Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Nopol was not mutagenic in an Ames test conducted according to Guideline OECD 471.

No chromosome aberrations were induced in a study conducted according to Guideline OECD 473.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 February to 31 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
None
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 (plate-incorporation method):
- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):
- TA1535, TA1537, TA98, TA100 and TA102: 25, 50, 100, 200, 400, 800 and 1600 μg/plate, with and without S9-mix

Experiment 3 (preincubation method):
- TA1535, TA1537, TA98, TA100 and TA102: 6.25, 12.5, 25, 50, 100, 200 and 400 μg/plate, with S9-mix
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Nopol was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 100 mg/mL. Therefore, DMSO was selected as vehicle.
- Test substance preparation: Test substance stock solutions were prepared by formulating Nopol under subdued lighting in DMSO with the aid of vortex mixing as required, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 4 h of initial formulation.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and conditions:
SOURCE OF TEST SYSTEM:
Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS:
- Vehicle and positive controls were included in quintuplicate and triplicate plates, respectively.
- Treatment (test item) groups were included in triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

OTHER:
- Strain characteristics: The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline). Checks were carried out according to Maron and Ames, 1983 and De Serres and Shelby, 1979.
- Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles, splits in the agar or contamination affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), 2-fold (in strains TA98 or TA100) or 3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance (OECD, 1997; ICH S2(R1), 2011).
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed on the test plates during scoring, in any of the experiments performed.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the laboratory’s historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Following the treatment, evidence of toxicity in the form of a slight thinning of the background bacterial lawn, was observed at 500 μg/plate in all strains in the absence and presence of S-9. A complete killing of the test bacteria was observed at 1600 μg/plate and above in all strains in the absence and presence of S-9. In addition, a reduction in revertant numbers was observed at 160 μg/plate in strain TA1537 only in the absence and presence of S-9.
- Experiment 2: Following the treatment, evidence of toxicity ranging from a diminution of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed in all strains from 800 and from 400 μg/plate in the absence and presence of S-9, respectively. In addition, a reduction in revertant numbers was observed at 400 μg/plate in strain TA1537 in the absence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for all tester strains due to toxicity, a further Experiment (Experiment 3) was performed.
- Experiment 3: Following the treatment, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a concurrent marked reduction in revertant numbers, to a complete killing of the test bacteria was observed at 200 μg/plate and above in all strains.

None

Conclusions:
The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains, with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to the test item, Nopol, at the concentrations below. 

Experiment 1 (plate-incorporation method):

- TA1535, TA1537, TA98, TA100 and TA102: 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, with and without S9-mix

 

Experiment 2 (plate-incorporation method without S9 mix; preincubation method with S9 mix):

- TA1535, TA1537, TA98, TA100 and TA102: 25, 50, 100, 200, 400, 800 and 1600 μg/plate, with and without S9-mix

 

Experiment 3 (preincubation method):

- TA1535, TA1537, TA98, TA100 and TA102: 6.25, 12.5, 25, 50, 100, 200 and 400 μg/plate, with S9-mix

 

Metabolic activation system was S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive controls were also included.

 

In Experiment 1, evidence of toxicity was observed at 160 and/or 500 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, evidence of toxicity was observed at 800 μg/plate and above in all strains in the absence of S-9 and at 400 μg/plate and above in the presence of S-9 in all strains and in strain TA1537 in the absence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for all tester strains due to toxicity, a further Experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 200 μg/plate and above in all strains.

 

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Therefore, the test item is not considered as mutagenic in this bacterial system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
None
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats
Test concentrations with justification for top dose:
Preliminary toxicity test: 3.265; 5.442; 9.070; 15.12; 25.19; 41.99; 68.98; 116.6; 194.4; 324.0; 540.0; 900.0 µg/mL

Main tests:
+/-S9 mix (3 hours): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180, 200, 300 µg/mL
+ S9 (3 hours) : 30, 50, 70, 90, 110, 125, 135, 145, 155, 165, 175, 185, 205 and 310 µg/mL
+S9 mix (20 hours): 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, µg/mL
+ S9 mix (3 hours) (additional test): 25, 50, 114, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulphoxide)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix: 0.3 and 0.4 μg/mL (3-hour treatment); 0.05 and 0.1 μg/mL (20-hour continuous treatment)
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix: 0.1 and 0.2 μg/mL
Details on test system and conditions:
PREPARATION OF CULTURES:
- Human blood was collected aseptically from three healthy, non-smoking, adult donors, and diluted with HEPES buffered RPMI medium. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 10 mL aliquots (in sterile universal containers) so that each contained blood , HEPES buffered RPMI medium and PHA solution (0.2 mL). All cultures were then incubated at 37±1 °C, and the cells resuspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HEPES buffered RPMI medium, supplemented with 10% inactivated foetal calf serum, 0.52% penicillin /streptomycin.

DURATION
- Exposure duration: Preliminary toxicity test: 3 h (±S9); 20 h continuous treatment (-S9); Main test: 3 h (±S9); 20 h continuous treatment (-S9)
- Fixation time (start of exposure up to harvest of cells): 20 h, with and without S9 mix in preliminary toxicity and main tests

SPINDLE INHIBITOR (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 1 µg/mL.

STAIN (for cytogenetic assays): Giemsa staining (4% (pH 6.8))

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Duplicate cultures were used for treatment with the vehicle, and test substance for each test condition.
- Main test: Duplicate cultures were used for treatment with, test substance and positive controls and quadriplate for the vehicle.

NUMBER OF CELLS EVALUATED:
- The proportion of mitotic cells per 1000 cells in each culture was recorded (except for when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
- One hundred metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 10 metaphases with structural aberrations (excluding gaps) were observed. In this study scoring was truncated only for the positive control cultures. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded, independently from the analysis for chromosome aberrations.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1.A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990).
Statistics:
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973). Statistical significance was declared with p-value .p inferior or egual to 0.05

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
At 200 and 300 µg/mL without S9 (3 hours)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No marked changes in osmolality or pH were observed at the highest concentration tested (900 µg/mL for 3+17 hour + S-9 cultures and 20+0 hour –S-9 cultures) as compared to the concurrent vehicle controls (individual data not reported).

PRELIMINARY TOXICITY TEST:
- In the absence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 33% of the vehicle control value at 116.6 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate may not have been detected.
- In the presence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 26% of the vehicle control value at 116.6 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.
- In the absence of S9 mix following 20-hour continuous treatment, test item caused a reduction in the mitotic index to 100% of the vehicle control value at 116.6 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.

MAIN TEST:

Cytotoxicity:
- In the absence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to 100% of the vehicle control value at 180 μg/mL. The concentrations selected for metaphase analysis were 80, 120, 140 and 150 μg/mL. . No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate have been detected at concentration of 120 µg/mL and above.
- In the presence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to72% of the vehicle control value at 205 μg/mL.
Since the required toxicity was not obtained in the 3+17 hour treatments in the presence of S-9, a further experiment was conducted.

- In the absence of S9 mix, 20-hour continuous treatment: Test item caused a reduction in the mitotic index to 82% of the vehicle control value at 100 μg/mL. The concentrations selected for metaphase analysis were 10, 25, 50 and 65 μg/mL. No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate may not have been detected.

A suitable toxicity range including a non-toxic concentration was not achieved therefore no metaphase analysis was conducted. No notable culture medium changes were observed, when compared to the vehicle control.
- Additional main test: 03-hour continuous treatment in the presence of S9 mix: Test item caused a reduction in the mitotic index to 99% of the vehicle control value at 225 μg/mL. The concentrations selected for metaphase analysis were 115, 145,175 and 195 μg/mL No notable culture medium changes were observed, when compared to the vehicle control.

Metaphase analysis:
3-hour treatment in the absence of S9 mix:
- No statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control.
- Sporadic increases in the frequency of cells with polyploidy, which exceeded the concurrent vehicle controls and the normal ranges, were observed in a single culture at 150 µg/mL following 3+17 hour treatments in the absence of S-9

3-hour treatment in the presence of S9 mix
- No statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control.
- Sporadic increases in the frequency of cells with polyploidy, which exceeded the concurrent vehicle controls and the normal ranges, were observed in a single culture at 175 µg/mL and at 195 µg/mL (duplicate cultures) .

20-hour continuous treatment in the absence of S9 mix
- No statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control.

Table 7.6.1/1: Summary of results

Experiment 1:


Treatment
(mg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

A/C

B/D

MIH*

A/C

B/D

MIH*

Vehicle

13.2/14.1

11.6/14.5

-

14.5/11.7

12.1/11.5

-

20.00

13.8

12.8

0

10.6

13.8

2

40.00

11.5

12.6

10

11.3

12.6

4

60.00

10.3

13.2

12

10.7

10.8

14 #

80.00

11.6

11.3

14 #

11.3

10.2

14

100.0

10.0

11.4

20

9.1

8.3

30 #

120.0

8.8

9.0

33 # P

12.0

10.0

12 P

130.0

8.7

9.0

34 P

10.4

7.5

28 P

140.0

8.2

7.5

41 # P

9.4

7.5

32 P

150.0

6.4

5.3

56 # P

11.3

8.7

20 P

160.0

4.3

3.4

71 P

6.9

7.8

41 # P

170.0

0.0

0.2

99 P

6.1

5.7

53 # P

180.0

0.0

0.0

100 P

4.2

5.7

60 P

200.0

T

T

- P

3.9

4.1

68 P

300.0

T

T

- P

0.6

0.5

96 P

T = Toxic

 *Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100 % (where T = treatment and C = vehicle control)

# Highlighted concentrations selected for analysis

Experiment 2:


Treatment
(mg/mL)

Mitotic index (%)

20+0 hours, -S-9

3+17 hours, +S-9

A/C

B/D

MIH*

A/C

B/D

MIH*

Vehicle

10.3/11.7

10.0/10.8

-

12.7/12.2

14.6/12.7

-

10.00

11.1

9.4

4 #

NT

NT

-

25.00

8.9

7.8

22 #

NT

NT

-

30.00

7.5

7.2

31

13.4

12.2

2

35.00

7.1

7.1

34

NT

NT

-

40.00

8.1

6.5

32

NT

NT

-

45.00

4.7

7.1

45

NT

NT

-

50.00

6.4

7.1

37 #

12.9

11.0

8

55.00

5.0

6.1

48

NT

NT

-

60.00

4.5

6.7

48

NT

NT

-

65.00

5.1

4.6

55 #

NT

NT

-

70.00

4.0

3.8

64

11.0

11.0

16

75.00

3.8

3.7

65

NT

NT

-

80.00

3.0

2.9

72

NT

NT

-

90.00

NT

NT

-

10.5

11.5

16

100.0

1.9

2.0

82

NT

NT

-

110.0

NT

NT

-

10.3

11.4

17

125.0

NT

NT

-

11.3

7.8

27

135.0

NT

NT

-

9.3

8.0

34

145.0

NT

NT

-

11.9

9.9

16

155.0

NT

NT

-

9.7

8.0

32

165.0

NT

NT

-

7.6

9.3

35

175.0

NT

NT

-

9.5

7.1

36

185.0

NT

NT

-

9.4

6.3

40

205.0

NT

NT

-

4.3

2.9

72

310.0

NT

NT

-

T

T

-

NT = Not tested 

T = Toxic

 *Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

 # Highlighted concentrations selected for analysis

 

Experiment 2 trial 2:


Treatment
(mg/mL)

Mitotic index (%)

3+17 hours, +S-9

A/C

B/D

MIH*

Vehicle

9.8/9.6

9.3/10.5

-

25.00

12.0

9.9

0

50.00

7.7

10.1

9

115.0

8.9

8.7

10 P#

125.0

7.7

7.8

21 P

135.0

7.0

8.1

23 P

145.0

6.9

7.5

27 P#

155.0

7.5

8.2

20 P

165.0

7.6

5.6

33 P

175.0

5.0

5.5

46 P#

185.0

5.7

7.4

33 P

195.0

4.9

4.0

55 P#

205.0

1.0

2.3

83 P

215.0

0.3

0.0

98 P

225.0

0.1

0.1

99 P

 

P = Precipitation observed at treatment

 *Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100% (where T = treatment and C = vehicle control)

 # Highlighted concentrations selected for analysis

Conclusions:
Nopol did not induce chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of a rat liver metabolic activation system (S-9).

 
Executive summary:

In an in vitro chromosome aberration test performed according to Guideline OECD 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the concentrations below.

Preliminary toxicity test: 3.265; 5.442; 9.070; 15.12; 25.19; 41.99; 68.98; 116.6; 194.4; 324.0; 540.0; 900.0  µg/mL

Main tests:

+/-S9 mix (3 hours): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160, 170, 180, 200, 300 µg/mL

+ S9 (3 hours) : 30, 50, 70, 90, 110, 125, 135, 145, 155, 165, 175, 185, 205 and 310 µg/mL

+S9 mix (20 hours): 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100,  µg/mL

+ S9 mix (3 hours) (additional test): 25, 50, 114, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225 µg/mL

 

Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 1 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats.

A preliminary toxicity test was performed using a dose range of 3.265 to 900 μg/mL. Cells were exposed for a 3-hour treatment in the absence and presence of S9 mix, and a 20-hour continuous treatment in the absence of S9 mix and mitotic index data were used to determine toxicity. Based on the mitotic index data, concentrations were selected for the main test.

 

In the main test, the mitotic index was assessed for all cultures treated with the test item and the vehicle control, Ethanol. Justification for the highest analysed concentration was determined by cytotoxicity. On the basis of these data, the following concentrations were selected for metaphase analysis.

In the absence of S9 mix, 3-hour treatment: 80, 120, 140 and 150 μg/mL.

In the presence of S9 mix, 3-hour treatment: 115, 145, 175 and 195 μg/mL.

In the absence of S9 mix, 20-hour continuous treatment: 10, 25, 50 and 65 μg/mL.

 

In the absence of S9 mix following a 3-hour treatment with the test item, no statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control. Sporadic increases in the frequency of cells with polyploidy, which exceeded the concurrent vehicle controls and the normal ranges, were observed in a single culture at 150 µg/mL following 3+17 hour treatment in the absence of S-9.

 

In the presence of S9 mix following a 3-hour treatment with the test item, no statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentrations, when compared to the vehicle control. Sporadic increases in the frequency of cells with polyploidy, which exceeded the concurrent vehicle controls and the normal ranges, were observed in a single culture at 175 µg/mL and at 195 µg/mL (duplicate cultures).

In the absence of S9 mix following a 20-hour continuous treatment, no statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control.

   

Therefore, exposure to the test item for 3 hours in the absence or presence of metabolic activation, did not induce statistically significant increases in numerical aberrations in the form of polyploidy in this in vitro cytogenetic test system.

It is concluded that Nopol did not induce chromosome aberrations in cultured human peripheral blood lymphocytes, when tested to the limit of cytotoxicity in both the absence and presence of a rat liver metabolic activation system (S-9).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to Guideline OECD 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to the test item, Nopol. Metabolic activation system in this test was S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive controls were also included.

 

In Experiment 1, evidence of toxicity was observed at 160 and/or 500 μg/plate and above in all strains in the absence and presence of S-9. In Experiment 2, evidence of toxicity was observed at 800 μg/plate and above in all strains in the absence of S-9 and at 400 μg/plate and above in the presence of S-9 in all strains and in strain TA1537 in the absence of S-9 only. Since mutation data were only available for four concentrations in the presence of S-9 for all tester strains due to toxicity, a further Experiment (Experiment 3) was performed. In Experiment 3, evidence of toxicity was observed at 200 μg/plate and above in all strains.

The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. Therefore, the test item is not considered as mutagenic in this bacterial system.

Justification for classification or non-classification

The test item is not considered as mutagenic in an Ames test conducted according to Guideline OECD 471.

No chromosome aberrations were induced in a study conducted according to Guideline OECD 473.

As the results obtained with nopol were negative in an Ames test and a chromosome aberration test, the test item is not classified according to CLP Regulation (EC) No. 1272/2008 and to GHS Regulation.