Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In several Ames test with and without prival modification conducted equivalent or according to OECD 471 no genotoxicity was seen. Also a mouse lymphoma assay and an UDS assay in rat hepatocytes yielded negative results. In two in vitro chromosome aberration assasy conducted equivalent or according to OECD guideline 473 no increase in structural chromosome aberrations was seen. Based on the data of these studies the test substance was not considered to be genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
oct - nov 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor induced rat liver and S9 mix from hamster supplemented with flavin mononucleotid
Test concentrations with justification for top dose:
0, 4, 20, 100, 500, 2500, 5000 microgramm/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMS
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
congo red
other: 2-Aminoanthracene (TA 98, TA 100, TA 1535, TA 1537, with S9 mix), 2-Aminoanthracene (Prival-Test: TA 100, TA 1535, TA 1537, with S9 mix), Benzidine (Prival-Test: TA 98, with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), preincubation (prival test)

DURATION
- Preincubation period: 30 min
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 2 independent experiments of each protocol

NUMBER OF CELLS EVALUATED: all colonies (his+ revertants) were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity tests were performed with all tester strains using two plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically.
Conclusions:
Interpretation of results:
negative

It is concluded the the test substance is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure. Also in the preincubation method to Prival the test compound is not mutagenic in the presence of hamster liver S-9.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
june - aug 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor induced rat liver and S9 mix from untreated syrien hamster liver supplemented with FMN (Riboflavin-5'- ate-sodium-salt)
Test concentrations with justification for top dose:
0, 4, 20, 100, 500, 2500, 5000 microgramm/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
congo red
other: 2-Aminoanthracene (TA 98, TA 100, TA 1535, TA 1537, with S9 mix), 2-Aminoanthracene (Prival-Test: TA 100, TA 1535, TA 1537, with S9 mix), Benzidine (Prival-Test: TA 98, with S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation at prival test

DURATION
- Preincubation period: 30 min
- Exposure duration: 48-72h

NUMBER OF REPLICATIONS: 2 independent experiments for each protocol

NUMBER OF CELLS EVALUATED: all revertant colonies were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative growth

Sterility check:
- Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S9 mix sterility check plates
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: starts at 500 microgramm/plate

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity tests were performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically.
Conclusions:
Interpretation of results:
negative

It is concluded that the test substance is not mutagenic in the absence and presence of rat S-9 Mix using the standard Ames Test procedure. Also in the presence of hamster liver S-9 Mix and preincubation the test compound did not induce a significant increase in the number of revertant colonies.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
october 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of Aroclor induced rat liver
Test concentrations with justification for top dose:
30, 150 and 300 microgramm/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18h
- Fixation time (start of exposure up to fixation or harvest of cells): 4.5 15.5 and 25.5 h after the start of the treatment colcemide was added (0.04 ug/ml culture medium), 2.5 h later the cells were trypsinised

SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 2 % orcein solution

NUMBER OF REPLICATIONS: singly

NUMBER OF CELLS EVALUATED: 100 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- The toxi city of the test substance was determi ned ina pre1iminary experiment by establishing the concentration-related plating efficiency
Evaluation criteria:
The evaluation of the results was performed as follows:
- the test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested
- the significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (Binomial statistic with Fisher's exact test)
- the test substance is cl ass ifi ed as mutagen i c if there is a reproducible concentration related increase in the aberration rate
- the test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.
Statistics:
Not necessary to perform as all mean chromosome aberration rates after treatment wit the test article were in the range of the negative control value.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity experiment was performed in order to select appropriate dose leve1s for the mutagenicity study. The test substance produced a signifiant cytotoxic effect (reduction of plating efficiency) with and without metabolic activation from 400 ug/ml up to the limit of solubility (500ug/ml).
Conclusions:
Interpretation of results:
negative

In conclusion the test substance does not induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, neither in the presence nor in the abse ce of a metabolic activation system, under the experimental conditions described.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from induced Hamster liver
Test concentrations with justification for top dose:
3 - 5000 microgramm/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
congo red
methylmethanesulfonate
other: 2-Aminoanthracene (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 48h, 37°C
- The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.

NUMBER OF REPLICATIONS: two independent experiments, in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in number of revertants
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experirnent.
A dose dependent increase in the number of revertant colanies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: from 800ug/plate onward

COMPARISON WITH HISTORICAL CONTROL DATA: yes, historical control data from January 2011 until December 2011, WP2 uvrA the historical data are based on approx. 200 experiments, TA 98 positive control with congo red historical data are based on approx. 50 experiments (2007 - 2010)

Table 1: Results Experiment I

S9 mix

Test group

Dose level [µg/plate]

Revertant Colony Counts [Mean ± SD]

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-

DMSO

/

13 ± 4

17 ± 4

32 ± 5

128 ± 13

51 ± 1

Untreated

17 ± 6

20 ± 4

26 ± 8

158 ± 21

52 ±11

Test item

3

10

33

100

333

1000

2500

5000

16 ± 4

12 ± 2

12 ± 5

13 ± 1

13 ± 4

13 ± 3P

12 ± 3P

10 ± 2P

15 ± 4

15 ± 2

14 ± 2

15 ± 1

17 ± 2

13 ± 6P

13 ± 2P

12 ± 4PCM

26 ± 3

25 ± 4

27 ± 4

31 ± 5

30 ± 6

34 ± 4P

28 ± 3P

43 ± 1P

125 ± 6

128 ± 21

126 ± 11

137 ± 4

129 ± 14

111 ± 16P

118 ± 8P

136 ± 3P

48 ± 3

52 ± 12

47 ± 15

48 ± 5

54 ± 8

60 ± 11P

65 ± 6P

86 ± 6PM

NaN3

10

2185 ± 111

/

/

2219 ± 117

/

4-NOPD

10

50

/

 

72 ± 1

296 ± 8

 

/

/

MMS

3

/

/

/

/

896 ± 36

+

DMSO

/

19 ± 5

28 ± 2

39 ± 7

122 ± 9

52 ± 7

Untreated

14 ± 2

29 ± 4

47 ± 7

116 ± 13

48 ± 5

Test item

3

10

33

100

333

1000

2500

5000

23 ± 7

18 ± 9

22 ± 5

22 ± 8

18 ± 7

20 ± 5P

20 ± 1P

20 ± 7P

29 ± 8

32 ± 7

27 ± 8

33 ± 2

32 ± 5

31 ± 8P

31 ± 5P

32 ± 6P

39 ± 2

39 ± 10

42 ± 1

37 ± 7

41 ± 13

43 ± 10P

51 ± 3P

46 ± 10P

123 ± 6

114 ± 6

116 ± 12

125 ± 13

114 ± 12

140 ± 23P

182 ± 17P

222 ± 10PM

58 ± 7

42 ± 11

58 ± 4

58 ± 13

56 ± 8

60 ± 19P

49 ± 4P

52 ± 10P

2-AA

2.5

2.5

10

655 ± 47

104 ± 17

/

2323 ± 13

 

 

 

 

304 ± 52

Congo red

500

/

/

1141 ± 233

/

/

NaN3sodium azide

2-AA 2-aminoanthracene

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

P Precipitate

M Manual count

C Contaminated

Table 2: Results Experiment II

S9 mix

Test group

Dose level [µg/plate]

Revertant Colony Counts [Mean ± SD]

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

-

DMSO

/

16 ± 2

22 ± 2

30 ± 9

167 ± 3

39 ± 5

Untreated

13 ± 4

21 ± 3

32 ± 9

167 ± 15

53 ± 8

Test item

8.2

20.5

51.2

128

320

800

2000

5000

11 ± 2

16 ± 3

16 ± 4

13 ± 6

13 ± 3

13 ± 2P

11 ± 3P

13 ± 3P

16 ± 3

22 ± 1

21 ± 2

20 ± 6

23 ± 4

14 ± 3P

16 ± 4P

15 ± 3P

33 ± 10

33 ± 3

29 ± 10

30 ± 10

27 ± 4

31 ± 3P

40 ± 6P

48 ± 5PM

149 ± 11

126 ± 15

128 ± 11

135 ± 21

120 ±8

120 ± 12P

112 ± 13P

123 ± 9P

44 ± 2

53 ± 9

43 ± 4

42 ± 1

49 ± 7

52 ± 9P

63 ± 10P

71 ± 8P

NaN3

10

1911 ± 25

/

/

2217 ± 28

/

4-NOPD

10

50

/

 

77 ± 7

310 ± 16

 

/

/

MMS

3

/

/

/

/

704 ± 29

+

DMSO

/

23 ± 3

25 ± 4

52 ± 7

136 ± 16

55 ± 4

Untreated

21 ± 7

26 ± 0

50 ± 6

132 ± 10

61 ± 8

Test item

8.2

20.5

51.2

128

320

800

2000

5000

20 ± 4

19 ± 2

20 ± 5

26 ± 3

24 ± 5

20 ± 4P

22 ± 6P

25 ± 8P

31 ± 2

26 ± 6

30 ± 6

36 ± 6

32 ± 5

31 ± 4P

32 ± 2P

32 ± 3P

55 ± 5

49 ± 10

48 ± 14

55 ± 7

49 ± 7

52 ± 5P

54 ± 2P

56 ± 9P

109 ± 15

127 ± 20

118 ± 15

108 ± 1

124 ± 19

126 ± 12P

153 ± 18P

204 ± 30P

56 ± 13

50 ± 6

53 ± 11

52 ± 6

56 ± 18

55 ± 3P

47 ± 7P

60 ± 12P

2-AA

2.5

2.5

10

436 ± 146

80 ± 1

/

2239 ± 33

 

 

 

 

215 ± 18

Congo red

500

/

/

976 ± 16

/

/

NaN3sodium azide

2-AA 2-aminoanthracene

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

P Precipitate

M Manual count

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
and guideline OECD 479
Principles of method if other than guideline:
NTP protocol and Galloway et al., 1985, 1987a
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of Aroclor induced rat liver
Test concentrations with justification for top dose:
0, 37.1, 50, 123.8 mug/ml without metabolic activation (chromosome aberration assay)
0, 50, 123.8, 250 mug/ml with metabolic activation (chromosome aberration assay)

0, 5, 16.7, 50 mug/ml without metabolic activation (SCE assay)
0, 50, 166.7, 500 mug/ml with metabolic activation (SCE assay)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
1. chromosome abarration assay:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without metabolic activation for 8h, with metabolic activation for 2h + 8h after washing
- Fixation time: 2-2.5h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per dose

NUMBER OF CELLS EVALUATED: 200

2. SCE assay

- exposure time: without metabolic activation 25h, with metabolic activation 2h
- BrdUrd: 10 muM were added 2h after dosing
- Fixation time: 2-2.5h
- total incubation time: 27-28h

NUMBER OF CELLS EVALUATED: 50
NUMBER OF CHROMOSOMES EXAMINED: app. 1000

fixation: 3:l methano1:glacial acetic acid
staining: Hoechst 33258
Evaluation criteria:
In the SCE assay an increase of 20% or greater increase in SCE per chromosome over the solvent control was considered significant. A significant increase in the aberration assay was based on a binomial sampling assumption [Margolin et al., 1986]
Trials with two or more significant doses were considered positive (+), and trials with one significant dose and a significant trend were judged as having weak evidence of a positive response (+ W). Trials with a significant response at one dose and no significant trend, and trials with no significant responses but having a significant trend were considered equivocal (?).
Statistics:
P values were adjusted according to Dunnett’s method to take into account multiple dose comparisons. The trend test for both assays used a linear regression analysis: SCE per chromosome vs. the log dose, and the percentage of cells with aberrations vs. the log dose
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not clastogenic, no sister chromatide exchange
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Chemicals were tested up to 5 mg/ml or as limited by solubility and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent (water, dimethyl sulfoxide, acetone, or ethanol, in that order of preference).

The pH of the test chemical solution diluted in the test culture media was measured and was found to be in the range of 7.0-7.5 for all chemicals.

Table 1: Summary of results SCE

S9 mix

Dose [µg/mL]

Total chromosomes

Total SCE

SCE per cell

-

0.0000

1018

419

8.36

5.0000

1010

399

7.98

16.7000

1002

417

8.34

50.0000

1016

399

7.98

MMC 0.0010

1015

500

10.00

MMC 0.0100

99

102

20.40

+

0.0000

1015

464

9.28

5.0000

1009

367

7.34

166.7000

992

386

7.72

500.0000

1008

455

9.10

CPA 0.4000

1019

612

12.24

CPA 2.0000

100

147

29.40

Table 2: Summary of results CA

S9 mix

Dose [µg/mL]

Cells

Cells with aberrations [%]

Total

Simple

Complex

-

0.0000

200

2.00

1.00

1.00

32.10

200

2.00

1.00

0.00

50.0000

200

3.00

2.00

1.00

127.0000

200

3.00

2.00

1.00

MMC 0.2500

200

7.00

5.00

3.00

MMC 0.7500

25

35.00

24.00

12.00

+

0.0000

200

4.00

3.00

1.00

50.0000

200

1.00

0.00

1.00

123.0000

200

3.00

2.00

1.00

250.0000

200

2.00

2.00

0.00

CPA 7.5000

200

8.00

5.00

3.00

CPA 37.5000

25

44.00

72.00

20.00

Conclusions:
Interpretation of results:
negative

D&C Red 9 did not induce SCE or chromosomal aberrations when tested to toxicity. Precipitate was evident at doses of 250 mug/ml and above.
Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
no
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
primary culture, other: rat hepatocyte
Test concentrations with justification for top dose:
0, 10, 100 and 1000 muM
Vehicle / solvent:
DMSO
final concentration did not exeed 1%
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
Acid Red 14 (Carmoisine)
Positive controls:
yes
Positive control substance:
other: Solvent Yellow 3 (o-aminoazotoluene)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Hepatocyte preparation: isolated and cultured by the two-step in situ liver perfusion method described by Maslansky and Williams [1982]. Approximately 2 x 10 viable hepatocytes were seeded into 25-mm wells and were allowed to attach to Thermanox@ plastic coverslides (Flow Labs, Rockville, MD) for 2 hr.

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): overnight

Analysis/measurement: quantified by the autoradiographic determination of incorporated [3H]-thymidin. Net nuclear grains (NNG) were determined by counting the number of grains in each nuelei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random seleetion of cells was used. The average NNG for 60 cells (±SD) as well as the percentage of cells with > 5 NNG was determined for each dye concentration.
Evaluation criteria:
Average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since diese differed from the control net nuclear counts by greater than 2 SD. Net nuclear grain counts below zero were considered negative responses. For those dyes that produced responses between zero and 5 average net nuclear grains, it was generally not possible to demonstrate a statistically significant difference from the control value within a given experiment. Therefore, these responses were judged to be equivocal, unless, in addition to an average net nuclear grain count between zero and 5, at least 25% of the cells examined contained > 5 net nuclear grains, in which case the response was considered weakly positive. Concentrations of the dyes that produced approximately 90% or greater detachment of die hepatocytes from the coverslips (as assessed visually by comparing to control stides) were assumed to be toxic and were not counted.
Species / strain:
primary culture, other: rat hepatocyte
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 muM
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver
Test concentrations with justification for top dose:
0, 1.25, 2.5, 5, 7.5 and 15 mug/ml trial 1 without metabolic activation
0, 1.25, 2.5, 5, 7.5, 10 and 15 mug/ml trial 2 without metabolic activation
0, 2, 3, 4, 5, 6 mug/ml trial 1 with metabolic activation
0, 2, 3, 4, 5, 6 and 8 mug/ml trial 2 with metabolic activation
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression: 2d, viable cell densities were determined by hemacytometer each day using trypan blue dye exclusion.

NUMBER OF REPLICATIONS: test substance and positive control tested in triplicate, 5 solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A chemical is evaluated for a test condition (-S9, +S9, +NS9) only if two or more acceptable experiments are available

1. Positive (+)
I. Replicate experiments are positive
II. Questionable experiments are reproducible

2. Questionable (?)
I. Replicate experiments yield results that just meet or just fail the tests for significance
II. Replicate experiments are evaluated as positive or not positive (= or -) and no reason exists to subordinate either evaluation

3. Negative (-1)
Replicate experiments are not positive (= or -)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary studies of test chemical solubility and cytotoxicity were conducted prior to performing the first mutation experiment. The solubility of the test chemical in treatment medium was examined carefully in clear tubes and without cells; centrifugation and microscopic examination were sometimes used to detect suspensions of fine particles. Changes in pH were noted by the color of the phenol red indicator in the medium, but, unless otherwise noted, no pH adjustments were made. Test chemical toxicity to 24-hr cell suspension growth was determined for 4-hr treatments with a range of doses up to a maximum of 5,000 pg/ml.

Table 1: Summary of results

Trial

S9 mix

Conc. [µg/mL]

CE

RTO

MC

MF

AVG MF

1

-

DMSO

0

 

 

70

61

83

83

103

92

98

107

62

67

59

63

27

28

24

25

26

1.25

75

85

73

63

75

67

82

100

60

35

39

28

34

2.5

105

84

91

79

70

71

95

76

59

30

30

33

31

5

98

101

105

115

82

98

53

88

97

18

29

31

26

7.5

93

91

104

85

75

89

95

114

88

34

42

28

35

10

92

104

83

83

64

64

23

20

22

15

85

86

86

74

85

77

71

59

89

27

23

27

26

MMS

35

40

51

20

30

40

164

634

611

155

524

403

361

+

DMSO

0

 

 

75

87

73

98

101

105

93

101

56

64

74

76

25

25

34

26

27

2

111

92

100

102

79

62

73

62

62

22

30

27

26

3

89

92

95

90

94

100

54

57

66

20

21

23

21

4

99

110

111

90

117

98

54

58

39

18

18

12

16

5

105

110

111

97

105

93

66

77

107

21

23

32

26

6

105

94

116

105

111

76

78

68

72

25

31

21

26

8

108

117

113

63

71

86

22

25

23

MCA

99

72

91

78

89

81

450

359

448

152

166

164

160

2

-

DMSO

0

 

 

88

93

87

96

87

115

91

107

88

94

85

72

34

34

33

25

31

1.25

76

83

91

83

103

83

69

84

86

30

34

32

32

2.5

94

94

96

94

105

85

96

84

109

34

30

38

34

5

92

96

99

90

86

111

93

119

70

34

41

24

33

7.5

109

103

96

88

90

84

141

126

137

43

41

48

44

10

104

99

99

95

70

71

139

141

119

44

47

40

44

15

89

113

121

88

96

97

101

112

143

38

33

40

35

MMS

44

78

51

24

51

32

439

517

399

335

221

263

273

+

DMSO

0

 

 

107

121

114

106

94

90

95

111

130

106

94

106

41

29

27

33

34

2

94

125

104

75

93

61

95

117

146

34

31

47

40

3

114

112

115

65

75

80

152

126

121

45

37

35

39

4

110

102

101

81

78

74

154

130

163

47

42

54

48

5

94

112

111

83

83

79

119

158

167

42

46

50

46

6

113

93

117

72

63

76

164

119

108

48

43

31

41

8

110

113

122

60

83

94

154

121

145

47

36

40

41

MCA

53

86

57

19

22

17

622

816

807

392

315

471

393

Conclusions:
Interpretation of results:
negative

D & C red 9 was not toxic or mutagenic to L5178Y cells, with or without the addition of S9 mix. No significant increases in the MF were observed. The solubility limit in Fischer’s medium was 7.5 microg/ml, but concentrations up to 15 microg/ml (without S9) were tested without any toxic effects.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo Micronucleus test according to OECD guideline 475 as well as a Drosophila SLRL and an UDS in vivo gave also no positive results. Based on the data of these studies the test substance was not considered to be genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: male from Canton-S, female from Basc stocks
Sex:
male/female
Details on test animals and environmental conditions:
Separate Canton-S and Basc stocks were maintained at Brown University and the University of Wisconsin. Males to be exposed were collected from theCanton-S stocks. The Basc stocks supplied the balancer X-chromosome. P1 females used in the SLRL test were collected as virgins from this stock.
Route of administration:
oral: feed
Vehicle:
DMSO
Details on exposure:
All nongaseous compounds were first tested by feeding exposure. Two or three glass fiber filter discs were saturated with the compound carried in a 5 % sucrose solution (or other control solution) at the bottom of a standard glass vial. Solutions were renewed at 24 hr and 48 hr. After 72 hr of exposure, surviving males were mated. If feeding exposures were found to be nonmutagenic, 2-3-day-old Canton-S males were injected with 0.7 % NaCl solution containing the test chemical. At 24 hr postinjection, toxicity was noted and survivors were mated. Concurrent control males were treated with the solution used to dissolve the test chemical.
Duration of treatment / exposure:
72h (oral), in addition non-mutated survivors were treated by injection
Frequency of treatment:
feed: permanent
injection: once daily
Post exposure period:
24h (after injection)
Dose / conc.:
2 500 ppm
Remarks:
nominal in diet
Dose / conc.:
1 000 ppm
Remarks:
adult injection
No. of animals per sex per dose:
P1: one male pared with 100 females (feed, 2500 ppm), 10 males and 20 females (injection, 1000 ppm)
Control animals:
yes, concurrent vehicle
Positive control(s):
4 substances were juged to be mutagenic in D. melanogaster (Gene-Tox report, EPA) and serve as positive control: 1,2-epoxypropane, ethylene bromide, and myleran (were mutagenic in this study), 2-chloro-l,3-butadiene (was not mutagenic in this study)
- Justification for choice of positive control(s): Gene-Tox report, EPA
- Route of administration:feed in diet
- Doses / concentrations: 2500 ppm
Evaluation criteria:
For a compound to be considered mutagenic, the mutant frequency in the treated series (treated frequency) must exceed 0.15 % with a P value of <0.05, or the treated frequency must exceed 0.10 % with a P value of <0.01. If the treated frequency is between 0.10 % and 0.15 % and the P value is between 0.1 and 0.01, or if the treated frequency is higher than 0.15 % and the P value is between 0.1 and 0.05, the result is considered equivocal. All other results are considered negative.
Translocation data for each treated sample were compared to the historical control data for that laboratory using a conditional binomial test [Kastenbaum and Bowman, 1970]. As a rule, at least two translocations are required among -5,000 tests in the treated series for a compound to be considered positive.
Statistics:
For the SLRL assay, a minimum of -5,000 chromosomes was scored from each of the treated and concurrent control groups unless the mutant frequency exceeded 1%. If two or more lethals were recovered among the progeny of one male, a Poisson analysis [Owen, 1962] was performed to
determine if these were part of a “cluster.” (A cluster is defined as a group of mutated sperm cells derived from a single mutational event occurring in a spermatogonial cell.) Clusters must be spontaneous in origin, because only meiotic and postmeiotic stages of spermatogenesis were treated. Therefore, in those cases in which a male was determined to have produced a cluster, the lethal and nonlethal tests for that PI male were removed from the data. The corrected treated and control data were compared using a normal approximation to the binomial distribution, as suggested by Margolin et al. [ 1983]. In addition, the treated data were compared to the historical control as described by Mason et al. [1992].
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
other: 3 of 4 positive controls were mutagenic
Additional information on results:
Chemicals were received coded from the NTP chemical repository. Solubility was determined by testing first in distilled water. If the chemical was not sufficiently soluble in distilled water, other solvents were tested in the following order of preference: ethanol, polysorbate, dimethyl sulfoxide (DMSO), rapeseed oil, or a mixture of these. In feeding exposures, palatability was noted based on feeding behavior and the presence of “fly specks” on the exposure vial.
Toxicity tests were run on a series of exposures and, if possible, an exposure level was chosen that resulted in -30% mortality after 72 hr of feeding or 24 hr after injections or inhalation.

Table 1: Results of the Sex-linked recessive lethal assay on coded chemicals

Dose

ROA

Mortality [%]

Sterility [%]

Lethals

Tests

Total lethals

Total tests

Lethals [%]

Br 1

Br 2

Br 3

Br 1

Br 2

Br 3

2.500

Feeding

0

0

3

3

0

2.860

2.185

1.608

6

6.653

0.09

0

2

2

1

2.691

2.262

1.616

5

6.569

0.08

1.000

Injection

15

4

2

3

3

2.214

1.592

1.449

8

5.255

0.15

0

4

1

0

2.045

1.933

1.475

5

5.453

0.09

 

Conclusions:
Interpretation of results:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Piebald Virol Glaxo, inbred hooded
Sex:
male
Details on test animals and environmental conditions:
Male PVG (Piebald Virol Glaxo, inbred hooded) rats were used aged between 8 and 10 weeks. These were provided by the Rodent Breeding Unit, Glaxo Group Research Ltd. The animals were maintained on a No. 1 SDS (Special Diet Services) diet and water ad libitum.
Route of administration:
oral: gavage
Vehicle:
The compound was not soluble in water, and so was suspended in corn oil with ultrasonication.
Details on exposure:
volume 10 ml/kg bw
Duration of treatment / exposure:
24h and 48h
Frequency of treatment:
once daily
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 per dose
11 for vehicle control
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CAS No. 6055-19-2; Sigma) dissolved in sterile water, 7.5 mg/kg bw, 24h exposure, 4 animals
Tissues and cell types examined:
24 or 48 h after a single oral dose, animals were killed by cervical dislocation and bone marrow smears made according to standard procedures (Pascoe and Gatehouse, 1986).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In both in vivo studies, the maximum dose administered, 2000 mg/kg, is the limit dose recommended for acute oral toxicity tests (OECD, 1987), and recently recommended in the UKEMS Guidelines for the micronucleus test (Richold et al., 1990). Although ASTM guidelines for the liver UDS assay suggest a limit dose of 500-1000 mg/kg (Butterworth et al., 1987), some azo dyes, e.g. butter yellow, may only show clear activity when tested at doses greater than this (Ashby and Keen, 1985).

DETAILS OF SLIDE PREPARATION: All slides were coded prior to analysis. Slides were stained with haematoxylin and eosin (Pascoe and Gatehouse, 1986), and for each animal 2000 polychromatic erythrocytes (PE) were analysed for the presence of micronuclei, whilst 500 erythrocytes per animal were scored to determine the percentage of PE among all erythrocytes.

METHOD OF ANALYSIS: Statistical analyses of the micronucleus frequencies were performed using the likelihood ratio test (Amphlett and Delow, 1984).
Statistics:
Statistical analyses of the micronucleus frequencies were performed using the likelihood ratio test (Amphlett and Delow, 1984).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Results of bone marrow micronucleus test after treatment of male PVG rats with D and C Red No. 9

Treatment

Dose [mg/kg]

Sample time [h]

No. of animals

MNPE/1000 PE

Mean ± SD

% PE ± SD

Control (corn oil)

10 mL/kg

24

11

0.86 ± 0.81

35 ± 7

D and C Red No. 9

500

1000

2000

24

24

24

7

7

7

1.29 ± 0.49

0.93 ± 0.36

0.93 ± 0.84

37 ± 5

31 ± 4

38 ± 4

Cyclophosphamide

7.5

24

5

7.20 ± 1.96

29 ± 2

Control (corn oil)

10 mL/kg

48

11

0.82 ± 0.51

34 ± 8

D and C Red No. 9

500

1000

2000

48

48

48

7

7

7

0.57 ± 0.61

1.21 ± 0.70

2.50 ± 0.85

32 ± 7

32 ± 4

46 ± 13

Conclusions:
Interpretation of results:
negative
Executive summary:

There were no significant increases in the frequency of micronuclei at any dose, or at either time point. Furthermore there was no effect upon the percentage of PE. The positive control compound, cyclophosphamide, gave the expected result, thus validating the assay.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Piebald Virol Glaxo, inbred hooded
Sex:
male
Details on test animals and environmental conditions:
Male PVG (Piebald Virol Glaxo, inbred hooded) rats were used aged between 8 and 10 weeks. These were provided by the Rodent Breeding Unit, Glaxo Group Research Ltd. The animals were maintained on a No. 1 SDS (Special Diet Services) diet and water ad libitum.
Route of administration:
oral: gavage
Vehicle:
The compound was not soluble in water, and so was suspended in corn oil with ultrasonication.
Duration of treatment / exposure:
16h
Frequency of treatment:
once daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 per dose
2 for vehicle control
1 for positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene (CAS No. 56-96-3; EGA-Chemie, Steinheim, Germany) suspended in corn oil. 100 mg/kg bw, 16h exposure, 1 animal
Tissues and cell types examined:
16 h after dosing animals were killed with CO2. Hepatocytes were isolated and cultured according to the procedure described by Ashby et al. (1985) with the inclusion of a 0.5 mM EGTA buffer before perfusions with buffers 1 and 2 as described by Butterworth et al. (1987).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In both in vivo studies, the maximum dose administered, 2000 mg/kg, is the limit dose recommended for acute oral toxicity tests (OECD, 1987), and recently recommended in the UKEMS Guidelines for the micronucleus test (Richold et al., 1990). Although ASTM guidelines for the liver UDS assay suggest a limit dose of 500-1000 mg/kg (Butterworth et al., 1987), some azo dyes, e.g. butter yellow, may only show clear activity when tested at doses greater than this (Ashby and Keen, 1985).

DETAILS OF SLIDE PREPARATION: Slides were coded and analysed using an AMS 40-10 image analyser. Slides were coded according to the criteria of Ashby et al. (1985). 50 cells were analysed per slide, 2 slides per animal.
Statistics:
Mean and SE of Net nuclear grain (NG) count (grains over the nucleus minus the number of grains over a nuclear-sized area of cytoplasm) for every animals was determined. Mean and SE of NG of pooled animal data was determined.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Table 1: Results of the in vivo rat liver UDS assay after treatment of male PVG rats with D and C Red No. 9

Treatment

Dose [mg/kg]

Exposure [h]

n

Mean NG ± SE

% Repair

Mean NG of cells in repair ± SE

Control (corn oil)

10 mL/kg

16

2

-      4.39 ± 0.25

1

11.00

D and C Red No. 9

1000

2000

16

16

3

3

- 4.57 ± 0.27

- 4.35 ± 0.28

0

1

5.00

7.00 ± 0.10

2AAF

100

16

1

+ 7.60 ± 0.68

65

11.26 ± 0.52

Conclusions:
Interpretation of results:
negative
Executive summary:

Uniformly negative results were obtained, indicating that D and C Red No. 9 did not induce DNA repair at 1000 or 2000 mg/kg. The animal treated with 2-acetylaminoflourene (100 mg/kg) gave a highly significant response with 65% of liver cells in repair. The mean net grain data for the vehicle control group fell within the historical control range for this strain of rat in this laboratory (-3.5 mean NG _+1.3, n = 61). These results confirm the earlier negative study of Kornbrust and Barfknecht (1985) in which lower doses of compound were used.

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

Additional information

Procedure and observations

Pigment red 53:1 has been tested for genotoxicity in a series of Ames tests with and without metabolic activation including the Prival test (Ciba 1985, Zeiger et al 1988, Brown et al 1979, Hoechst 1985, Hoechst 1985, Hoechst 1989), in the Cytogenetic assay with V 79 cells and with ovary cells of Chinese hamsters (CHO) (Ivett et al 1989, Hoechst 1989), in the Mouse lymphoma assay (Myhr et al 1991), in the Sister chromatid exchange assay with ovary cells of Chinese hamsters (CHO) (Ivett et al 1989) and the Unscheduled DNA synthesis in rat hepatocytes (Kornbrust et al 1985). In all these in vitro studies pigment red 53:1 gave negative results. Pigment red 53:1 was also assayed for genotoxicity in vivo using the rat micronucleus test (Westmoreland and Gatehouse 1992), a rat ex vivo liver UDS assays and the SLRL-test in Drosophila. Uniformly negative results were obtained in all assays, even though large oral doses were used (up to 2 g/kg).

Discussion

In various in vitro and in vivo studies pigment red 53:1 proved to be non-genotoxic. The results suggest that the tumorigenic effects of this compound in rats are mediated through a non-genotoxic rather than a genotoxic mechanism.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.