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 environment.

Bisphenol A was determined to be 3500 times less potent that 17β-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 innoculum 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: 3
TEST 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 Assay

• 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 ßgalactosidase).

• 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: 17β-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 unrealistic to test at concentrations in excess 

of 100 mg/l.

HUMAN HEALTH ENDPOINTS (NON SIDS)

OESTROGENIC ACTIVITY
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 environment.

Bisphenol A was determined to be 3500 times less potent that 17β-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 innoculum 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 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: 15 May 2002

• Order number for sorting: 2
TEST 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 guinea 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 (grade 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 the 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 HEALTH ENDPOINTS (NON SIDS)

SKIN SENSITIZATION
Desquamation 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 

precluded 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 animals 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 noted 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 observation. 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: Cardolite 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 assessment of cross-sensitisation potential with poison ivy oil, Cardolite NC513, 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: None
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: Algae

Remarks: None

RESULTS

• Unit: mg/L

• EC50 at 96 hours: 0.00011 – 0.00034 (Cardanol)

0.00031 – 0.00096 (Cardol) 

Remarks: The predicted EC50 values vary with the degree of unsaturation in the alkyl side 

chain of Cardanol and Cardol as follows:

EC50, 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, Environmental 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 replicates: 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 metabolic activation: >25 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 a significant increase in the 

frequency of cells with chromosome aberrations or polyploid cells in either the presence 

or absence of a liver enzyme metabolizing system.

Experiment 1: Harvest Time 20 hours, without metabolic activation

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

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

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

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 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 ** represents p ≤ 0.01 

Experiment 2: Harvest Time 20 hours, without metabolic activation

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

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 15

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

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

nt

Group

Replicat

e

ID

No. Cells

Scored

Total

Gaps Breaks Exchange

s

Breaks Exchange

s

X (+Gaps) (-Gaps) (+Gaps) (-Gaps)

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

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

(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, without metabolic activation

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

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

Treatme Chromatid Chromosome Others Total Aberrations Aberrant Cells

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 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 included in total aberrations) Figures in brackets = aberrations per 100 cells

Experiment 1: Mean Frequency of Polyploid Cells (%)

Dose Level 20 Hours

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 500 0.0 -

CP 25 - 0.0

Experiment 2: Mean Frequency of Polyploid Cells (%)

Dose Level Without S9 With S9

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)

Dose Without S9 With S9

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 S9
Level

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 conducted 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 chromosome 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 testing: 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)

• Statistical 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-nitroquinoline-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 metabolic 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

12 (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 

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 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 TA1537
0

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 

precipitation 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 3.5
50

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)

40p 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. 

Figures 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 5.5
150

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

Positive 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: ‘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 

precipitation was observed.

Positive Control: 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 the 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, 1995
Kirkland, D.J., (Ed), Statistical Evaluation of Mutagenicity Test Data, UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989), Cambridge 

University Press

OTHER

• 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

• Temperature 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 Abwasserforschung (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 the 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 by 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 test (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

• LC50 at 96 hours: 0.002 – 0.005 (Cardanol)

0.005 – 0.011 (Cardol)

Remarks: The predicted LC50 values vary with the degree of unsaturation in the alkyl side 

chain of Cardanol and Cardol as follows:

LC50, mg/L

unsaturated monoene diene triene

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 fish.

DATA QUALITY

• Reliabilities: 4, Not Assignable

Remarks:

Estimation using ECOSAR v0.99e

REFERENCES

ECOTOXICITY ENDPOINTS

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

Research Corporation, Environmental 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

• 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

RESULTS

• Unit: mg/L

• LC50 at 48 hours: 0.024 – 0.040 (Cardanol)

0.039 – 0.066 (Cardol)

Remarks: The predicted LC50 values vary with the degree of unsaturation in the alkyl side 

chain of cardanol and cardol as follows:

LC50, mg/L

unsaturated monoene diene triene

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 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 476

• Test Type: Forward Mutation Assay

• System of testing: Non bacterial

• 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 control: 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 activation: 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: 

Dose Level Without S9 With S9

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/106 viable cells

Experiment 2: 

Dose Level Without S9 With S9

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/106 viable cells

Remarks:

CONCLUSIONS

Remarks: The test material was found to be non-mutagenic to CHO cells at the HGPRT 

locus under the conditions of this 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: CHO HGPRT Forward 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: