Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A series of key studies using different methods demonstrate that HAB is not mutagenic. The first study examined the mutagenic potential of HAB using the Ames test. Salmonella typhimurium strains TA1538, TA1537, TA1535, TA98, and TA100, were exposed to concentrations of 8, 40, 200, 1000, 5000 ug/plate of test substance in acetone in both the presence and absence of S9. Positive control substances were nitroflourene, sodium azide, or aminoacridine. Cultures were tested in triplicate for mutation frequency. No treatment cultures showed mutation frequencies significantly greater than negative controls. Positive controls responded as expected, therefore the test was valid. The test substance is not mutagenic in either the presence or absence of metabolic activation.

In the second study, HAB concentrations of 0.001, 0.004, 0.02, 0.10, 0.30, 1.00 mg/plate were added to bacterial cultures of Salmonella strains TA 1535, TA 1537, TA 100, and TA 98. The positive control substances were 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, NaNO2, 2-aminoanthracene, and 9 -aminoacridine. Acetone was used as a solvent. The test substance was also tested for cytotoxicity at concentrations up to 10 mg/plate, higher than the solubility limit of 1 mg/plate. Results show the test substance was not cytotoxic even at 10 mg/plate. Results also show no statistically significant increase in the number of revertants/plate in any of the strains at any concentration. The test substance is not mutagenic either in the presence or absence of metabolic activation.

The third study examined the potential for HAB to cause chromosomal aberrations. In accordance with standard OECD guidelines, Chinese hamster ovary (CHO) cells were exposed to concentrations of 5.0 - 80.0 nl/ml (ppm) of test substance both in the presence and absence of metabolic activation. Preparation of chromosomes was done 7 hours (high dose), 24 hours (low, medium, and high dose) and 30 hours (high dose) after start of treatment with the test material. The treatment interval was 4 hours. Treatment was performed with the following test concentrations, with and without S9 activation:

7h: 10, 30, 60, 80 nL/mL

24h: 1, 5, 10, 30, 60, 80 nL/mL

30h: 10, 30, 60, 80 nL/mL

In each experimental group two parallel cultures were used. After the exposure period, the cells were examined for chromosomal aberrations. Ethylmethanesulfonate and cyclophosphamide were used as positive control substances. No increases in chromosomal aberrations were seen in either the presence or absence of metabolic activation. The test substance is not mutagenic.

The fourth study examined the potential for the test substance to be clastogenic. Chinese hamster ovary (CHO) cells were exposed to concentrations of 50, 100, 500, 1000, 4000 ug/ml of test substance for 24 hrs. After the exposure period, the cells were fixed, and examined under a microscope for cytotoxicity and chromosomal aberrations. The only positive result in exposure groups was at the 1000 ug/ml dose. Since a positive result was not seen at the 4000 ug/ml dose level, the test substance was considered not clastogenic. These findings were confirmed by an independent lab. No cytotoxicity was seen at any dose level.

The fifth study examined the potential of the test substance to be mutagenic in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0, 100, 250, 500, 750, and 1000 ug/ml of test substance in both the presence and absence of metabolic activation. Benzo(a)pyrene and ethylmethylsulfonate were used as positive control substances. No biologically significant results were seen in any treatment groups. The test substance was not mutagenic in either the presence or absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov. 13, 1989-Jan. 19, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
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 from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
5.0 - 80.0 nl/ml
Vehicle / solvent:
ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate, cyclophosphamide
Details on test system and experimental conditions:
The study was conducted using standard OECD protocols. Preparation of chromosomes was done 7 hours (high dose), 24 hours (low, medium, and high dose) and 30 hours (high dose) after start of treatment with the test material. The treatment interval was 4 hours. Treatment was performed with the following test concentrations, with and without S9 activation:

7h: 10, 30, 60, 80 nL/mL
24h: 1, 5, 10, 30, 60, 80 nL/mL
30h: 10, 30, 60, 80 nL/mL

In each experimental group two parallel cultures were used.
Evaluation criteria:
Per culture 100 metaphases were scored for structural chromosomal aberrations.
Statistics:
Statistical significance was evaluated using the chi-square test (p<0.05).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant differences between aberration rates in the treatments vs. the controls was observed. The mitotic index was not, or only slightly, reduced after treatment with the highest dose level.
Remarks on result:
other: all strains/cell types tested

Results of Cytogenicity Assay in CHO Cells

Dose per ml

S9

% aberrant cells including gaps

% aberrant cells including gaps

Cell exchanges

7 hrs

Solvent

No

3.50

1.50

0.00

80 nl

No

5.00

2.00

0.50

Solvent

Yes

15.00

5.00

1.00

80 nl

Yes

10.00

3.50

0.00

24 hrs

Control

No

7.00

2.50

1.50

Solvent

No

4.00

3.00

1.00

EMS 0.72 mg

No

17.00

16.00

11.00

5.0 nl/ml

No

2.50

2.00

0.00

30 nl/ml

No

2.50

2.00

0.50

80 nl/ml

No

4.50

3.50

0.50

Control

Yes

9.50

4.50

1.00

Solvent

Yes

3.00

3.00

0.50

CPA 4.2 ug

Yes

60.50

58.50

33.00

5.0 nl

Yes

5.50

2.00

1.50

30 nl

Yes

4.50

2.50

1.00

80 nl

Yes

4.00

3.00

0.00

30 hrs

Solvent

No

6.00

3.50

1.00

60.0 nl

No

4.50

0.00

0.00

Solvent

Yes

8.50

5.00

0.50

80.0 nl

Yes

2.00

0.00

0.00

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was not mutagenic in either the presence or absence of metabolic activation.
Executive summary:

This study examined the potential for the test substance to cause mutations. Chinese hamster ovary (CHO) cells were exposed to concentrations of 5.0 - 80.0 nl/ml of test substance both in the presence and absence of metabolic activation. After the exposure period, the cells were examined for chromosomal aberrations. Ethylmethanesulfonate and cyclophosphamide were used as positive control substances. No increases in chromosomal aberrations were seen in either the presence or absence of metabolic activation. The test substance is not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov. 25, 1992-Dec. 21, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Qualifier:
according to
Guideline:
other: The study was conducted following the standard test method based on Guideline 84/449/EWG B.14, with and without Arochlor-derived metabolic activation.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98, and TA100
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver
Test concentrations with justification for top dose:
8, 40, 200, 1000, 5000 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: nitroflourene, sodium azide, or aminoacridine
Details on test system and experimental conditions:
DURATION
- Preincubation period: 30 minutes at 30 ± 1°C
- Exposure duration: 96 hr, 37°C


NUMBER OF REPLICATIONS: 3

Evaluation criteria:
Dose-related response with significant increase over negative controls. Test is valid if there is a significant increase in mutation frequency in positive controls as compared to negative controls.
Statistics:
Statistics were determined using software from BIOSYS.
Species / strain:
other: TA1535, TA1537, TA1538, TA98, and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Precipitation: precipitation was observed at the 5000 µg/plate concentration
Remarks on result:
other: all strains/cell types tested

Results for Experiment I (Mean Revertants/plate (SD)) ¿ Without S9

Concentration (ug/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Water

31  (4)

152 (3)

15 (4)

20 (6)

25 (4)

Acetone

42 (2)

165 (5)

13 (6)

11 (2)

26 (2)

8

38 (8)

154 (26)

15 (6)

11 (4)

24 (1)

40

36 (2)

140 (26)

12 (9)

11 (1)

30 (4)

200

39 (6)

162 (6)

9 (1)

17 (4)

25 (4)

1000

31 (2)

149 (15)

14 (4)

10 (1)

22 (1)

5000

36 (2)

136 (6)

10 (3)

6 (2)

25 (3)

Nitrofluorene 2.5

101 (12)

-

477 (12)

-

62 (12)

Sodium Azide 2.5

-

651 (36)

-

-

-

Aminoanthracene 10

-

135 (7)

-

-

-

Aminoacridine 25

-

-

-

67 (19)

-

Results for Experiment I (Mean Revertants/plate (SD)) ¿ With S9

Concentration (ug/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Water

54  (9)

126 (34)

13 (2)

17 (1)

34 (8)

Acetone

54 (3)

155 (25)

14 (3)

18 (1)

38 (6)

8

50 (3)

160 (6)

17 (8)

22 (6)

47 (6)

40

45 (2)

164 (24)

19 (4)

23 (6)

41 (13)

200

46 (3)

140 (29)

11 (3)

23 (4)

39 (4)

1000

57 (1)

140 (11)

20 (3)

24 (4)

32 (10)

5000

57 (3)

162 (8)

14 (5)

22 (2)

38 (3)

Nitrofluorene 2.5

-

-

-

-

-

Sodium Azide 2.5

-

-

-

-

-

Aminoanthracene 10

-

649 (51)

-

-

-

Aminoacridine 25

-

-

-

-

-

Results for Experiment II (Mean Revertants/plate (SD)) ¿ Without S9

Concentration (ug/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Water

35  (4)

109 (6)

10 (1)

13 (3)

27 (3)

Acetone

33 (1)

104 (7)

14 (5)

15 (1)

27 (6)

8

37 (4)

113 (12)

9 (2)

15 (1)

36 (8)

40

37 (1)

106 (4)

11 (2)

12 (1)

28 (2)

200

40 (9)

117 (8)

12 (6)

14 (6)

31 (3)

1000

44 (2)

107 (7)

11 (5)

11 (3)

37 (5)

5000

33 (4)

108 (5)

17 (4)

16 (3)

22 (4)

Nitrofluorene 2.5

90 (11)

-

-

-

84 (13)

Sodium Azide 2.5

-

550 (57)

440 (24)

-

-

Aminoanthracene 10

-

122 (4)

-

-

-

Aminoacridine 25

-

-

-

66 (17)

-

Results for Experiment II (Mean Revertants/plate (SD)) ¿ With S9

Concentration (ug/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

Water

51  (8)

114 (6)

15 (5)

21 (3)

38 (10)

Acetone

47 (12)

124 (10)

13 (3)

23 (2)

39 (7)

8

40 (4)

112 (11)

13 (4)

15 (3)

42 (4)

40

33 (4)

112 (10)

18 (7)

16 (2)

43 (2)

200

43 (2)

117 (13)

14 (3)

16 (4)

46 (5)

1000

39 (3)

118 (5)

9 (3)

14 (3)

42 (5)

5000

44 (5)

103 (4)

15 (4)

15 (2)

40 (4)

Nitrofluorene 2.5

-

-

-

-

-

Sodium Azide 2.5

-

        -

-

-

-

Aminoanthracene 10

-

437 (48)

-

-

-

Aminoacridine 25

-

-

-

-

-

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in either the presence or absence of metabolic activation.
Executive summary:

This study examined the mutagenic potential of the test substance. Salmonella typhimuirum strains TA1538, TA1537, TA1535, TA98, and TA100, were exposed to concentrations of 8, 40, 200, 1000, 5000 ug/plate of test substance in acetone in both the presence and absence of S9. Positive control substances were nitroflourene, sodium azide, or aminoacridine. Cultures were tested in triplicate for mutation frequency. No treatment cultures showed mutation frequencies significantly greater than negative controls. Positive controls showed significantly greater mutation frequencies over negative controls, therefore the test was valid. The test substance is not mutagenic in either the presence or absence of metabolic activation.

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: Guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 from livers of aroclor 1254 induced rats
Test concentrations with justification for top dose:
0.001, 0.004, 0.02, 0.10, 0.30, 1.00 mg/plate
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, NaNO2, 2-aminoanthracene, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
0.1 ml of bacterial culture, test solution, and, for tests with metabolic activation, 0.5 ml S-9 mix, were mixed with 2 ml top agar, then poured into minimal glucose agar plates.

DURATION
- Exposure duration: 48 hrs, incubated at 37°C

NUMBER OF REPLICATIONS: 3


OTHER: For plates with more than 500 revertant,colonies/plates, number of revertant colonies were counted using a stereomicroscope. An Artek model 880 automatic colony counter was used for other plates. Visual examination was used for plates with <10 revertants/plate.
Evaluation criteria:
A positive response was considered to be three treatment levels with revertants/plate greater than solvent control and a significant positive dose response (p<0.01).
Statistics:
Analyses included Bartlett's test, one-sided t-test, Grubb's test, and regression analysis.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance was not soluble at concentrations above 1 mg/plate

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity tests show the test substance was not cytotoxic at concentrations up to 10 mg/plate. This was higher than the solubility of the test substance.
Remarks on result:
other: all strains/cell types tested

Results for TA1538 and TA1537 ¿ Experiment I (Revertants/plate (SD))

Concentration (mg/plate)

TA1535 ¿ Without S9

TA1535 ¿ With S9

TA1537 ¿ Without S9

TA1537 ¿ Without S9

0.001

17.3  (6.8)

7.0 (3.6)

7.0 (4.0)

4.3 (2.1)

0.004

15.0 (2.6)

9.3 (3.1)

6.3 (3.2)

7.0 (3.0)

0.02

18.0 (3.0)

9.0 (2.6)

6.3 (0.6)

7.0 (1.7)

0.10

13.7 (4.0)

8.3 (0.6)

6.0 (2.0)

6.7 (3.1)

0.30

18.0 (5.3)

10.3 (4.0)

3.7 (2.9)

5.7 (1.5)

1.00

18.7 (6.1)

11.0 (5.2)

3.0 (2.0)

3.3 (1.2)

Solvent Control

12.4 (4.9)

9.4 (1.9)

7.7 (2.5)

7.6 (3.6)

Non-solvent Control

9

3

7

4

Positive Control 0.001-0.5

100

51

8

29

Positive Control 0.005-2.5

286

271

5

138

Positive Control 0.01-5

347

304

46

Toxicity Observed

Results for TA1538 and TA1537 ¿ Experiment II (Revertants/plate (SD))

Concentration (mg/plate)

TA1535 ¿ Without S9

TA1535 ¿ With S9

TA1537 ¿ Without S9

TA1537 ¿ Without S9

0.001

18.3  (4.2)

10.3 (1.2)

8.0 (1.0)

9.7 (4.7)

0.004

19.7 (3.5)

13.3 (1.5)

6.0 (2.6)

9.7 (5.5)

0.02

14.3 (3.2)

13.0 (3.5)

7.0 (1.7)

13.3 (0.6)

0.10

15.3 (9.2)

10.7 (1.5)

10.3 (3.1)

11.0 (2.6)

0.30

18.7 (1.5)

9.0 (1.7)

6.7 (1.5)

12.0 (4.6)

1.00

19.7 (5.5)

10.7 (2.3)

10.0 (4.4)

13.0 (2.6)

Solvent Control

16.6 (2.1)

10.8 (2.7)

8.6 (2.9)

11.6 (3.0)

Non-solvent Control

17

12

9

8

Positive Control 0.001-0.5

101

77

12

43

Positive Control 0.005-2.5

421

262

21

237

Positive Control 0.01-5

2100

399

180

Toxicity Observed

Results for TA98 and TA100 ¿ Experiment I (Revertants/plate (SD))

Concentration (mg/plate)

TA98 ¿ Without S9

TA98 ¿ With S9

TA100 ¿ Without S9

TA100 ¿ Without S9

0.001

13.7  (4.6)

40.7 (6.4)

85.7 (5.9)

92.3 (1.5)

0.004

13.3 (1.5)

30.3 (10.0)

84.3 (15.0)

95.0 (15.9)

0.02

13.7 (1.5)

38.0 (4.6)

84.3 (5.1)

87.0 (11.1)

0.10

13.3 (4.0)

31.0 (7.0)

92.5 (3.5)

86.3 (14.5)

0.30

11.7 (1.2)

29.7 (6.4)

97.7 (8.4)

96.0 (4.6)

1.00

15.7 (5.3)

40.0 (10.4)

93.3 (2.9)

104.0 (21.7)

Solvent Control

12.4 (4.9)

39.6 (8.4)

84.2 (8.3)

91.4 (9.2)

Non-solvent Control

20

39

100

106

Positive Control 0.002-0.01

33

287

118

204

Positive Control 0.001-0.05

30

1800

145

805

Positive Control 0.02-0.1

69

3800

204

1200

Results for TA98 and TA100 ¿ Experiment II (Revertants/plate (SD))

Concentration (mg/plate)

TA98 ¿ Without S9

TA98 ¿ With S9

TA100 ¿ Without S9

TA100 ¿ Without S9

0.001

15.0  (2.0)

35.0 (3.0)

82.7 (2.1)

89.0 (11.5)

0.004

20.0 (4.4)

40.0 (6.1)

83.0 (3.6)

84.7 (2.1)

0.02

18.0 (6.6)

37.0 (2.0)

92.7 (16.8)

88.3 (8.1)

0.10

23.3 (3.2)

33.3 (8.0)

84.0 (21.5)

93.7 (12.2)

0.30

19.0 (1.0)

30.3 (1.2)

89.0 (7.8)

97.7 (15.2)

1.00

19.0 (2.6)

36.3 (7.1)

98.7 (15.6)

101.3 (5.7)

Solvent Control

19.2 (4.1)

35.0 (5.3)

83.2 (9.3)

78.9 (10.6)

Non-solvent Control

20

31

95

108

Positive Control 0.002-0.01

26

413

86

230

Positive Control 0.001-0.05

45

900

157

1200

Positive Control 0.02-0.1

73

1500

152

1900

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not cytotoxic or mutagenic either in the presence or absence of metabolic activation.
Executive summary:

This study examined the potential mutagenicity of the test substance Therminol 55 Heat-Transfer Medium. Concentrations of 0.001, 0.004, 0.02, 0.10, 0.30, 1.00 mg/plate of test substance were added to bacterial cultures of Salmonella strains TA 1535, TA 1537, TA 100, and TA 98. 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, NaNO2, 2-aminoanthracene, and 9 -aminoacridine were used as positive control substances. Acetone was used as a solvent. The test substance was also tested for cytotoxicity at concentrations up to 10 mg/plate, higher than the solubility limit of 1 mg/plate. Results show the test substance was not cytotoxic even at 10 mg/plate. Results also show no statistically significant increase in the number of revertants/plate in any of the strains at any concentration. The test substance is not mutagenic either in the presence or absence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
according to
Guideline:
other: Preston, RJ, Au, W, Bender, MA, Brewen, JG, Carrano, AV, Heddle, JA, McFee, AF, Wolff, S, and Wassom, JS. 1981. Mammalian in vivo and in vitro cytogenetics assays: A report of the US Gene-Tox Program. Mutation Res. 87: 143-188.
GLP compliance:
yes (incl. certificate)
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 from aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Study I - 50, 100, 500, 1000, 4000 µg/ml
Study II - 600, 1000, 4000 µg/ml
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: 500,000 cells were seeded in flasks and pre-incubated for 18-24 hrs. Medium and test solution were added on the day of the test. Cells were incubated for 24 hrs.


DURATION
- Preincubation period: 18-24 hrs
- Exposure duration: 24 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24.25 hrs

STAIN (for cytogenetic assays): Hoescht and Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 500 for mitotic index, 100 for average cell generation time


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
The statistical significance level was p <=0.05.
Statistics:
Number of cells with structural aberrations: Chi-square analysis
Structural aberrations per cell: Dunnett's t-test
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A statistically significant increase in structural aberrations was seen at the 1000 µg/ml dose level. However, no increase in structural aberrations was seen at the 4000 µg/ml dose level. Due to the errant results at the 1000 µg/ml dose level, the slides from the experiment were sent to an independent lab, Pharmakon Research International, for independent verification of the results. This lab re-analyzed the slides for chromosome aberrations, and performed an independent statistical analysis based on their results. The conclusion of the independent lab was that since the only positive result was at the 1000 ug/ml dose, and no positive results were seen at the 4000 µg/ml dose, there was no dose-response relationship, and the test substance is not clastogenic.
Remarks on result:
other: all strains/cell types tested

Results of Cytogenicity Assay in CHO Cells at 24 hrs- With Metabolic Activation

Dose

No. Aberrations

No. Cells with Aberrations

% Aberrant Cells

Aberrations/Cell

% Mean Mitotic Index

Acetone

3

1

0.5

0.015

3.7

500 ug/ml

6

4

2.0

0.025

4.2

1000 ug/ml

11

9

4.5

0.055

5.1

4000 ug/ml

4

4

2.0

0.020

5.9

Cyclophosphamide

1319

199

99.5

6.595

1.6

Results of Cytogenicity Assay in CHO Cells at 24 hrs- Without Metabolic Activation

Dose

No. Aberrations

No. Cells with Aberrations

% Aberrant Cells

Aberrations/Cell

% Mean Mitotic Index

Acetone

6

6

2.5

0.030

6.8

500 ug/ml

8

8

4.0

0.040

4.6

1000 ug/ml

4

4

2.0

0.020

7.3

4000 ug/ml

2

2

1.0

0.010

6.7

MMS

372

151

75.5

1.860

6.1

Conclusions:
The test substance Therminol 55 is not clastogenic either in the presence or absence of metabolic activation.
Executive summary:

This study examined the potential for the test substance Therminol 55 to be clastogenic. Chinese hamster ovary (CHO) cells were exposed to concentrations of 50, 100, 500, 1000, 4000 ug/ml of test substance for 24 hrs. After the exposure period, the cells were fixed, and examined under a microscope for cytotoxicity and chromosomal aberrations. The only positive result in exposure groups was at the 1000 ug/ml dose. Since a positive result was not seen at the 4000 ug/ml dose level, the test substance was considered not clastogenic. Theses findings were confirmed by an independent lab. No cytotoxicity was seen at any dose level.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
100, 250, 500, 750, 1000 ug/ml
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene, ethylmethylsulfonate
Details on test system and experimental conditions:

0.5 x 10^6 CHO cells/plates were seeded in culture flasks with growth medium 18-24 hrs before the start of the test. On the day of the test, medium was changed to Ham's F12 medium without serum. Test substance was then added. Plates were incubated for 3 hrs at 37.5 +/- 2 degree. The cells were washed, and counted. Three aliquots of 200 cells were plated, then incubated for 6-9 days, and then counted for cloning efficiency. 10^6 cells per sample were supplemented with 10% newborn calf serum. Cells were subcultured every 2-3 days for 7-9 days. 5 plates of 2 x 10^5 cells were made to examine for mutagenicity.
Statistics:
Snee, RD, and Irr, JD (1981). Design of a statistical method for the analysis of mutagenesis a the hypoanthine guanine phosphoribosyl transferase locus of cultured Chinese hamster ovary cells. Mutat. Res., 85: 77-93.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A statistically significant increase in mutations was seen at the 750 ug/ml without S9 dose level in the confirmation experiment. The mean mutant frequency was less than half the maximum acceptable background level at this dose, so this result was not considered biologically significant.
Remarks on result:
other: all strains/cell types tested

Results of Mutagenicity Assay in CHO Cells - Initial Experiment

Dose

Cytotoxicity

Mean Mutant Frequency x 10-6

Without S9

Acetone

-

12.0

250 ug/ml

1.29

8.0

500 ug/ml

0.96

5.6

1000 ug/ml

1.03

9.7

EMS 200 ug/ml

-

177.9

1 % S9

Acetone

-

4.6

250 ug/ml

1.02

9.9

500 ug/ml

1.10

7.4

1000 ug/ml

0.90

7.4

B(a)P 1ug/ml

-

201.6

2 % S9

Acetone

-

8.8

250 ug/ml

0.99

18.3

500 ug/ml

0.61

28.1

1000 ug/ml

0.96

2.9

B(a)P 1ug/ml

-

296.2

5 % S9

Acetone

-

26.1

250 ug/ml

1.18

17.5

500 ug/ml

1.20

14.5

1000 ug/ml

1.05

7.9

B(a)P 1ug/ml

-

185.5

10 % S9

Acetone

-

1.7

250 ug/ml

1.35

11.1

500 ug/ml

1.27

11.8

1000 ug/ml

1.32

12.2

B(a)P 1ug/ml

-

80.6

Results of Mutagenicity Assay in CHO Cells - Confirmatory Experiment

Dose

Cytotoxicity

Mean Mutant Frequency x 10-6

Without S9

Acetone

-

2.4

100 ug/ml

1.04

3.3

250 ug/ml

0.95

0.5

500 ug/ml

0.98

2.3

750 ug/ml

1.22

9.0

1000 ug/ml

1.04

1.9

EMS 200 ug/ml

-

212.2

5% S9

Acetone

-

3.1

100 ug/ml

1.00

0.5

250 ug/ml

1.14

2.3

500 ug/ml

1.21

2.6

750 ug/ml

1.22

1.4

1000 ug/ml

1.32

2.8

B(a)P 1ug/ml

-

273.2

Conclusions:
Interpretation of results (migrated information):
negative

The test substance is not mutagenic in CHO cells in either the presence or absence of metabolic activation.
Executive summary:

This study examined the potential of the test substance to be mutagenic in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0, 100, 250, 500, 750, and 1000 ug/ml of test substance in both the presence and absence of metabolic activation. Benzo(a)pyrene and ethylmethylsulfonate were used as positive control substances. No biologically significant results were seen in any treatment groups. The test substance is not mutagenic in either the presence or absence of metabolic activation.

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

Additional information

Additional information from genetic toxicity in vitro:

In a series of reliable mutagenicity studies, HAB was determined to be not mutagenic in any of the in vitro studies conducted. In vivo data were not available (scientifically not justified because acute dermal studies indicate no adverse effects up to and surpassing 2000 mg/kg bw).


Justification for selection of genetic toxicity endpoint
Experimental results. In vivo genetic toxicity data not available (scientifically not justified).

Justification for classification or non-classification

In a series of reliable mutagenicity studies, HAB was determined to be not mutagenic in any of thein vitrostudies conducted.