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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In Vitro (Mutagenic effects - bacterial): OECD 471; Bacterial reverse mutation assay. Negative. Reliability = 1. In Vitro (Clastogenic effects - mammalian): OECD 473; Chromosome aberrations in human lymphocytes. Negative. Reliability = 1 In Vivo (Clastogenic effects - mammalian): OECD 474; in vivo rat micronucleus study; Negative at concentrations up to 50000 ppm (282233 mg/m3). Reliability = 1.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Remarks:
Conducted according to guideline dated 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine (S. typhumurium)
tryptophan (E. coli)
Species / strain / cell type:
other: TA 1535, TA 97a, TA 98, TA 100 and E. coli WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Trial 1 (with activation): 0.00%, 9.13%, 19.6%, 41.5%, 65.3%, and 92.8%
Trial 1 (without activation): 0.00%, 20.3%, 42.0%, 66.6%, and 90.4%

Trial 2 (with activation): 0.00%, 9.85%, 25.3%,48.3%,74.0%, and 102.1 %
Trial 2 (without activation): 0.00%, 10.1%, 24.4%, 46.9%, 73.5%, and 102%

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Air
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
other: 2-aminoanthracene [2AA] (CAS 613-13-8; +S9: TA100, TA1535, TA97a, TA98, WP2 uvrA)
Positive control substance:
other: 2-nitrofluorene [2NF] (CAS 607-57-8; -S9: TA98); sodium azide [NAAZ] (CAS 26628-22-8; -S9: TA100 and TA 1535); ICR-191 Acridine (CAS 17070-45-0; -S9: TA97a); methylmethanesulfonate [MMS] (CAS 66-27-3; -S9: WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Treatments with activation were conducted by adding 0.5 mL of S9 mix and 0.1 mL of an overnight culture containing 1e8 bacteria to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCI (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium strains. These components were mixed and poured onto a plate containing approximately 25 mL of Davis minimal agar with dextrose. Treatments without activation were identical to those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile phosphate buffered saline. For positive controls, 0.1 mL of the positive indicators were added to the above mixes for both activated and non-activated treatments.

Specially designed glass chambers (6 L) were used to expose the bacterial cultures to the test gas. A flow rate of approximately 10 L/minute for approximately 3 minutes was used to create approximately 5 volume changes within the chambers to insure homogenous concentrations. Chambers were closed and at least 3 samplings of each chamber were taken and analysed by gas chromatography to determine the initial concentration of the test substance. Chambers were placed into an incubator at ~37°C for ~48 hours. Chambers were again sampled and analysed to determine test substance concentration at the end of incubation. Chambers were flushed with at least 5 chamber volumes of filtered air and then refrigerated until counted. Plates were then removed for an evaluation of background lawns and colony formation.

DURATION
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 10e8

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (average number of revertants)
Evaluation criteria:
An individual trial must have included at least five test concentrations (of which at least four must have been acceptable), a solvent control, and a positive indicator for each selectee: tester strain. Data acceptability criteria were as follows:
• The density of each overnight bacterial culture was at least 1e9 cells/mL.
• A single data point may have been rejected if contamination or excessive toxicity was seen on a treatment plate. A single data point may also have been rejected if excessive precipitate on the plate prevented accurate colony counting.
• A negative control data point may have been rejected if it fell outside the acceptable spontaneous mutation range.
• A concentration level was rejected if there were less than two data points at the treatment level or if the data point values were judged by the study director to be too divergent.
• A trial for the affected strain was rejected if the negative control was rejected or if there was no evidence of mutagenic activity on any positive indicator plate or if the tester strains failed to exhibit the appropriate phenotypes.
• Only those trials which met the criteria of acceptability were included in this report.
Statistics:
For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.
Species / strain:
other: S. typhimurium TA 1535, TA 97a, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytotoxicity as measured by a significant reduction (greater than 50%) in the number of revertants was observed at the highest concentration in Salmonella typhimurium strain T A98 and Escherichia coli strain WP2 uvrA (pKMIOl) in both Trials 1 and 2 with and without activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Trial #1 A. (without Activation) Revertants per Plate

(% R116)

Strain T100

Strain T1535

Strain TA97a

Strain TA98

WP2 uvrA (pKM101)

Concentration

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

0

174, 140, 134

15, 24, 28

119, 135, 151

24, 34, 33

156, 183, 157

20.3

126, 131, 133

13, 9, 18

135, 131, 130

19, 22, 25

122, 118, 145

42

119, 116, 131

19, 23, 15

103, 89, 109

21, 19, 9

107, 108, 102

66.6

101, 103, 103

9, 19, 10

87, 78, 99

10, 10, 12

69, 78, 66

90.4

120, 95, 106

15, 19, 19

80, 87, 71

8, 6, 9

5, 7, 4

Control Info

NAAZ (2 µg/plate)

NAAZ (2 µg/plate)

ICR (2 µg/plate)

2NF (25 µg/plate)

MMS (1000 µg/plate)

 

752, 825, 809

547, 563, 554

1827, 1995, 1883

1541, 1929, 1910

1938, 2090, 2113

Note: Control abbreviations are 2AA =2-aminoanthracene, 2NF=2-nitrofluorene, NAAZ=sodium azide, 

ICR=ICR -191 Acridine, MMS=methylmethanesulfonate.

B. Trial #1 (with Activation) Revertants per Plate

(% R116)

Strain T100

Strain T1535

Strain TA97a

Strain TA98

WP2 uvrA (pKM101)

Concentration

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

0

152, 167, 159

15, 11, 23

118, 125, 95

29, 19, 21

134, 147, 130

9.13

134, 136, 126

17, 22, 28

130, 116, 126

24, 26, 19

147, 144, 147

19.6

153, 129, 142

24, 21, 20

165, 99, 145

19, 25, 24

110, 138, 115

41.5

149, 104, 115

19, 18, 22

92, 131, 102

18, 33, 20

98, 81, 74

65.3

112, 88, 126

17, 18, 18

127, 133, 112

12, 13, 19

65, 51, 60

92.8

117, 95, 82

11, 10, 12

79, 81, 68

5, 6, 8

2, 4, 14

Control Info

2AA (1 µg/plate)

2AA (2 µg/plate)

2AA (1 µg/plate)

2AA (2 µg/plate)

2AA (25 µg/plate)

 

1403, 1397, 1214

393, 371, 440

889, 942, 866

2611, 2491, 2470

1841, 2226, 2071

Table 2. Trial #2 A. (without Activation) Revertants per Plate

(% R116)

Strain T100

Strain T1535

Strain TA97a

Strain TA98

WP2 uvrA (pKM101)

Concentration

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

0

119, 107, 102

10, 12, 10

101, 97, 80

19, 21, 22

129, 141, 125

10.1

119, 114, 102

8, 9, 10

81, 84, 81

19, 16, 22

136, 148, 130

24.4

111, 107, 91

11, 13, 8

120, 97, 90

22, 21, 24

120, 127, 128

46.9

93, 83, 99

6, 13, 10

93, 107, 63

16, 19, 23

126, 125, 107

73.5

77, 73, 92

15, 11, 8

96, 77, 100

14, 18, 19

78, 84, 60

102

78, 73, 60

12, 5, 5

60, 82, 68

6, 4, 6

11, 4, 0

Control Info

NAAZ (2 µg/plate)

NAAZ (2 µg/plate)

ICR 191 (2 µg/plate)

2NF (25 µg/plate)

MMS (1000 µg/plate)

 

811, 827, 828

563, 554, 554

2058, 1914, 2000

1572, 1593, 1566

2326, 2289, 2103

B. Trial #2 (with Activation) Revertants per Plate

(% R116)

Strain T100

Strain T1535

Strain TA97a

Strain TA98

WP2 uvrA (pKM101)

Concentration

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

Plates 1, 2, 3

0

110, 124, 115

10, 7, 9

122, 97, 105

41, 25, 26

145, 126, 142

9.85

100, 101, 116

6, 6, 9

82, 87, 81

25, 30, 28

122, 126, 127

25.3

100, 83, 87

9, 14, 7

101, 99, 87

22, 22, 25

117, 130, 125

48.3

78, 83, 92

7, 12, 7

94, 107, 117

30, 29, 24

118, 118, 104

74

87, 73, 80

8, 10, 8

77, 100, 96

18, 22, 17

57, 75, 48

102

43, 62, 64

8, 13, 6

64, 64, 54

8, 5, 8

16, 7, 0

Control Info

2AA (1 µg/plate)

2AA (2 µg/plate)

2AA (1 µg/plate)

2AA (2 µg/plate)

2AA (25 µg/plate)

 

942, 957, 1011

281, 376, 337

599, 691, 720

1607, 1747, 1747

2335, 2423, 2472

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. In this study, the test substance was negative.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was evaluated for mutagenicity in  Salmonella typhimurium  strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli  strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9).

In Trial 1, cells were exposed to average concentrations of 0.00, 9.13, 19.6, 41.5, 65.3, and 92.8% with metabolic activation. Without metabolic activation, average concentrations of 0.00, 20.3, 42.0, 66.6, and 90.4% were analysed. No test substance was detected after the 48 hour incubation at the lowest targeted concentration (10%) in the treatment without activation. A leak in the chamber was suspected as the cause.

In Trial 2, cells were exposed to average concentrations of 0.00, 9.85, 25.3, 48.3, 74.0, and 102.1 % with metabolic activation. Without metabolic activation, cells were exposed to average concentrations of 0.00, 10.1, 24.4, 46.9, 73.5, and 102%. Plates were compared to negative (filtered air) controls.

Cytotoxicity as measured by a significant reduction (greater than 50%) in the number of revertants was observed at the highest concentration in  Salmonella typhimurium  strain TA98 and Escherichia coli   strain WP2 uvrA (pKM101) in both Trials 1 and 2 with and without activation.

No mutagenic activity was observed in either Trial 1 or 2.

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

Additional information

Additional information from genetic toxicity in vitro:

Negative results were observed in an in vitro mutagenicity study in bacterial cells (Salmonella typhimurium and Escherichia coli)and in an in vitro clastogenicity study in human lymphocytes. Negative results were also observed in an in vivo micronucleus (rat bone marrow) study. Overall, the test substance is considered negative for genotoxicity.

Justification for classification or non-classification

The test substance did not produce mutagenicity or clastogenicity when evaluated in in vitro systems or in laboratory animals. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for germ cell mutagenicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.