### HUMAN HEALTH ENDPOINTS (NON SIDS)

OESTROGENIC ACTIVITY

TEST SUBSTANCE

l 

### Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NX 4708 (distilled cashew nut shell liquid)

Source: Cardanol Chemicals N.V., Lot No. LT-0481

METHOD

.

. 

### Statistical Methods: None Method: Routledge and Sumpter (1996)

Test Type: Recombinant Yeast Screen A ssay

System of testing: Non bacterial

GLP: Yes

Year: 1999

Species/Strain: Saccharomyces cerevisiae, recombinant strain containing the human

oestrogen receptor (hER) and the reporter gene lac -Z(encoding for the enzyme 13.

galactosidase).

Concentrations tested: 0.049, 0.098, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50,

100 mg/l

Remarks:

- Test Design:

. Number of replicates: 2

. Frequency of dosing: Single

. Positive control: 17P-estradiol, Bisphenol A

- Solvent: Ethanol

RESULTS

l 

### Result: Negative

Remarks: None

CONCLUSIONS

Remarks:

No oestrogenic activity was observed at all concentrations tested

In 

### accordance with current regulatory guidelines for the environmental classification of

chemicals it was considered unnecessary and unrealistic to test at concentrations in excess

of 100 mg/l.Care should be taken in the interpretation of these results, as a negative result in this in

vivo study does not necessarily indicate that the material will not have an oestrogenic effect

in the e nvironment.

Bisphenol A was determined to be 3500 times less potent that 17 β

- estradiol.

The response of the recombinant yeast screen to both of the positive control materials was

comparable to published results thereby confirming the suitability of the i nnoculum and

culture conditions.

REFERENCES (Free Text)

SafePharm Laboratories Ltd., Cardolite NX4708: Assessment of oestrogenic activity using a

recombinant yeast screen assay, Report No. 814/005, June 1999

Routledge EJ and Sumpter JP, 1996, Estrogenic activity of surfactants and some of their

degradation products assessed using a recombinant yeast screen, Environmental

Toxicology and Chemistry 15: 241

- 248

OTHER

• Last Changed: 20 May 2002

• Order number for sorting: 3TEST SUBSTANCE

• Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 700 (distilled cashew nut shell liquid)

Source: Cardanol Chemicals N.V., Lot No. GT457

METHOD

• Method: Routledge and Sumpter (1996)

• Test Type: Recombinant Yeast Screen Assa y

• System of testing : Non bacterial

• GLP: Yes

• Year: 1999

• Species/Strain: Saccharomyces cerevisiae, recombinant strain containing the human

oestrogen receptor (hER) and the reporter gene lac

- Z (encoding for the enzyme ß

galactosidase).

• Concentrations tested: 0.049, 0.098, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50,

100 mg/l

• Statistical Methods: None

Remarks:

- Test Design:

• Number of replicates: 2

• Frequency of dosing: Single

• Positive control: 17 β

- estradiol, Bisphenol A

- Solvent: Ethanol

RESULTS

• Result: Negative

Remarks: None

CONCLUSIONS

Remarks:

No oestrogenic activity was observed at all concentrations tested.

In accordance with current regulatory guidelines for the environmental classification of

chemicals it was considered unnecessary and unrea listic to test at concentrations in excess

of 100 mg/l. HUMAN HEALTH ENDPOINTS (NON SIDS)

OEST ROGENIC ACTIVITYCare should be taken in the interpretation of these results, as a negative result in this in

vivo study does not necessarily indicate that the material will not have an oestrogenic effect

in the envi ronment.

Bisphenol A was determined to be 3500 times less potent that 17 β

- estradiol.

The response of the recombinant yeast screen to both of the positive control materials was

comparable to published results thereby confirming the suitability of the inno culum and

culture conditions.

REFERENCES (Free Text)

SafePharm Laboratories Ltd., Cardolite NC700: Assessment of oestrogenic activity using a

recombinant yeast screen assay, Report No. 814/004, June 1999

Routledge EJ and Sumpter JP, 1996, Estrogenic act ivity of surfactants and some of their

degradation products assessed using a recombinant yeast screen, Environmental

Toxicology and Chemistry 15: 241

- 248

OTHER

• Last Changed: 15 May 2002

• Order number for sorting: 2TEST SUBSTANCE

• Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 700 (distilled cashew nut shell liquid)

Source: Cardolite Corporation, Lot No.: EQ

- 1

METHOD

• Method: OECD 406, ‘Skin Sensitisation’.

• Species/strain: Albino Dunkin Hartley gu inea pigs.

• Concentration

- Intradermal induction: 1% w/v in liquid paraffin

1% w/v in a mixture of Freund’s Complete Adjuvant plus

distilled water (1:1)

- Topical induction: 25% v/v in liquid paraffin

- Topical challenge: 5% and 2% v/v in liquid paraffin

• No of animals/sex/dose: 20 females/dose

• Vehicle: Liquid Paraffin BP

• GLP: Yes

• Year: 1996

Remarks: None

RESULTS

• Sensitization rate: 14/20 (70%) sensitised

• Result: Positive

Remarks:

Skin reactions observed after intradermal induction: Well

- defined erythema (g rade 2) was

commonly noted at the intradermal injection sites at the 24

- hour observation. Incidents of

moderate to severe erythema were also noted at this time. Well

- defined erythema persisted

at all intradermal injection sites at the 48

- hour observation

.

Skin reactions observed after topical induction: Very slight or well

- defined erythema (grades

1 or 2) with or without very slight oedema (grade 1), was commonly noted at the topical

induction sites at the 1

- hour observation. Incidents of fissuring of t he skin, or bleeding

were also noted at this time. The bleeding was probably caused by self

- inflicted scratching

of the skin.

Skin reactions observed after topical challenge with 5% v/v Cardolite NC

- 700: Very slight or

well

- defined erythema (grade 1 or 2 ) was noted at the challenge sites of eleven animals at

the 24

- hour observation. Very slight oedema (grade 1) was also noted at five of these sites

at this observation. Very slight erythema (grade 1) was noted at the challenge sites of 14

animals at the 48

- hour observation, with very slight oedema (grade 1) at two of these sites. HUMAN H EALTH ENDPOINTS (NON SIDS)

SKIN SENSITIZATIONDesquamation was seen at the challenge sites of seven animals. No evidence of erythema

or oedema was seen at the 72

- hour observation, although the presence of desquamation

prec luded evaluation of erythema at the challenge sites of none animals at this time.

Skin reactions observed after topical challenge with 2% v/v Cardolite NC

- 700: Very slight or

well

- defined erythema (grade 1 or 2) was noted at the challenge sited of six ani mals at the

24

- hour observation. Very slight oedema (grade 1) was also noted at one of these sites at

this observation. Very slight erythema (grade 1) was noted at the challenge sited of five

animals at the 48

- hour observation. No skin reactions were no ted at the challenge sites of

two of these animals at the 24

- hour observation. Desquamation was noted at one challenge

site at the 48

- hour observation. Very slight erythema (grade 1) persisted at the challenge

site of one animal at the 72

- hour observatio n. Desquamation was noted at the challenge

sites of three animals at this time.

Clinical observations: All animals showed an expected gain in bodyweight over the study

period. No signs of ill

- health were noted in any animal.

CONCLUSIONS

Remarks: Cardo lite NC

- 700 produced a 70% (14/20) sensitisation rate in this study and

was classified as a strong as a strong sensitiser.

REFERENCES (Free Text)

SafePharm Laboratories Ltd., Determination of the skin sensitisation potential of Cardolite

NC

- 700 and asse ssment of cross

- sensitisation potential with poison ivy oil, Cardolite NC

513, Cardolite NC

- 514, Cardolite NC

- 541 and Cardolite NC0558, Report No. 661/010,

February 1996

OTHER

• Last Changed: 20 May 2002

• Order number for sorting: 1

Remarks: NoneTEST SUBSTANCE

• Identity: Cashew Nut Shell Liquid

Remarks: Test substance: Cardanol (CAS No. 37330

- 39

- 5), Cardol (CAS No. 57486

- 25

- 6)

METHOD

• Method: Calculation using ECOSAR v.0.99e

• Type: N/A

• GLP: No

• Year: 2002

• Species: Algae

Remarks: None

RESULTS

• Unit: mg/L

• EC 50 at 96 hours: 0.00011

– 0.00034 (Cardanol)

0.00031

–

0.00096 (Cardol)

Remarks: The predicted EC 50 values vary with the degree of unsaturation in the alkyl side

chain of Cardanol and Cardol as follows:

EC 50 , mg/L

unsaturated monoene diene triene

Cardanol 0.00011 0.00017 0.00026 0.00034

Cardol 0.00031 0.00048 0.00072 0.00096

CONCLUSIONS

Remarks:

Estimation using ECOSAR v0.99e predict Cardanol and Cardol, the two major components

of distilled and technical grade Cashew Nut Shell Liquid, to be toxic to algae.

DATA QUALITY

Reliabilities: 4, Not Assignable

Remarks:

Estimation using ECOSAR v.0.99e

REFERENCES (Free Text) ECOTOXICITY ENDPOINTS

11. TOXICITY TO AQUATIC PLANTS (E.G., ALGAE)ECOSAR v0.99e. EPIWIN modelling program. Meylan, W. & Howard, P. (1999), Syracuse

Research Corporation, Envir onmental Science Center, 6225 Running Ridge Road, North

Syracuse, NY 13212

- 2510

OTHER

• Last Changed: 24 April 2002

• Order number for sorting: 1

Remarks:TEST SUBSTANCE

• Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 511 (distilled cashew nut shell liquid)

Source: Cardolite Corporation, Lot No.: LP

- 2

METHOD

• Method: OECD 473

• Test Type: Chromosomal aberration test

• System of testing: Non bacterial

• GLP: Yes

• Year: 1995

• Species/Strain: Human Lymphocytes

• Metabolic activation: S9

- mix, Rat liver cells, Aroclor induced, 1 ml

• Concentrations tested: Expt. 1 (20h harvest): 0, 6.25, 12.5, 25 μg/ml (

- S9)

0, 3.125, 6.25, 12.5 μg/ml (+S9)

Expt. 2 (20h harvest): 12.5, 25, 37.5 μg/ml (

- S9)

0.78, 1.56, 3.125, μg/ml (+S9)

Expt. 2 (44h harvest): 25 μg/ml (

- S9)

3.125 μg/ml (+S9)

• Statistical Methods: Fisher's Exact test or Chi

- squared test

Remarks:

- Test Design

• Number of re plicates: 2

• Positive control: Ethyl methanesulphonate (EMS) (

- S9), cyclophosphamide (+S9)

• Negative control: Solvent vehicle

- Solvent: Dimethylsulfoxide

RESULTS

• Result: Negative

• Cytotoxic concentration

- With metabolic activation: 12.5 μg/ml

- Without metabo lic activation: >25 μg/ml

• Genotoxic effects

- With metabolic activation: None

- Without metabolic activation: None HUMAN H EALTH ENDPOINTS

15. GENETIC TOXICITY IN VITRO (GENE MUTATIONS)• Statistical results: The test material did not induce a significant increase in the

frequency of cells with chromosome aberrations or polyploi d cells in either the presence

or absence of a liver enzyme metabolizing system.

Experiment 1: Harvest Time 20 hours, without metabolic activation

Chromatid Chromosome Others Total Aberrations Aber rant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Scored Total

Gaps Breaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps)

A 100 0 0 1 0 0 0 1 1 1 1

B 100 0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 1 1 1 1 Vehicle

Control

Total 200

(0.0) (0.0) (0.5) (0.0) (0.0) (0.0) (0.5) (0.5) (0.5) (0.5)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 1 0 1 0 6.25

μg/ml

Total 200

(0.5) (0.0) (0.0) (0.0) (0.0) (0.0) (0.5) (0.0) (0.5) (0.0)

A 100 2 1 0 0 0 0 3 1 3 1

B 100 0 0 0 1 0 0 1 1 1 1

2 1 0 1 0 0 4 2 4 2 12.5

μg/ml

Total 200

(1.0) (0.5) (0.0) (0.5) (0.0) (0.0) (2.0) (1.0) (2.0) (1.0)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 1 1 0 0 0 0 2 1 2 1

2 1 0 0 0 0 3 1 3 1 25

μg/ml

Total 200

(1.0) (0.5) (0.0) (0.0) (0.0) (0.0) (1.5) (0.5) (1.5) (0.5)

A 50 31 14 7 2 1 0 55 24 33 21

B 50 13 18 8 2 0 0 41 28 29 24

100 44 32 15 4 1 0 96 52 62*** 45*** Positive

Control

EMS

500

μg/ml Total

(44.0) (32.0) (15.0) (4.0) (1.0) (0.0) (96.0) (52.0) (62.0) (45.0)

X = > 10 aberrations per cell (not included in total aberrations) Figures in brackets =

aberrations per 100 cells *** represents p ≤ 0.001

Experiment 1: Harvest Time 20 hours, with metabolic activation

Chromatid Chromosome Others Total Aberrations Aberrant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Scored Total

Gaps Br

eaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps)

A 100 0 0 0 0 0 0 0 0 0 0

B 100 0 1 0 0 0 0 1 1 1 1

0 1 0 0 0 0 1 1 1 1 Vehicle

Control

Total 200

(0.0) (0.5) (0.0) (0.0) (0.0) (0.0) (0.5) (0.5) (0.5) (0.5)

A 100 0 0 0 0 0 0 0 0 0 0

B 100 0 1 0 0 0 0 1 1 1 1

0 1 0 0 0 0 1 1 1 1 1.56

μg/ml

Total 200

(0.0) (0.5) (0.0) (0.0) (0.0) (0.0) (0.5) (0.5) (0.5) (0.5)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 1 0 0 0 0 0 1 0 1 0 3.125

μg/ml

Total 200 2 0 0 0 0 0 2 0 2 0(1.0) (0.0) (0 .0) (0.0) (0.0) (0.0) (1.0) (0.0) (1.0) (0.0)

A 100 0 0 0 0 0 0 0 0 0 0

B 100 4 0 0 0 0 0 4 0 4 0

4 0 0 0 0 0 4 0 4 0 6.25

μg/ml

Total 200

(2.0) (0.0) (0.0) (0.0) (0.0) (0.0) (2.0) (0.0) (2.0) (0.0)

A 100 4 0 0 1 0 0 5 1 5 1

B 100 1 2 0 2 0 0 5 4 4 3

200 5 2 0 3 0 0 10 5 9** 4 Positive

Control

CP

25

μg/ml Total

(2.5) (1.0) (0.0) (1.5) (0.0) (0.0) (5.0) (2.5) (4.5) (2.0)

X = > 10 aberrations per cell (not included in total aberrations) Figures in brackets =

aberrations per 100 cells ** rep resents p ≤ 0.01

Experiment 2: Harvest Time 20 hours, without metabolic activation

Chromatid Chromosome Others Total Aberrations Aberrant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Scored Total

Gaps Breaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps)

A 100 2 0 0 0 0 0 2 0 2 0

B 100 0 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 2 0 2 0 Vehicle

Control

Total 200

(1.0) (0.0) (0.0) (0.0) (0.0) (0.0) (1.0) (0.0) (1.0) (0.0)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 1 0 1 0 12.5

μg/ml

Total 200

(0.5) (0.0) (0.0) (0.0) (0.0) (0.0) (0.5) (0.0) (0.5) (0.0)

A 100 1 1 0 0 0 0 2 1 2 1

B 100 0 0 0 0 0 0 0 0 0 0

1 1 0 0 0 0 2 1 2 1 25

μg/ml

Total 200

(0.5) (0.5) (0.0) (0.0) (0.0) (0.0) (1.0) (0.5) (1.0) (0.5)

A 100 1 1 0 0 0 0 2 1 2 1

B 100 0 0 0 0 0 0 0 0 0 0

1 1 0 0 0 0 2 1 2 1 37.5

μg/ml

Total 200

(0.5) (0.5) (0.0) (0.0) (0.0) (0.0) (1.0) (0.5) (1.0) (0.5)

A 100 6 9 4 0 0 0 19 13 13 11

B 100 16 17 2 1 0 0 36 20 26 1 5

200 22 26 6 1 0 0 55 33 39*** 26*** Positive

Control

EMS

500

μg/ml Total

(11.0) (13.0) (3.0) (0.5) (0.0) (0.0) (27.5) (16.5) (19.5) (13.0)

X = > 10 aberrations per cell (not included in total aberrations) Figures in brackets =

aberrations per 100 cells *** represents p ≤ 0.001

Experiment 2: Harvest Time 20 hours, with metabolic activation

Chromatid Chromosome Others Total Aberrations Aberrant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Scored Total

Gaps Breaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps )

A 100 1 1 1 0 0 0 3 2 3 2 Vehicle

Control B 100 0 1 0 0 0 0 1 1 1 11 2 1 0 0 0 4 3 4 3 Total 200

(0.5) (1.0) (0.5) (0.0) (0.0) (0.0) (2.0) (1.5) (2.0) (1.5)

A 100 0 0 0 0 0 0 0 0 0 0

B 100 0 3 0 0 0 0 3 3 3 3

0 3 0 0 0 0 3 3 3 3 0.78

μg/ml

Total 200

(0.0) (1.5) (0.0) (0.0) (0.0) (0.0) (1.5) (1.5) (1.5) (1.5)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 1 0 1 0 1.56

μg/ml

Total 200

(0.5) (0.0) (0.0) (0.0) (0.0) (0.0) (0.5) (0.0) (0.5) (0.0)

A 100 0 0 0 0 0 0 0 0 0 0

B 100 1 0 0 0 1 0 2 1 2 1

1 0 0 0 1 0 2 1 2 1 3.125

μg/ml

Total 200

(0.5) (0.0) (0.0) (0.0) (1.0) (0.0) (1.0) (0.5) (1.0) (0.5)

A 100 5 4 0 1 0 0 10 5 9 5

B 100 6 0 2 1 0 0 9 3 8 3

200 11 4 2 2 0 0 19 8 17** 8 Positive

Control

CP

25

μg/ml Total

(5.5) (2.0) (1.0) (1.0) (0.0) (0.0) (9.5) (4.0) (8.5) (4.0)

X = > 10 aberrations per cell (not included in total aberrations) Figures in brackets =

aberrations per 100 cells ** represents p ≤ 0.01

Experiment 2: Harvest Time 44 hours, with out metabolic activation

Chromatid Chromosome Others Total Aberrations Aberrant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Scored Total

Gaps Breaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps)

A 100 0 3 0 0 0 0 3 3 3 3

B 100 0 0 0 0 0 0 0 0 0 0

0 3 0 0 0 0 3 3 3 3 Vehicle

Control

Total 200

(0.0) (1.5) (0.0) (0.0) (0.0) (0.0) (1.5) (1.5) (1.5) (1.5)

A 100 1 0 0 0 0 0 1 0 1 0

B 100 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 1 0 1 0 25

μg/ml

Total 200

(0.5) (0.0) (0.0) (0.0) (0.0 ) (0.0) (0.5) (0.0) (0.5) (0.0)

X = > 10 aberrations per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells

Experiment 2: Harvest Time 44 hours, with metabolic activation

Chromatid Chromosome Others Total Aberrations Aberrant Cells Treatme

nt

Group Replicat

e

ID No. Cells

Score d Total

Gaps Breaks Exchange

s Breaks Exchange

s X (+Gaps) (

- Gaps) (+Gaps) (

- Gaps)

A 100 0 0 0 1 0 0 1 1 1 1

B 100 0 1 0 1 0 0 2 2 2 2

0 1 0 2 0 0 3 3 3 3 Vehicle

Control

Total 200

(0 .0) (0.5) (0.0) (1.0) (0.0) (0.0) (1.5) (1.5) (1.5) (1.5)

A 100 2 1 0 1 0 0 4 2 4 2

B 100 1 0 0 0 0 0 1 0 1 0 25

μg/ml

Total 200 3 1 0 1 0 0 5 2 5 2(1.5) (0.5) (0.0) (0.5) (0.0) (0.0) (2.5) (1.0) (2.5) (1.0)

X = > 10 aberrations per cell (not in cluded in total aberrations) Figures in brackets = aberrations per 100 cells

Experiment 1: Mean Frequency of Polyploid Cells (%)

20 Hours Dose Level

μg/ml Without S9 With S9

0 0.0 0.0

1.56

- 0.5

3.125

- 0.0

6.25 0.0 0.0

12.5 0.0

-

25 0.0

-

EMS 5 00 0.0

-

CP 25

- 0.0

Experiment 2: Mean Frequency of Polyploid Cells (%)

Without S9 With S9 Dose Level

μg/ml 20 hours 44 hours Dose Level

μg/ml 20 hours 44 hours

0 0.0 0.5 0 0.0 1.0

12.5 0.5

- 0.78 0.0

-

25 0.0 0.5 1.56 1.0

-

37.5 0.0

- 3.125 1. 0 0.0

EMS 500 0.0

- CP 25 0.5

-

Remarks:

Experiment 1: Mitotic Index (20

- hour harvest)

Without S9 With S9 Dose

Level

μg/ml A B Mean % of

Control A B Mean % of

Control

0 5.80 6.25 6.03 100 3.10 2.40 2.75 100

0.78

- - - -

1.56

- - - - - - - -

3.125

- - - - 3.60 3.60 3.60 131

6.25 4.90 7.80 6.35 105 1.15 2.25 1.70 62

12.5 6.70 6.50 6.60 109 0.85 0.55 0.70 25

25 8.30 4.30 6.30 104

- - - -

50 NM NM

- -

EMS

500 3.40 4.30 3.85 64

CP 25

- - - - 1.40 2.45 1.93 70

- = not assessed NM = no scorable metaphases

Experiment 2: Mitotic Index (20

- hour harvest)

Dose

Level Without S9 With S9Level

μg/ml A B Mean % of

Control A B Mean % of

Control

0 8.55 7.90 8.23 100 3.00 3.25 3.13 100

0.39

- - - -

0.78 1.80 3.35 2.58 82

1.56 2.50 2 .80 2.65 85

3.125

- - - - 1.35 1.90 1.63 52

6.25 7.20 6.75 6.98 85 0.45 0.45 0.45 14

9.38 NM NM

- -

12.5 7.75 9.45 8.60 104

25 6.00 9.45 7.73 94

37.5 3.25 3.65 3.45 42

50 NM NM NM

-

EMS

500 4.70 7.95 4.83 59

- - - -

CP 25

- - - - 1.60 1.45 1.53 49

- = not assessed NM = no scorable metaphases

CONCLUSIONS

Remarks: The substance was found to be non

- clastogenic under the conditions of the test.

DATA QUALITY

• Reliabilities

: 1, Reliable without restriction

Remarks: Study conduct ed under GLP to OECD test guideline by SafePharm Laboratories

Ltd.

REFERENCES (Free Text)

Safepharm Laboratories Ltd., Cardolite NC 511: Chromosome Aberration Test in Human

Lymphocytes In Vitro, Report No. 814/002, 1995

Scott, D., Et al, Metaphase chrom osome aberration assays in vitro. In: Kirkland, D.J., Basic

mutagenicity tests: UKEMS recommended procedures. Report. Part 1 revised. Cambridge

University Press, 1990:62

- 84

OTHER

• Last Changed: 25 April 2002

• Order number for sorting: 2

Remarks :TEST SUBSTANCE

• Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 511 (distilled cashew nut shell liquid)

Source: Cardolite Corporation, Lot No.: LP

- 2

METHOD

• Method: OECD 471

• Test Type: Reverse Mutation Assay (Ames Test)

• System of testi ng: Bacterial

• GLP: Yes

• Year: 1995

• Species/Strain: Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 & TA100)

• Metabolic activation: S9

- mix, Rat liver cells, 0.5 ml, Aroclor induced

• Concentrations tested: 50, 150, 500, 1500, 5000 μg/plate (±S9)

• Statistica l Methods: Dunnett’s method of linear regression

Remarks:

- Test Design

• Number of replicates: 3

• Positive controls: N

- ethyl

- N'

- nitro

- N

- nitrosoguanidine (

- S9, TA100 & TA1535)

9

- aminoacridine (

-

S9, TA 1537)

4

- nitro

- o

- phenylenediamine (

- S9, TA1538)

4

- nitr oquinoline

- 1

-

oxide (

- S9, TA98)

2

- aminoanthracene (+S9, TA98, TA100, TA1535, TA1537 &

TA1538)

• Negative control: Solvent vehicle

- Solvent: Acetone

RESULTS

• Result: Negative

• Cytotoxic concentration

- With metabolic activation: >5000 μg/plate

- Without metaboli c activation: >5000 μg/plate

• Genotoxic effects

- With metabolic activation: None

- Without metabolic activation: None HUMAN HEALTH ENDPOINTS

15. GENETIC TOXICITY IN VITRO (GENE MUTATIONS)• Statistical results: No significant increase in the frequency of revertant colonies was

recorded for any of the bacterial strains with any dose of the test material, either with or

without metabolic activation.

Experiment 1

– Without Metabolic Activation

Number of revertants (Number of colonies per plate)

Base

- pair

substitution type Frameshift type Test Substance

Concentration

(μg/plate)

TA100 TA1535 TA1538 TA98 TA1537

0 115

117 (110)

97 11.0 28

25 (21)

9 10.2 34

25 (25)

17 8.5 36

30 (30)

25 5.5 10

17 (15)

18 4.4

50 149

118 (131)

127 15.9 20

19 (19)

18 1.0 17

27 (23)

24 5.1 17

24 (21)

23 3 .8 19

18 (18)

18 0.6

150 118

120 (120)

121 1.5 12

10 (12)

15 2.5 9

28 (16)

11 10.4 24

17 (18)

14 5.1 15

18 (17)

18 1.7

500 121

115 (116)

111 5.0 17

20 (16)

12 4.0 13

30 (18)

12 10.1 17

33 (25)

25 8.0 10

1 2 (12)

14 2.0

1500 115p

107p

(117)

128p

10.6 8p

22p (16)

17p 7.1 15p

17p (15)

14p 1.5 22p

25p (27)

34p 6.2 13p

10p (12)

13p 1.7

5000 122p

85p

(106)

111p

19.0 7p

15p (10)

8p 4.4 25p

15p (19)

18p 5.1 29p

22p (23)

17p 6.0 12p

19p (16)

18p 3.8

Positive Control ENNG ENNG 4NOPD 4NQO 9AA

Concentration

(μg/plate) 3 5 5 0.2 80

Number of

colonies per

plate 670

933

(729)

583

182.2 198

213 (224)

260 32.3 470

474

(479)

494

12.9 168

152

(156)

149

10.2 76

208

(152)

172

68.2

Key to Table: ‘number of revertants’

– observed values and average values (in parentheses) are shown at each dose.

Figures immediately below average values refer to standard deviation. The letter ‘p’ following a number indicates

precip itation was observed.

Positive controls: ENNG (N

- ethyl

- N’

- nitro

- N

- nitroguanidine), 4NOPD (4

- nitro

- σ

- phenylenediamine), 4NQO (4

-

nitroquinloine

- 1

- oxide), 9AA (9

- aminoacridine)

Experiment 1

–

With Metabolic Activation

Number of revertants (Number of colonies per plate)

Base

- pair substitution

type Frameshift type Test Substance

Concentration

(μg/plate)

TA100 TA1535 TA1538 TA98 TA15370 131

108

(113)

101

15.7 19

17 (17)

15 2.0 39

35 (33)

24 7.8 24

38 (32)

35 7.4 17

18 (16)

14 2.1

50 118

129

(125)

127 5.9 18

13 (15)

15 2.5 34

24 (32)

39 7.6 29

34 (33)

35 3.2 17

13 (16)

17 2.3

150 121

111

(117)

120 5.5 13

19 (16)

17 3.1 32

33 (33)

33 0.6 33

33 (35)

39 3.5 10

15 (15)

19 4.5

500 111

143

(115)

91

26.2 10

15 (15)

19 4.5 18

35 (29)

34 9.5 35

38 (33)

27 5.7 12

15 (12)

10 2.5

1500 98p

133p

(114)

112p

17.6 20p

17p (19)

20p 1.7 25p

28p (27)

28p 1.7 28p

28p (30)

33p 2.9 13p

18p (15)

13p 2.9

5000 128p

112p

(117)

112p

9.2 12p

17p (14)

13p 2.6 30p

18p (24)

25p 6.0 29p

28p (29)

30p 1.0 14p

19p (17)

17p 2.5

Positive Control 2AA 2AA 2AA 2AA 2AA

Concentration

(μg/plate) 1 2 0.5 0.5 2

Number of

colonies per

plate 1332

1615(138

2)

1200

212.0 64

69 (66)

64 2.9 353

323 (361)

408 43.1 219

453 (329)

315

117.6 194

252

(232)

250

32.9

Key to Table: ‘number of revertants’

– observed values and average values (in parentheses) are shown at each dose.

Figures immediately below average values refer to standard deviation. The letter ‘p’ following a number indicates

precip itation was observed.

Positive control: 2AA (2

- aminoanthracene)

Experiment 2

–

Without Metabolic Activation

Number of revertants (Number of colonies per plate)

Base

- pair substitution

type Frameshift type Test Substance

Concentration

(μg/plate)

TA100 TA1535 TA1538 TA98 TA1537

0 107

132 (116)

110 13.7 14

18 (17)

19 2.6 29

22 (21)

12 8.5 17

23 (21)

24 3.8 19

19 (17)

13 3.550 149

133 (140)

138 8.2 12

18 (21)

32 10.3 41

22 (29)

23 10.7 34

30 (32)

0 10

20 (14)

13 5.1

150 133

118 (129)

137

10.0 18

24 (24)

29 5.5 10

22 (17)

19 6.2 35

33 (32)

28 3.6 14

10 (12)

12 2.0

500 134

139 (131)

121 9.3 13

13 (15)

20 4.0 20

14 (21)

30 8.1 23

34 (25)

19 7.8 13

19 (19)

24 5.5

1500 117p

117p

(109)

92p

14.4 12p

10p (12)

14p 2.0 22p

23p (21)

18p 17p

20p (26)

4 0p 12.5 18p

12p (15)

14p 3.1

5000 107p

121p

(108)

95p

13.0 10p

25p (19)

23p 8.1 19p

19p (20)

23p 2.3 20p

24p (28)

39p 10.0 14p

12p (12)

10p 2.0

Positive Control ENNG ENNG 4NOPD 4NQO 9AA

Concentration

(μg/plate) 3 5 5 0.2 80

Number of

colonies per

plate 916

711 (711)

740

110.9 514

504 (498)

477

19.1 406

499 (455)

459

46.7 177

203 (196)

208

16.6 638

656 (589)

474

100.3

Key to Table: ‘number of revertants’

– observed values and average values (in parentheses) are shown at each dose.

Figure s immediately below average values refer to standard deviation. The letter ‘p’ following a number indicates

precipitation was observed.

Positive controls: ENNG (N

- ethyl

- N’

- nitro

- N

- nitroguanidine), 4NOPD (4

- nitro

- σ

- phenylenediamine), 4NQO (4

-

nitroquinloine

- 1

- oxide), 9AA (9

- aminoacridine)

Experiment 2

–

With Metabolic Activation

Number of revertants (Number of colonies per plate)

Base

- pair substitution

type Frameshift type Test Substance

Concentration

(μg/plate)

TA100 TA1535 TA1538 TA98 TA1537

0 137

139

(131)

117

12.2 25

20 (20)

15 5.0 35

28 (34)

39 5.6 24

28 (32)

44 10.6 22

18 (18)

13 4.5

50 133

138

(128)

112

13.8 24

22 (21)

18 3.1 31

33 (32)

32 1.0 38

33 (34)

32 3.2 19

24 (19)

13 5.5150 108

120

(115)

118 6.4 23

30 (25)

22 4.4 25

35 (34)

43 9.0 29

36 (33)

35 3.8 14

22 (17)

14 4.6

500 122

142

(125)

110 16.2 23

24 (23)

23 0.6 23

30 (26)

25 3.6 28

24 (32)

44 10.6 13

13 (16)

22 5.2

1500 129p

120p

(123)

121p

4.9 13p

15p (19)

30p 9.3 25p

22p (25)

28p 3.0 28p

13p (26)

36p 11.7 17p

17p (16)

14p 1.7

5000 128p

170p

(135)

106p

32.5 18p

20p (18)

15p 2.5 32p

17p (29)

38p 10.8 36p

35p (36)

36p 0.6 15p

15p (15)

14p 0.6

Positi ve Control 2AA 2AA 2AA 2AA 2AA

Concentration

(μg/plate) 1 2 0.5 0.5 2

Number of

colonies per

plate 1398

1553(140

6)

1268

142.7 102

139 (114)

102 21.4 276

256 (273)

286 15.3 159

293

(243)

278

73.4 255

250

(254)

258 4.0

Key to Table: ‘num ber of revertants’

– observed values and average values (in parentheses) are shown at each dose.

Figures immediately below average values refer to standard deviation. The letter ‘p’ following a number indicates

precipitation was observed.

Positive Contro

l: 2AA (2

- aminoanthracene)

Remarks: A precipitate was observed at and above 1500 μg/plate, however this did not

interfere with the scoring of revertant colonies.

CONCLUSIONS

Remarks: The substance was found to be non

- mutagenic under the conditions of th e test.

DATA QUALITY

• Reliabilities

: 1, Reliable without restriction

Remarks: Study conducted under GLP to OECD test guideline by SafePharm Laboratories

Ltd.

REFERENCES (Free Text)

Safepharm Laboratories Ltd., Cardolite NC 511: Reverse Mutation Assay ' Ames Test' Using

Salmonella Typhimurium, Report No. 814/001, 1995Kirkland, D.J., (Ed), Statistical Evaluation of Mutagenicity Test Data, UKEMS Sub

committee on Guidelines for Mutagenicity Testing, Report

- Part III (1989), Cambridge

University Press

OTH ER

• Last Changed: 25 April 2002

• Order number for sorting: 1

Remarks :TEST SUBSTANCE

• Identity: Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 511 (distilled cashew nut shell liquid)

Source: Cardolite Corporation. Lot No.: LP

- 2

METHOD

• Method: OECD Method 302D

• Test Type: aerobic

• GLP: Yes

• Year: 1993

• Contact time: 28 (days)

• Innoculum: activated sludge

Remarks:

• Innoculum: Fresh activated sludge from a municipal biological sewage treatment plant.

30 mg suspended solids/l of test medium.

• Concentration of test chemical: 6.01

– 6.39 mg, direct addition

• Temperat ure of incubation: 20°C

• Dosing procedure: Test substance weighed on a piece of glass to an amount of

about 20 mg ThOD (or COD) and added directly to the test flask.

• Sampling frequency: 0,7,14,21 & 28 days

• Controls: Sodium acetate used as positive control, innoculum used as blank.

• Analytical method used to measure biodegradation: The COD of the poorly soluble

substance was determined in a variation of ISO Method 6060 (closed system with a

pressure equaliser / Kelkenberg method, Z.f. Wasser und Abwasserforsch ung (1975)

146). Oxygen determination was performed using an oxygen electrode (WTW;FRG;

Microprocessor oximeter OXI 2000 with electrode model TriOxmatic EO 200).

• Method of calculating measured concentrations: Arithmetic mean

RESULTS

• Degradation % after time: 96% after 28 days

• Results: Readily biodegradable

• Kinetic:

Day % Degradation

Sample Positive control

7 46 75

14 72 86

21 86 91

28 96 97 ENVIRONMENTAL FATE ELEMENTS AND PATHWAYS

9. BIODEGRADATION• Breakdown products: Not determined

Remarks: None

CONCLUSIONS

Remarks:

According to the author of t he study, based on the data (i.e. 96% degradation after 28

days) Cardolite NC

- 511 can be regarded as very highly biodegradable.

DATA QUALITY

• Reliabilities: 1, Reliable without restriction

Remarks: Study conducted under GLP to recognised test method b y Henkel KGaA

REFERENCES

Henkel KGaA, Cardolite NC

- 511 Ultimate biodegradability in the BODIS

- Test, Report No.

RE930104, 1993

OTHER

Last Changed: 23 April 2002

Order number for sorting: 1

Remarks:

The test method used was based on the closed bottle t est (OECD test method 302D) and

the RDA

- Blok test, previously published (Blok, J., A Repetitive Die Away (RDA) Test

Combining Several Biodegradability Test Procedures, Int. Biodeterior. Bull., 15 (1979) 57

- 63) and ring

- tested by the OECD (1988 ring test on ready biodegradability).TEST SUBSTANCE

• Identity: Cashew Nut Shell Liquid

Remarks: Test substance: Cardanol (CAS No. 37330

- 39

- 5), Cardol (CAS No. 57486

- 25

- 6)

METHOD

• Method: Calculation using ECOSAR v.0.99e

• Type: N/A

• GLP: No

• Year: 2002

• Species: Fish

Remarks: None

RESULTS

• Unit: mg/L

• LC 50 at 96 hours: 0.002

– 0.005 (Cardanol)

0.005

– 0.011 (Cardol)

Remarks: The predicted LC 50 values vary with the degree of unsaturation in the alkyl side

chain of Cardanol and Cardol as follows:

LC 50 , mg/L

unsaturated monoene diene tri ene

Cardanol 0.002 0.003 0.004 0.005

Cardol 0.005 0.007 0.009 0.011

CONCLUSIONS

Remarks:

Estimation using ECOSAR v0.99e predict Cardanol and Cardol, the two major components

of distilled and technical grade Cashew Nut Shell Liquid, to be toxic to fi sh.

DATA QUALITY

• Reliabilities: 4, Not Assignable

Remarks:

Estimation using ECOSAR v0.99e

REFERENCES ECOTOXICITY ENDPOINTS

10. ACUTE TOXICITY TO FISHECOSAR v0.99e. EPIWIN modelling program. Meylan, W. & Howard, P. (1999), Syracuse

Research Corporation, Environmental Science Center, 6225 Running Ri dge Road, North

Syracuse, NY 13212

- 2510

OTHER

• Last Changed: 24 April 2002

• Order number for sorting: 1

Remarks:TEST SUBSTANCE

• Identity: Cashew Nut Shell Liquid

Remarks: Test substance: Cardanol (CAS No. 37330

- 39

- 5), Cardol (CAS No. 57486

- 25

- 6)

METHOD

• Method: Calculation using ECOSAR v.0.99e

• Type: N/A

• GLP: No

• Year: 2002

• Species: Daphnia

Remarks: None

RESULT S

• Unit: mg/L

• LC 50 at 48 hours: 0.024

– 0.040 (Cardanol)

0.039

– 0.066 (Cardol)

Remarks: The predicted LC 50 values vary with the degree of unsaturation in the alkyl side

chain of cardanol and cardol as follows:

LC 50 , mg/L

unsaturated monoene diene t riene

Cardanol 0.024 0.029 0.035 0.040

Cardol 0.039 0.048 0.058 0.066

CONCLUSIONS

Remarks:

Estimation using ECOSAR v0.99e predicts Cardanol and Cardol, the two major components

of distilled and technical grade Cashew Nut Shell Liquid, to be toxic to Daphnia.

DATA QUALITY

• Reliabilities: 4, Not Assignable

Remarks:

Estimation using ECOSAR v.0.99e

REFERENCES ECOTOXICITY ENDPOINTS

12. TOXICITY TO AQUATIC INVERTIBRATES (E.G., DAPHNIA)ECOSAR v0.99e. EPIWIN modelling program. Meylan, W. & Howard, P. (1999), Syracuse

Research Corporation, Environmental Science Center, 6225 Run ning Ridge Road, North

Syracuse, NY 13212

- 2510

OTHER

• Last Changed: 24 April 2002

• Order number for sorting: 1

Remarks:TEST SUBSTANCE

• Cashew Nutshell Liquid

Remarks: Test substance: Cardolite NC 511 (distilled cashew nut shell liquid)

Source: Cardolite Corporation, Lot No.: LP

- 2

METHOD

• Method: OECD 476

• Test Type: Forward Mutation Assay

• System of testing: Non bact erial

• GLP: Yes

• Year: 1996

• Species/Strain: Chinese Hamster Ovary CHO

- KI BH4

• Metabolic activation: S9

- mix, Rat liver cells, Aroclor induced

• Concentrations tested: Expt. 1: 0, 0.75, 1.5, 3, 6, 12 μg/ml (

- S9)

0, 1.5, 3, 6, 12, 18 μg/ml (+S9)

Expt. 2: 0, 0.75, 1.5, 3, 6, 9 μg/ml (

- S9)

0, 3, 6, 12, 18, 24 μg/ml (+S9)

• Statistical Methods: Cochran

- Armitage test for trend analysis, Fisher

- Irwin exact test for

group comparisons for proportions.

Remarks:

- Test Design

• Number of replicates: 2

• Positive co ntrol: Ethyl methanesulphonate (EMS) (

- S9), 3

- methylcholanthrene (3

- MC) (+S9)

• Negative control: Solvent vehicle

- Solvent: Dimethylsulfoxide

RESULTS

• Result: Negative

• Cytotoxic concentration

- With metabolic activation: 47.19 μg/ml

- Without metabolic activat ion: 47.19 μg/ml

• Genotoxic effects

- With metabolic activation: None

- Without metabolic activation: None HUMAN HEALTH ENDPOINTS

15. GENETIC TOXICITY IN VITRO (GENE MUTATIONS)• Statistical results: The test material did not induce significant or dose

- related increases

in mutant frequency per survivor in either the presence or absence of metabolic

activation in either of the two experiments.

Summary of Results:

Experiment 1:

Without S9 With S9 Dose Level

μg/ml A B Mean

MFS Dose Level

μg/ml A B Mean

MFS

0 3.4 0.7 2.05 0 3.5 3.3 3.4

0.75 1.4

- 1.4 1.5 2.9 0.7 1.80

1.5 2.0 0.0 1.00 3.0 0.6 1.4 1.00

3 0.0 0.0 0.0 6.0 2.9 0.0 1.45

6 0.0 0.0 0.0 12 1.4 6.3 3.85

12 0.0 6.3 3.15 18 0.7 8.6 4.65

EMS 200 154.

5 189.9 172.20 24

- - -

3

- MC 4 238.

8 285.9 262.35

MFS = 6

- TG resistant mutants/10 6 viable cells

Experiment 2:

Without S9 With S9 Dose Level

μg/ml A B Mean

MFS Dose Level

μg/ml A B Mean

MFS

0 0.0 0.6 0.30 0 8.1 0.8 4.45

0.75 0.4 7.6 4.00 3 1.3 0.0 0.65

1.5 3.2 0.9 2.05 6 0.9 0.0 0.45

3 0.6 6.2 3.40 2 0.0 0.0 0.00

6 0.5 1.7 1.10 18 0.0 0.0 0.00

9 0.6 0. 0 0.30 24 TOXIC

EMS 200 158.

3 149.1 153.70

3

- MC 4 284.

6 278.1 281.35

MFS = 6

- TG resistant mutants/10 6 viable cells

Remarks:

CONCLUSIONS

Remarks: The test material was found to be non

- mutagenic to CHO cells at the HGPRT

locus under the condit ions of this test.

DATA QUALITY

• Reliabilities

: 1, Reliable without restrictionRemarks: Study conducted under GLP to OECD test guideline by SafePharm Laboratories

Ltd.

REFERENCES (Free Text)

Safepharm Laboratories Ltd., Cardolite NC 511: CHO HGPRT For ward Mutation Assay,

Report No. 814/003, 1996

Cole, J., et al, (1990): Gene Mutation in Cultured Mammalian Cells. In ‘Basic Mutagenicity

Tests: UKEMS Recommended Procedures’, (ed D.J. Kirkland), Cambridge University Press,

New York,

OTHER

• Last Changed: 26 April 2002

• Order number for sorting: 3

Remarks :