cranium (observed in four animals that died 

spontaneously during the study), pigmentation of the brain, thymus and liver, urinary bladder dilatation 

and an enlarged thymus. No treatment-related histopathological changes were observed in the tissues 

examined from rats in the 45 mg/kg group; all findings were considered spontaneous or incidental 

changes commonly observed in rats of this age and strain. 

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for parental systemic 

toxicity of NTAN was 10 mg/kg/day. 

111
7.6 Genetic toxicity 

Endpoint summary: Genetic toxicity 

UUID IUC5-2be2f523-0fe5-4bcb-9e5c-f86d6da8cf38 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-11 16:31:55 CET 

Remarks 

 

Administrative Data 

Short description of key information 

Two in vitro genotoxicity studies were available, an Ames test and a Chromosome Aberration test in CHO 

cells. 

Key value for chemical safety assessment 

Genetic toxicity 

negative 

Discussion 

Both in vitro genotoxicity studies, viz. the Ames test and the chromosome aberration test showed that 

NTAN was not mutagenic. 

Justification for classification or non-classification 

Because both the Ames test and the chromosome aberration test showed that NTAN was not mutagenic, 

no classification for genotoxicity is needed. 

7.6.1 Genetic toxicity in vitro 

Endpoint study record: 7327-60-8, Genetic toxicity in vitro, Ames, Stankowski 2009, RS 

UUID IUC5-eedd3cf8-1939-475b-bdc3-ae1d64899cc0 

Dossier UUID 0 

Author LondenK / Akzo Nobel N.V. / Arnhem / Netherlands

Date 2012-01-30 09:56:53 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period May-August 2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study, according to GLP 

Data source 

Reference 

Referenc

e type 

Author Yea

r 

Title Bibliographi

c source 

Testing 

laborator

y 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Stankows

ki LF 

200

9 

Bacterial 

reverse 

mutation 

assay with 

a 

confirmator

y assay; 

NTAN 

 Covance 

Lab. Inc., 

Vienna, 

Virgina 

22182-

1699, 

USA 

821150

6 

Akzo 

Nobel 

Functiona

l 

Chemical

s LLCChelates, 

Chicago, 

 2009-

08-11 

112
Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Type of genotoxicity 

gene mutation 

Type of study 

bacterial reverse mutation assay (e.g. Ames test) 

Test guideline 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Details on test material 

Appearance: off-white crystalline powderPurity: 99.6 +/- 1.5% Other:- water: 0.4 +/- 0.2% 

Method

Target gene 

reverse mutations at the histidine locus and tryptophan locus 

Species/strain 

Species/strain S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

Aroclor 1254-induced rat liver homogenate 

Species/strain E. coli WP2 uvr A 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

Aroclor 1254-induced rat liver homogenate 

Test concentrations 

In initital test: 1.60, 5.00, 16.0, 50.0, 160, 500, 1600, 5000 μg/plate with and without S9-mixIn 

confirmatory test: 50.0, 160, 500, 1600, 5000 μg/plate with and without S9-mix 

Vehicle

113
DMSO, purity 99.94% 

Controls 

 

Solvent / 

vehicle 

controls 

yes (DMSO) 

Positive 

controls 

yes 

Positive 

control 

substance 

other: 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide 

Remarks without S9-mix 

 

Solvent / 

vehicle 

controls 

yes (DMSO) 

Positive 

controls 

no (benzo(a)pyrene, 2-aminoanthracene) 

Remarks with S9-mix 

Details on test system and conditions 

Plating Procedures: Each plate was labeled with a code that identified the test article, test phase, tester 

strain, activation condition and dose level. The S9 mix and dilutions of the test article were prepared 

immediately prior to their use. Treatments were performed by adding 100 μL tester strain and 50 μL test 

or controlarticle to 2.5 mL molten selective top agar (maintained at 45 ± 2°C). After the required 

components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal 

bottom agar in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and 

incubated for 52 ± 4 hours at 37 ± 2°C. Cultures were treated in the presence of S9 in an identical 

manner, except using 2.0 niL undiluted molten selective top agar and adding 500 μL S9 mix.Scoring the 

Plates: Plates which were not evaluated immediately following the incubation period were held at >0 to 

10°C until such time that colony counting and bacterial background lawn evaluation could take 

place.Bacterial Background Lawn Evaluation: The condition of the bacterial background lawn was 

evaluated macroscopically and microscopically (using a dissecting microscope) for indications of 

cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control 

plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were 

scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), 

absent (A), or enhanced (E); contaminated plates (C) were also noted.Counting Revertant Colonies: 

Revertant colonies were counted by automated colony counter and by hand. 

Evaluation criteria 

See below at remarks 

Statistics 

Not done 

Any other information on materials and methods incl. tables 

Assay Acceptance Criteria: Before assay data were evaluated, the criteria for a valid assay had to be 

met. The following criteria were used to determine a valid assay: 

Tester Strain Integrity 

- Histidine/Tryptophan Auxotrophy (his-/trp- Mutation): Salmonella tester strains must exhibit the presence 

of the his mutation, as demonstrated 

by the ability to grow on minimal bottom agar plates supplemented with histidine/biotin (but not 

tryptophan/biotin). Tester strain WP2uvrA must exhibit the presence of the trp mutation, as demonstrated 

114
by the ability to grow on bottom agar plates supplemented with tryptophan/biotin (but not histidine/biotin). 

- rfa Cell Wall Mutation: Salmonella tester strains must exhibit the presence of the rfa mutation, as 

demonstrated by sensitivity to crystal violet. Tester strain WP2uvrA must exhibit resistance to crystal 

violet. 

- pKM101 Plasmid: Tester strains TA98 and TA100 must exhibit the presence of the pKM101 plasmid by 

demonstrating resistance to ampicillin. In contrast, tester strains TA1535, TA1537, and WP2uvrA must be 

sensitive to ampicillin. 

- uvrA and uvrB Mutation: All tester strains must exhibit the presence of the uvrA or uvrB mutations, as 

demonstrated by their sensitivity to ultraviolet light. 

Characteristic Number of Spontaneous Revertants 

The average revertants/plate of the vehicle control cultures must be within the following acceptance limits 

which are based upon historical data and published reports. 

TA98: 8 - 60 

TAI00: 60 - 240 

TA1535: 4 - 45 

TA1537: 2 - 25 

WP2uvrA: 5 - 40 

Tester Strain Culture Density 

Each culture must reach a target 0D (650(1:4)) = 0.4 to 0.6, which has been demonstrated to be 

representative of cultures in the late exponential or early stationary phase with ≥l0E9 cells/mL. 

Positive Control Values 

The positive controls must induce a ≥3-fold increase in revertants/plate as compared to the concurrent 

vehicle controls. A 3-fold increase in the absence of S9 indicates that the tester strain can identify a 

mutagen, while a 3-fold increase in the presence of S9 will be considered to have demonstrated the 

integrity of the S9 mix as well as the ability of the tester strain to detect a mutagen. 

Number of Dose Levels 

A minimum of three non-toxic doses is required to evaluate assay data. 

Maximum Dose Level 

The highest dose evaluated was the limit dose of 5000 μg/plate. 

Assay Evaluation Criteria 

Once the criteria for a valid assay had been met, responses observed in the assay were evaluated. 

Criteria for a Positive Response 

A test article is considered to have produced a positive response if it induces a dose-dependent increase 

in revertant frequency that is ≥2.0-fold vehicle control values for tester strains TA98, TA100, and 

WP2uvrA, or ≥3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any 

response should be reproducible. 

Criteriafor a Negative Response 

A test article is considered to have produced a negative response if no dose-dependent, ≥2.0-fold or ≥3.0-

fold increases are observed in tester strains TA98, TA 100, and WP2uvrA, or TA1535 and TA1537, 

respectively. 

Criteriafor an Equivocal Response 

Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly 

negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are 

reproducible). In those rare instances, the test article may be considered to have produced an equivocal 

response. Other criteria also may be used in reaching a conclusion about the study results (e.g., 

comparison to historical control values, biological significance, etc.). In such cases, the Study Director will 

use sound scientific judgment and clearly report and describe any 

such considerations. 

Results and discussions 

Test results 

Species/strain S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 

Metabolic 

activation 

with and without 

Test system all strains/cell types tested 

115
Genotoxicity negative 

Cytotoxicity no, but tested up to limit concentrations 

Vehicle 

controls valid 

yes 

Positive 

controls valid 

yes 

Species/strain E. coli WP2 uvr A 

Metabolic 

activation 

with and without 

Genotoxicity negative 

Cytotoxicity no, but tested up to limit concentrations 

Vehicle 

controls valid 

yes 

Positive 

controls valid 

yes 

Additional information on results 

NTAN was found to form a transparent, non-viscous, colorless solution in DMSO at a concentration of 

approximately 100 mg/mL, which was the highest doseformulation prepared for treatment; it remained 

freely soluble at all succeeding lower dilutions prepared assay. 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, Genetic toxicity in vitro, Ames, Stankowski 2009, ref.docx / 38.52 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

negative 

Conclusions 

Under the conditions of this test, NTAN did not show mutagenic effects. 

Executive summary 

The objective of this study was to evaluate the test article, NTAN (nitrilotriacetonitrile;CAS No.: 7327-60-

8), and/or its metabolites for their ability to induce reverse mutations at the histidine locus in several 

strains of Salmonella (TA98, TAI00, TA1535, and TA1537), and at the tryplophan locus of E. coli strain 

WP2uvrA, in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

NTAN was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.60, 5.00, 

16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9. All doses of the test article, as well as 

the concurrent positive and vehicle controls were evaluated in duplicate plates. Normal growth was 

observed in all five tester strains, and the test article was found to be freely soluble in the aqueous top 

agar, at all doses evaluated with and without S9. Revertant frequencies for all doses ofNTAN, in all tester 

strains with and without S9, approximated those observed in the concurrent vehicle control cultures. 

NTAN was re-evaluated in the confirmatory mutagenicity assay at doses of 50.0, 160, 500, 1600, and 

5000 μg/plate with and without 89. All doses of the test article, as well as the concurrent positive and 

vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester 

strains, and the test article again was found to be freely soluble, at all doses evaluated with and without 

S9. Revertant frequencies for all doses of NTAN, in all tester strains with and without 89, again 

approximated control values. All positive and vehicle control values were within acceptable ranges, and 

all criteria for a valid study were met. 

These results indicate NTAN was negative in the Bacterial Reverse Mutation Assay with a Confirmatory 

116
Assay under the conditions, and according to the criteria, of the test protocol. 

Endpoint study record: 7327-60-8, Genetic toxicity in vitro, CAT, Murli, 2009, RS 

UUID IUC5-10407bb7-94ed-476d-b431-393e13bcf509 

Dossier UUID 0 

Author GeltenP / Akzo Nobel T&E / Arnhem / Netherlands

Date 2010-12-21 15:10:50 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental result Study period May-August 2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. deficiencies Well conducted and reported study according to 

GLP 

Data source 

Reference 

Referenc

e type 

Autho

r 

Yea

r 

Title Bibliographi

c source 

Testing 

laboratory 

Report 

no. 

Owner 

company

Compan

y study 

no. 

Repor

t date 

study 

report 

Murli 

H 

200

9 

Chromosom

al 

aberrations 

in Chinese 

hamster 

ovary (CHO) 

cells - NTAN 

 Covance 

Laboratorie

s Inc., 

Vienna, 

Virginia, 

USA 

821150

5 

Akzo 

Nobel 

Functiona

l 

Chemical

s LLCChelates 

 2009-

11-02 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Materials and methods 

Type of genotoxicity 

chromosome aberration 

Type of study 

in vitro mammalian chromosome aberration test 

Test guideline 

Qualifier Guideline Deviations 

according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

117
CAS number 7327-60-8 

Details on test material 

Batch no.: CFC 9161Expiry date: 2 June 2010Purity: 99.6 +/- 1.5%Water: 0.4 +/- 0.2% 

Method 

Target gene 

Chromosome aberration 

Species/strain 

Species/strain Chinese hamster Ovary (CHO) 

Details on 

mammalian 

cell lines (if 

applicable)

permament cell line (University of California, San Francisco, USA) 

Additional 

strain 

characteristics 

not applicable 

Metabolic 

activation 

with and without 

Metabolic 

activation 

system 

Aroclor 1254-induced rat liver (S9-mix) 

Test concentrations 

Initital test: - concentrations tested: 18-2700 μg/mL- concentrations evaluated: 1320, 1890, 2700 μg/mL (-

S9 mix)- concentrations evaluated: 926, 1320, 1890 μg/mL (+ S9 mix)Confirmatory test: - concentrations 

tested: 61-2620 μg/mL- concentrations evaluated: 1940, 2280, 2620 μg/mL (-S9 mix)- concentrations 

evaluated: 1940, 2280, 2620 μg/mL (+ S9 mix) 

Vehicle 

DMSO 

Controls 

Negative 

controls 

no 

Solvent / 

vehicle 

controls 

yes 

True 

negative 

controls 

no 

Positive 

controls 

yes 

Positive 

control 

substance 

other: mitomycin (-S9 mix) cyclophosphamide (+S9 mix) 

Details on test system and conditions 

Initial test: 3 h exposure, harvest at ~20 h (both with and without S9 mix)Confirmatory test: 20 h exposure, 

harvest at ~20 h (without S9 mix); 3 h exposure, harvest at ~20 h (with S9 mix) 

Evaluation criteria 

See below 

Statistics 

118
See below 

Any other information on materials and methods incl. tables 

Data Evaluation 

Mitotic index was evaluated from the vehicle controls and a range of concentrations by analyzing the 

number of mitotic cells in 1000 cells, if possible, and the ratio expressed as a percentage of mitotic cells. 

When analysis for aberrations was performed, 100 cells, if possible, from each replicate culture from three 

concentrations of the test article, vehicle controls, and one dose of the positive control cultures were 

analyzed for the different types of chromosomal aberrations. At least 25 cells were analyzed from those 

cultures that had greater than 25% of cells with one or more aberrations. Cells were selected for good 

morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 (range 19-

23) were analyzed. Percent polyploidy and endoreduplication were also analyzed from the dose levels 

selected for analysis and controls by evaluating at least 100 metaphases, if possible, and tabulated. For 

control of bias, all slides were coded prior to analysis. Cells with aberrations were recorded on the data 

sheets by the microscope stage location. 

Data Presentation 

Data were summarized in tables showing mitotic index and percent cells with polyploidy and 

endoreduplication. For structural aberrations, the aberrant cells were presented in two categories: the 

number and percentages of aberrant cells excluding gaps (-g), and the number and percentages of 

aberrant cells including gaps (+g). 

Assay Acceptance Criteria 

An assay was considered acceptable for evaluation of test results only if all of the following criteria were 

satisfied. The metabolic activation and nonactivation sections of the aberrations assay were independent 

units and would be repeated independently, as needed, to satisfy the acceptance criteria. 

Acceptable Controls 

The vehicle control cultures must contain less than —5% cells with aberrations. The positive control result 

must be significantly higher (p<=0.01) than the vehicle controls. 

Acceptable High Dose 

If the aberration results are negative and there is no significant reduction (~>=50%) in mitotic index, the 

assay must include the highest applicable dose (a target dose of 10mM or 5 mg/mL, whichever is lower) 

or a dose exceeding the solubility limit in culture medium. 

Acceptable Number of Doses 

The assay must include at least three analyzable concentrations. 

Assay Evaluation Criteria 

After completion of microscopic analysis for aberrations, data were decoded. The following factors are 

taken into account in evaluation of the test article data: 

• The number and percentages of aberrant cells excluding gaps (-g). 

• The number and percentages of aberrant cells including gaps (+g). 

• Evidence of a dose-response relationship. 

The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the 

basis for evaluation. Statistical analysis employed a Cochran Armitage test for linear trend and Fisher’s 

Exact Test to compare the percentage of cells with aberrations in treated cells to the results obtained for 

the vehicle controls. Statistical analysis was also performed for cells exhibiting polyploidy and/or 

endoreduplication in order to detect significant (p ≤ 0.01) increases in these events as indicators of 

possible induction of numerical aberrations. 

Evaluation of a Positive Response 

A test article was considered positive for inducing chromosomal aberrations if a significant increase (the 

difference was considered significant when p≤0.01) in the number of cells with chromosomal aberrations 

is observed at one or more concentrations. The linear trend test evaluated the dose-responsiveness. A 

dose-response should be observed if a significant increase was seen at one or more concentrations. 

Evaluation of a Negative Response 

A test article was considered negative for inducing chromosomal aberrations if no significant increase 

was observed in the number of cells with chromosomal aberrations at any of the concentrations. 

Equivocal Evaluation 

Although most assays give clearly positive or negative results, in rare cases the data set would preclude 

making a defmitive judgment about the activity of the test article. Results might remain equivocal or 

119
questionable regardless of the number of times the assay is repeated. Although the evaluation criteria 

provided here is normally sufficient, the Study Director might use additional considerations to obtain a 

fmal evaluation of the test article based upon the Study Director’s scientific judgment. 

Results and discussions 

Test results 

Species/strain Chinese hamster Ovary (CHO) 

Metabolic 

activation 

without 

Genotoxicity negative 

Cytotoxicity yes at 2620 μg/mL (20 h treatment) 

Vehicle 

controls valid 

yes 

Negative 

controls valid 

not examined 

Positive 

controls valid 

yes 

Species/strain Chinese hamster Ovary (CHO) 

Metabolic 

activation 

with 

Genotoxicity negative 

Cytotoxicity yes (at >= 2280 μg/mL in confirmatory test) 

Vehicle 

controls valid 

yes 

Negative 

controls valid 

not examined 

Positive 

controls valid 

yes 

Overall remarks, attachments 

Attached background material 

Attached document Remarks 

7327-60-8, genetic toxicity in vitro, CAT, Murli, 2009, ref.docx / 38 KB 

(application/vnd.openxmlformats-officedocument.wordprocessingml.document) 

 

Applicant's summary and conclusion 

Interpretation of results 

negative 

Conclusions 

NTAN was considered negative for inducing chromosomal aberrations in CHO cells with and without 

metabolic activation. 

Executive summary 

The objective of this in vitro assay was to evaluate the ability of NTAN (Nitrilotriacetonitrile; CAS No.: 

7327-60-8) to induce chromosomal aberrations in 

cultured Chinese hamster ovary (CHO) cells without and with an exogenous metabolic activation system. 

Dimethylsulfoxide (DMSO) was the vehicle for the study. The highest concentration tested in the assay 

was 2700 μgImL, which was above the solubility limit of the formulated NTAN after dosing into culture 

medium. The stock formulations and their dilutions were dosed using a dosing volume of 1% (10.0 μL/mL) 

and the vehicle control cultures were treated with 10.0 μL/mL of DMSO. In the initial chromosomal 

120
aberrations assay, the treatment period was for 3 hours without and with metabolic activation and the 

cultures were harvested ~20 hours from the initiation of treatment. Concentrations of 18.3, 26.2, 37.4, 

53.4, 76.3, 109,156, 222, 318, 454, 648, 926, 1320, 1890, and 2700 μg/mL were tested without and with 

metabolic 

activation. Cultures treated with concentrations of 1320, 1890, and 2700 μtg/mL without metabolic 

activation and 926, 1320, and 1890 μtg/mL with metabolic activation were analyzed for chromosomal 

aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or 

endoreduplication was observed in the cultures analyzed. 

In the confirmatory chromosomal aberrations assay, the treatment period was for ~ 20 hours without 

metabolic activation and 3 hours with metabolic activation and the cultures were harvested ~ 20 hours 

from the initiation of treatment. Concentrations of 60.7, 121, 243, 485, 728, 971, 1210, 1460, 1940, 2280, 

and 2620 μg/mL were tested without metabolic activation and 485, 971, 1210, 1460, 1940, 2280, and 

2620 μg/mL were tested with metabolic activation. Cultures treated with concentrations of 1940, 2280, 

and 2620 μg/mL without and with metabolic activation were analyzed for 

chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or 

endoreduplication was observed in the cultures analyzed. 

The vehicle control cultures were in the historical control range for cells with chromosomal aberrations 

and the positive control cultures had significant increase in cells with chromosomal aberrations as 

compared with the vehicle control cultures. The high doses selected for analysis in the assay had >50% 

reduction in mitotic index as compared to the vehicle control cultures and/or a precipitate at the end of the 

treatment period as recommended for this assay by the OECD Testing Guidelines. 

The test article, NTAN, was considered negative for inducing chromosomal aberrations in cultured CHO 

cells without and with metabolic activation. 

7.8 Toxicity to reproduction 

Endpoint summary: Toxicity to reproduction, 

UUID IUC5-6c49f5a5-b56d-4c65-8ec0-f6054f181b96 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-11 16:32:34 CET 

Remarks 

 

Administrative Data 

Effects on fertility 

Short description of key information 

A combined oral repeated dose (subacute) and reproductive/developmental toxicity screening study was 

available. 

Discussion 

Oral administration of NTAN to Sprague-Dawley rats at doses of 1, 10 and 45 mg/kg/day resulted in overt 

signs of parental toxicity at the 45 mg/kg/day dose including death, clinical signs of toxicity (including 

convulsions, labored breathing, etc.), decreased food consumption, body weight, body weight gain, and 

motor activity changes (see also the 28-day repeated dose study). Overall mating, pregnancy, offspring 

survival and pup body weights were not notably affected by treatment with NTAN. Based on the results of 

this study, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity of NTAN was 10 

mg/kg/day and the NOAEL for reproductive/developmental toxicity was at least 45 mg/kg/day. 

Developmental toxicity / teratogeniticy 

Short description of key information 

In the combined oral repeated dose (subacute) and reproductive/developmental toxicity screening study, 

it was noted (see above) that offspring survical and pup body weights were not notably affected by 

treatment with NTAN. Gross external observations, following parturition on lactation day 0, were low in 

incidence and were noted in 4-6 fetuses in each treatment group, including the vehicle control group. All 

gross external morphology observations were common observations likely associated with delivery and 

consisted mainly of bruising, pale skin and a lesion/scab on the neck. None of the gross external 

121
observations were attributed to NTAN treatment.Visceral examinations were performed on dead pups, 

when feasible. No visible abnormalities were noted during the visceral examinations. 

Discussion 

Although not a full-blown developmental toxicity study was available, the information described above 

indicates that NTAN did not induce developmental toxicity at a dose level up to 45 mg/kg bw when given 

during 2 weeks prior to mating, during mating and during the complete pregnancy period. 

Toxicity to reproduction: other studies 

Short description of key information 

No info 

Justification for classification or non-classification 

NTAN did not show reproduction/developmental toxicity at a level up to 45 mg/kg bw, although this level 

resulted in severe parental toxicity, including death. As such no classification for reproduction toxicity is 

warranted. 

7.8.1 Toxicity to reproduction 

Endpoint study record: 7327-60-8, Toxicity to reproduction, Doyle, 2010, RS 

UUID IUC5-7ea14391-1a96-492e-b395-77b478fb53d2 

Dossier UUID 0 

Author ArtsJ / Akzo Nobel Functional Chemicals BV / Amersfoort / Netherlands

Date 2010-11-10 12:10:27 CET 

Remarks 

 

Administrative Data 

 

Purpose flag key study; robust study summary 

Study result type experimental 

result 

Study 

period 

September-November 

2009 

Reliability 1 (reliable without restriction) 

Rationale for reliability incl. 

deficiencies 

Well conducted and reported study under GLP 

Data source 

Reference 

Referen

ce type 

Auth

or 

Yea

r 

Title Bibliograp

hic source 

Testing 

laborato

ry 

Repo

rt no. 

Owner 

compan

y 

Compa

ny 

study 

no. 

Repo

rt 

date 

study 

report 

Doyle 

A 

201

0 

Combined oral 

repeated dose and 

reproductive/developm

ental toxicity screening 

test of 

Nitrilotriacetonitrile 

(NTAN) in rats 

 Experim

ur, 

Chicago, 

IL, USA 

09-

574 

Akzo 

Nobel 

Function

al 

Chemica

ls LLCChelates 

 2010-

10-19 

Data access 

data submitter is data owner 

Data protection claimed 

yes, but willing to share 

Cross-reference to same study 

122
Stusy also used for repeated exposure and developmental toxicity 

Materials and methods 

Test type 

screening 

Limit test 

no 

Test guideline 

Qualifier Guideline Deviations 

according 

to 

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 

Reproduction / Developmental Toxicity Screening Test) 

 

GLP compliance 

yes 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

yes 

Test material identity 

Identifier Identity 

CAS number 7327-60-8 

Details on test material 

Batch no.: CFC 9161Expiry date: 02 June 2012Purity: 99.6 +/- 1.5%Water: 0.4 +/- 0.2% 

Test animals 

Species 

rat 

Strain 

Sprague-Dawley 

Sex 

male/female 

Details on test animals and environmental conditions 

See further 

Administration / exposure 

Route of administration 

oral: gavage 

Vehicle 

corn oil 

Details on exposure 

Dose formulations of the test material were prepared approximately every 2 weeks as suspensions in a 

vehicle of corn oil at graded concentrations of 0.5, 5.0 and 22.5 mg/ml, which corresponded to target 

doses of 1, 10 and 45 mg/kg/day, when administered at a dosing volume of 2 ml/kg. Dose levels of the 

test material were selected by the Sponsor based on the results of a 14-day range-finding study 

(Experimur Study No.: 09-570). Test material formulations of NTAN were previously shown to be stable 

for a minimum of 4 weeks when stored at room temperature. Test material formulations were stored at 

room temperature (approximately 22 ± 3 degrees C), and thoroughly mixed on a stir plate prior to and 

during dosing. 

Details on mating procedure 

One male was housed with one female of the same dose level for up to 2 weeks. Daily vaginal smears, 

taken from the female rats during the mating period, were evaluated for the presence of sperm as well as 

the stage of the estrus cycle. The presence of sperm in the smear or a sperm-plug indicated a positive 

123
mating, and that day was designated as gestation day 0. Ten (10) spermpositive female rats per group 

were selected to produce the F1 generation. 

Analytical verification of doses or concentrations 

yes 

Details on analytical verification of doses or concentrations 

Samples of the test material formulations were collected from the first preparation (i.e., prior to dosing, 

week 0), mid-way through the study (approximately week 3), and towards the end of the study (week 4), 

and analyzed for concentration. Samples for homogeneity (homogeneity is performed on duplicate 

samples collected from the top, middle and bottom of the test material formulation) were collected from 

the low and high dose formulations of the 1st preparation and analyzed concurrently with the first 

concentration analyses. A validated gas chromatography (GC) with flame ionization detection (FID) 

method was used to determine concentration and homogeneity of the NTAN dose formulations. The 

analytically-determined concentrations met the acceptance criterion of ± 15% of the target concentration 

as specified in the study protocol. Samples for homogeneity were collected from the low and high dose 

formulations at study initiation and were found to be within the protocol-specified acceptance criteria of ± 

15%. 

Duration of treatment / exposure 

2 weeks prior to mating, during mating, and until sacrifice (males: 31 doses; females: a minimum of 48 

doses) 

Frequency of treatment 

Daily 

Details on study schedule 

See above 

Doses / concentrations 

1, 10, 45 mg/kg bw/day 

Basis actual ingested 

No. of animals per sex per dose 

10 rats/sex main study, 5 additional rats/sex in the control and high dose groups (recovery animals kept 

for 14 days after the last treatment; these animals were not mated) 

Control animals 

yes, concurrent vehicle 

Positive control 

Not used 

Examinations 

Parental animals: Observations and examinations 

CAGE SIDE OBSERVATIONS: Yes DETAILED CLINICAL OBSERVATIONS: Yes BODY WEIGHT: Yes 

FOOD CONSUMPTION: YesWATER CONSUMPTION: Nosee for furthet details and outcome, the 

repeated dose toxicity study (28-day). 

Estrous cyclicity (Parental animals) 

Daily vaginal smears were collected over 3 days prior to cohabitation to ensure and evaluate cyclicity. 

Vaginal smears were collected during the mating period until the presence of sperm was detected. 

Sperm parameters (Parental animals) 

No 

Litter observations 

Physical Examination of Rat Pups: Rat pups (F1 generation) were sexed and given a gross external 

physical examination on postnatal day 0 and again on postnatal day 4. Dead or stillborn pups were 

examined for gross external anomalies, and, when feasible (for fresh un-autolyzed pups), a visceral 

examination was performed. 

124
Postmortem examinations (Parental animals) 

All animals, including those found dead, received a complete necropsy. Necropsy included examination of 

the external surface of the body, all orifices, the cranial, thoracic, and peritoneal cavities, and their 

contents. The uterus of each rat was examined for evidence of implantation sites and the number of sites 

was recorded; in addition, the corpora lutea were counted. Rats scheduled for necropsy were fasted 

overnight and were euthanized using sodium pentobarbital. Seevral tissues were collected. In addition, a 

bone marrow smear was collected and fixed for possible future evaluation. Testes and epididymides were 

fixed in Bouin’s solution, and the eyes and associated tissues (e.g., optic nerve and Harderian gland) 

were fixed in Davidson’s solution; all other tissues were fixed in10% neutral buffered formalin. 

Postmortem examinations (Offspring) 

Only in case of dead or stillborn pups (see above). 

Statistics 

See below 

Reproductive indices 

See at results 

Offspring viability indices 

See at results 

Any other information on materials and methods incl. tables 

Data were manually collected, entered into Excel spreadsheets, and means and standard deviations were 

calculated using the same software. Data were analyzed for homogeneity of variance using Levene's test. 

When the variances were homogeneous (p > 0.001), the data were further analyzed by one-way analysis 

of variance (ANOVA). When a significant F value was observed (p<=0.05) each treatment group was 

compared to the vehicle control group using Dunnett’s two-tailed t-test. Statistical significance was 

declared at p<=0.05 for Dunnett’s test. 

When Levene’s test was significant (p<=0.001), an appropriate transformation was applied to the data 

(e.g., log-transformation), and the analyses were performed on the transformed data. When the variancestabilizing transformation was not achieved, Kruskal-Wallis test was performed followed 

by Mann-Whitney U (MWU) statistic. Minimal statistical significance was declared at p<=0.05). ChiSquare analysis was not warranted for the 

reproductive performance parameters as mating incidence was relatively identical across groups. 

Minor differences during recalculation of results, as presented in this report, may be noted due to digital 

storage of numbers in computers or calculators as well as software calculating or rounding issues. 

Results and discussions 

Effect levels 

Endpoint Generation Sex Effect level Based 

on 

Basis for effect level / 

Remarks 

NOAEL P male/female >= 45 mg/kg bw/day (actual 

dose received) 

test mat. No effects on reproduction 

observed 

Results of examinations: parental animals 

Clinical signs (parental animals) 

yes 

Body weight and food consumption (parental animals) 

yes 

Test substance intake (parental animals) 

no effects 

Reproductive function: estrous cycle (parental animals) 

not examined 

Reproductive function: sperm measures (parental animals) 

not examined 

125
Reproductive performance (parental animals) 

no effects 

Organ weights (parental animals) 

no effects 

Gross pathology (parental animals) 

yes 

Histopathology (parental animals) 

no effects 

Details on results (parental animals) 

See for general toxicity, the repeated dose study (28-day). No differences were detected in the 

percentage of breeding pairs that mated across the groups (100% in all groups). All females were 

detected as sperm positive during the mating period, with the exception of animal #575 in the 1 mg/kg 

group for which no evidence of mating was detected due tomisdiagnosis of a vaginal smear; this animal 

was pregnant, underwent successful parturition and was therefore considered to be sperm positive. 

Thenumber of dams that successfully underwent parturition was 9-10 in each group. One female (#558) 

in the vehicle control group was detected as sperm positive, but did not show any evidence of parturition 

or pregnancy. Another female (#581) in the 10 mg/kg group was pregnant (as detected byimplantation 

sites in the uterus), but never showed signs/evidence of parturition. The average number of days to 

achieve sperm positive status ranged from 3.5-4.1 days across treatment groups. The length of gestation 

was similar across all groups and averaged approximately 22 days. No significant differences in the mean 

number of corpora lutea/dam or total implantation sites/dam were observed. 

Results of examinations: offspring 

Viability (offspring) 

no effects 

Clinical signs (offspring) 

no effects 

Body weight (offspring) 

no effects 

Sexual maturation (offspring) 

not examined 

Organ weights (offspring) 

not examined 

Gross pathology (offspring) 

not examined 

Histopathology (offspring) 

not examined 

Details on results (offspring) 

Evaluations of litter survival and offspring body weights were limited to those dams that underwent 

successful parturition. No statistically significant differences in mean litter size were detected across 

groups. The number of dead fetuses that were observed (ranged from 2-5/group) and the percentage of 

pups surviving to lactation day 4 were comparable across groups. In addition, no significant differences in 

pre-implantation, pre-natal or post-natal loss of offspring were observed across groups. Mean sex ratios 

(males/females) for days 0 and 4 were also unaffected by treatment. No significant differences in 

offspring body weights were observed. Overall mating, pregnancy, offspring survival and pup body 

weights were not notably affected by treatment with NTAN.Gross external observations, following 

parturition on lactation day 0, were low in incidence and were noted in 4-6 fetuses (of the F1 generation) 

in each treatment group, including the vehicle control group. One control fetus was noted with a blunted 

tail on lactation days 0 and 4. All other gross external morphology observations were common 

observations likely associated with delivery and consisted mainly of bruising, pale skin and a lesion/scab 

126
on the neck. Two pups in the 1 mg/kg group were noted as cannibalized on day 0; one of which was 

missing its left hindlimb and tail (most likely due to cannibalization). None of the gross external 

observations were attributed to NTAN treatment.Visceral examinations were performed on dead pups, 

when feasible. No visible abnormalities were noted during the visceral examinations. The stomach of one 

pup from dam# 590 in the 45 mg/kg group was noted to be devoid of milk; however, this observation was 

not attributed to NTAN treatment. 

Applicant's summary and conclusion 

Conclusions 

Oral administration of NTAN to Sprague-Dawley rats at doses of 1, 10 and 45 mg/kg/day resulted in overt 

signs of parental toxicity at the 45 mg/kg/day dose including death, clinical signs of toxicity (including 

convulsions, labored breathing, etc.), decreased food consumption, body weight, body weight gain, and 

motor activity changes (see also the 28-day repeated dose study). Overall mating, pregnancy, offspring 

survival and pup body weights were not notably affected by treatment with NTAN. Based on the results of 

this study, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity of NTAN was 10 

mg/kg/day and the NOAEL for reproductive/developmental toxicity was at least 45 mg/kg/day. 

Executive summary 

Nitrilotriacetonitrile (NTAN) was administered to male and female Sprague-Dawley rats by oral gavage at 

concentrations of 0.5, 5.0 or 22.5 mg/ml equivalent to doses of 1, 10 or 45 mg/kg/day, when administered 

at a constant dosing volume of 2 ml/kg. The vehicle control group received vehicle (corn oil) only. Ten 

rats/sex/group were selected for cohabitation (i.e., core/parental animals) and 5 additional rats/sex in the 

control and high dose groups were designated as recovery animals in order to evaluate reversibility of 

any NTAN-induced effects. The parental rats were administered NTAN for 2 weeks prior to mating, during 

the mating period, and until sacrifice (males: 31 doses; females: a minimum of 48 doses). Recovery 

animals were treated for the same duration as the parental rats, but were not mated, and were held for 14 

days after treatment to evaluate recovery from any adverse treatment-related effects. Due to the 

spontaneous deaths of 2 high dose recovery females, only 3 unmated recovery females remained. In 

order to increase the size of the female high dose recovery group, one core parental high dose dam was 

held without treatment 

for approximately 14 days and was necropsied with the recovery animals. By doing this, there was one 

additional high dose female (n = 4 instead of n = 3) to evaluate for recovery potential from treatmentrelated effects. Body weights, body weight gain and food consumption were measured during the study. 

In addition, clinical observations, a functional observational battery with automated motor activity, organ 

weights, and clinical and 

diagnostic pathology were evaluated. 

Four rats in the 45 mg/kg/day group died spontaneously during the study; male #536 was found dead on 

study day 19 (during treatment/ cohabitation), female #592 was found dead on day 3 (during 

treatment/pre-cohabitation), female #599 (designated as a recovery animal) was 

found dead on day 30 (during the treatment period), and female #586 was found dead on lactation day 3. 

All four deaths were considered to be treatment-related, although microscopically the cause of death for 

these animals was undetermined. Treatment-related clinical signs of toxicity were limited to animals in the 

45 mg/kg group (6/15 females) and were often noted within a few hours following dose administration 

(see also the 28 -day repeated toxicity study). Treatment-related decreases in mean food consumption 

were observed for male and female rats in the 45 mg/kg group during the study. These reductions in food 

consumption began over study days 0-4 for both sexes and continued for the remainder of the treatment 

period for females. Corollary reductions in body weight were also observed: mean body weight and/or 

body weight gain were reduced compared to concurrent control values in both sexes in the 45 mg/kg 

group beginning over study days 1-4 and these changes generally persisted for males and females 

throughout the treatment phase of the study. No treatment-related changes in clinical chemistry or 

hematology profiles were observed in animals treated with NTAN (see also the 28 -day repeated toxicity 

study). 

No differences were detected in the percentage of breeding pairs that mated across the groups (100% in 

all groups). The number of dams that successfully underwent parturition was 9-10 in each group. No 

gross external or visceral observations were attributed to NTAN treatment. Overall mating, pregnancy, 

offspring survival and pup body weights were not notably affected by treatment with NTAN. 

127
Absolute and relative organ weights were not considered to be affected by treatment with NTAN. 

Treatment-related gross necropsy findings were low/singular in incidence, limited to the 45 mg/kg group 

and consisted of red fluid/blood in the cranial cavity/cranium (observed in four animals that died 

spontaneously during the study), pigmentation of the brain, thymus and liver, urinary bladder dilatation 

and an enlarged thymus. No treatment-related histopathological changes were observed in the tissues 

examined from rats in the 45 mg/kg group; all findings were considered spontaneous or incidental 

changes commonly observed in rats of this age and strain. 

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for parental systemic 

toxicity of NTAN was 10 mg/kg/day and the NOAEL for reproductive/developmental toxicity was 45 

mg/kg/day. 

128