Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100: negative with and without metabolic activation (similar to OECD TG 471)
Mammalian cytogenicity (UDS): primary rat hepatocytes: negative (similar to OECD TG 482)
Mammalian mutagenicity (HGPRT Test): Chinese hamster Ovary (CHO): negative with and without metabolic activation (similar to OECD TG 476)


Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 1979 to 17 November 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study predates the appropriate OECD test guideline, but was conducted in compliance with GLP. Nevertheless, the study was conducted similar to the OECD TG 482 with acceptable restrictions. The restrictions were that less cells were evaluated than demanded in the guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Version / remarks:
(1986)
Deviations:
yes
Remarks:
(only 30 cells evaluated)
GLP compliance:
yes
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
not applicable
Species / strain / cell type:
hepatocytes: primary hepatocytes from one male Sprague Dawley rat (8-12 weeks of age; approximately 221 g bw)
Details on mammalian cell type (if applicable):
- Type and identity of media: Williams Medium E supplemented with 10% foetal calf serum- Properly maintained: yes- Periodically checked for Mycoplasma contamination: not applicable- Periodically checked for karyotype stability: not applicable- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Metabolic activation system:
primary hepatocyte cultures have sufficient metabolic capacity
Test concentrations with justification for top dose:
10 and 1 mM, 100, 10, and 1 µM, and 100, 10, and 1 nM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: cell culture medium- Due to limited solubility of the positive control substance, cell culture medium additional supplemented with 0.1% (v/v) DMSO was used for the positive control.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
(cell culture medium with 0.1% DMSO with respect to the positive control)
True negative controls:
no
Positive controls:
yes
Remarks:
diluted in cell culture medium with 0.1% DMSO
Positive control substance:
2-acetylaminofluorene
Remarks:
10, 1, and 0.1 µM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium containing 3H-Thymidine (10 µCi/mL) and dexamethasone (1 µM in 0.1% ethanol final concentration)DURATION- Exposure duration: 18 h- Fixation time (start of exposure up to fixation of cells): 20 hNUMBER OF REPLICATIONS: 1NUMBER OF CELLS EVALUATED: 30DETERMINATION OF CYTOTOXICITY- Method: morphological examination (detachment and/or rounded granular appearence of the cells)
Evaluation criteria:
The net number of grains in treated cells were compared to the appropriate control. Net nuclear labelling is often observed in control cells, but rarely exceeds a mean of 5 grains per nucleus. Therefore, a mean of 6 or more grains per nucleus (this may vary with experimental conditions) and statistical significance from control is required for an unequivocal positive result. In cases where nuclear labelling is very intense (approximately greater than 75 grains per nucleus) the results are reported as the best approximation of the number of grains present. In addition to statistical significance from control, a dose response relationship should exist.
Statistics:
The net number of grains in treated cells were compared to the appropriate control by analysis of variance and Dunnett's t-test, with statistical significance from control at p ≤ 0.05.
Species / strain:
hepatocytes: primary hepatocytes from one male Sprague Dawley rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1 mM and above clearly cytotoxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- none stated in the study reportADDITIONAL INFORMATION ON CYTOTOXICITY:- The test item was cytotoxic to the rat hepatocyte cultures at concentrations of 10 and 1 mM as indicated by the detachment and/or rounded granular appearence of the cells after chemical treatment, however, enough cells were present after processing for UDS evaluation. The appearence of the cultures improved with decreasing concentration of the test material so that cultures treated with 0.1 mM to 1 nM approached and matched the appearence of control cultures.

Tab. 1: The effects of the test item in the Rat Hepatocyte Unscheduled DNA Synthesis (UDS) Assay

Treatment

Concentration

Net nuclear grains*

(mean±SD)

Negative control

-

-

-2±3

Test item

10 mM

1550 µg/mL

0±1 #

1 mM

155 µg/mL

1±2 #

100 µM

15.5 µg/mL

-3±4

10µM

1.55 µg/mL

-2±3

1 µM

0.155 µg/mL

-2±3

100 nM

0.0155 µg/mL

-2±2

10 nM

0.00155 µg/mL

-2±3

1 nM

0.000155 µg/mL

-3±3

Positive control

(2-AAF)**

10 µM

2.2 µg/mL

40±10***

1 µM

0.22 µg/mL

40±12***

100 nM

0.022 µg/mL

33±12***

Solvent control (with 0.1% DMSO)

-

-

-3±4

* net nuclear grains = nuclear grains-background cytoplasm grains

**2 -AAF = acetylaminofluorene

*** positive responses, 6 or more net nuclear grains and p ≤ 0.05

# treatment related cytotoxicity was observed

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February 1980 to 25 April 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study predates the appropriate OECD test guideline and GLP. Nevertheless, the study was conducted similar to the OECD TG 471 with acceptable restrictions. The restrictions are that only 4 strains were tested and only 2-AA was assayed as positive control for the test with metabolic activation system. However, the test meets the scientific standards accepted at that time.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
(only 4 bacterial strains tested and 2-AA was the only positive control assayed for the test with metabolic activation)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate from Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
0.7, 1.39, 2.09, and 2.78 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
ethylmethanesulphonate
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 2 hNUMBER OF REPLICATIONS: - TA 1535 and TA 1537: 1 trial with 2 replicates with and without metabolic activation each; 3 plates each replicate for mutagenicity testing and 2 plates for cytotoxicty testing- TA 98 and TA 100: 3 trials with 2 replicates with metabolic activation and 2 trial with 2 replicates without metabolic activation; 3 plates each replicate for mutagenicity testing and 2 plates for cytotoxicity testingDETERMINATION OF CYTOTOXICITY- Method: relative total growth The cytotoxicity of the test item was analysed concurrently with the mutagenicity experiments. Treated bacteria were cultured in agar supplemented with histidine and incubated in parallel with the samples of the mutagenicity testing (48 h at 37°C).
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
up to cytotoxic concentrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
decreased growth <50% of the concurrent solvent control was obtained for TA 1535: ≥1.39 mM +S9 and ≥1.74 mM -S9; TA 1537: ≥2.78 mM + S9 and not cytotoxic -S9; TA 98: ≥0.7 mM + S9 and ≥1.39 mM -S9; TA 100: ≥1.39 mM +S9 and ≥3.48 mM -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- none stated in the study reportRANGE-FINDING/SCREENING STUDIES:- On the basis of the results obtained over a wider range of concentrations in Experiment 1 in strain TA 100 with and without metabolic activation, concentrations which gave an sufficient number of survivors were chosen for subsequent experiments. Concentrations were adjusted in subsequent experiments in other strains due to the differential sensitivity of the strains to the toxicty of the test item.

Tab. 1: Mutagenic activity in Salmonella Typhimurium

Strain

Trial No.

Treatment

Conc.

[mM]

S9

Mean revertants/

plate

Mean survivors/

plate

Revertants/

1E+8 survivors

TA 1535

1

Acetone

-

+

8.7

9.0

2392.5

2413.0

1.8

1.9

Test item

0.70

6.3

9.7

1117.5

1494.5

2.8

3.2

1.39

8.3

8.0

1930.0

423.0

2.2

9.5

2.09

4.7

8.3

378.5

636.5

6.2

6.5

2.78

2.7

5.0

133.0

311.5

10.2

8.0

2AA

0.05

137.7

400.7

946.0

657.5

72.8

304.7

Acetone

-

-

10.3

11.3

2098.0

2719.5

4.9

4.2

Test item

0.70

5.0

7.7

1224.0

1499.5

4.1

5.1

1.74

4.3

6.0

1115.5

1088.0

3.9

5.5

3.48

2.7

5.3

231.5

395.0

11.7

13.4

MNNG

0.01

3143.0

3079.3

1353.0

1225.0

2323.0

2513.7

TA 1537

1

Acetone

-

+

4.3

6.0

460.5

418.5

4.7

7.2

Test item

0.70

4.0

4.3

1506.3

1048.0

1.3

2.0

1.39

4.0

2.7

900.0

1004.0

2.2

1.3

2.09

5.7

3.3

305.0

1104.0

9.3

1.5

2.78

0.7

3.7

0.0

32.0

Ct.

Ct.

2AA

0.05

149.3

361.7

59.5

60.5

1254.6

2989.3

Acetone

-

-

11.3

7.7

418.0

550.0

27.0

14.0

Test item

0.70

7.7

5.3

1205.0

1598.0

6.4

3.3

1.74

5.3

6.0

540.5

483.5

9.8

12.4

9AAc

0.13

3234.3

2814.3

1019.5

750.0

3172.4

3752.4

TA 98

1

Acetone

-

+

18.7

13.3

1998.0

1605.5

4.7

4.1

Test item

0.70

13.0

16.3

795.0

913.5

8.2

8.9

1.74

23.7

18.7

193.5

136.5

61.2

68.5

3.48

11.0

10.7

20.0

0.0

Ct.

Ct.

2AA

0.05

910.3

798.7

1691.0

1952.5

269.2

204.5

2

Acetone

-

11.3

9.0

1946.5

1526.5

2.9

2.9

Test item

0.70

16.3

8.7

750.0

926.5

10.9

4.7

1.39

22.3

19.0

498.5

32.0

22.4

Ct.

2.09

13.7

11.3

18.0

5.0

Ct.

Ct.

2AA

0.05

870.3

921.7

1684.0

1803.0

258.3

255.3

3

Acetone

-

7.7

12.7

1346.0

1175.0

2.9

5.4

Test item

0.70

18.0

21.0

967.5

544.0

9.3

19.3

1.04

22.7

27.7

397.0

409.0

28.6

33.9

1.39

27.3

13.7

305.5

0.5

44.7

Ct.

1.74

9.0

13.3

0.0

49.0

Ct.

Ct.

2AA

0.05

1539.7

1611.7

1047.0

413.0

735.3

1951.2

1

Acetone

-

-

12.3

6.0

503.5

454.0

12.2

6.6

Test item

0.70

11.7

11.0

729.5

637.5

8.0

8.6

1.74

11.3

5.7

572.5

23.5

9.9

Ct.

2NF

0.06

1135.3

1078.0

476.0

533.5

1192.5

1010.3

2

Acetone

-

13.0

9.7

415.5

409.0

15.6

11.9

Test item

0.70

10.0

12.0

873.5

649.5

5.7

9.2

1.04

10.7

14.0

401.0

621.5

13.3

11.3

1.39

6.3

5.7

167.5

12.0

18.8

Ct.

2NF

0.06

1228.3

973.0

233.5

616.0

2201.9

789.8

TA 100

1

Acetone

-

+

34.0

33.0

2493.0

1433.0

6.8

11.5

Test item

0.007

39.3

39.0

1567.0

1223.0

12.7

16.1

0.07

38.7

43.7

1361.0

1819.0

14.2

12.0

0.7

44.7

44.3

991.0

1043.5

22.6

21.2

6.96

37.7

33.0

64.0

17.5

450.8

Ct.

2AA

0.05

1489.7

1449.7

508.5

576.0

1464.8

1258.4

2

Acetone

-

33.7

36.3

1034.0

862.5

16.3

21.0

Test item

0.70

51.7

48.7

403.0

434.5

64.1

56.0

1.74

76.0

65.7

220.0

286.0

172.7

114.9

3.48

62.3

80.3

146.5

221.0

212.6

181.7

5.22

39.3

61.7

6.0

164.5

Ct.

187.5

2AA

0.05

662.7

736.7

897.0

966.5

369.4

382.2

3

Acetone

-

45.0

37.0

884.0

654.5

25.5

27.8

Test item

0.70

41.0

37.7

463.0

443.0

44.3

42.6

1.39

65.7

103.7

319.0

364.5

103.0

142.2

2.09

73.3

71.0

220.5

320.5

166.2

110.8

2.78

53.3

54.7

18.0

201.0

Ct.

136.1

2AA

0.05

649.7

379.7

673.0

947.0

482.7

200.5

1

Acetone

-

-

54.7

48.3

663.5

716.0

41.2

33.7

Test item

0.007

46.0

42.3

772.3

635.0

29.8

33.3

0.07

61.3

41.3

782.5

517.0

39.2

39.9

0.70

38.3

34.7

360.0

458.0

53.2

37.9

6.96

29.0

31.7

65.0

9.5

223.1

Ct.

EMS

45.7

2921.7

2743.0

457.0

447.0

3196.6

3068.2

2

Acetone

-

62.3

57.3

732.0

875.0

42.6

32.7

Test item

0.70

35.0

51.0

516.5

569.0

33.9

44.8

1.74

32.3

37.0

469.5

420.5

34.4

44.0

3.48

41.3

25.7

336.0

366.0

61.5

35.1

5.22

40.7

29.0

52.0

60.5

391.3

239.7

MNNG

0.01

2381.0

2240.0

451.5

469.0

2636.8

2258.1

Conclusions:
Interpretation of results: negativeThe authors concluded the test item to be not mutagenic to S. typhimurium strains TA 1535 and TA 1537 with and without metabolic activation system, and in TA 98 and TA100 in the absence of a metabolic activation system. However, the authors also concluded the test item to be mutagenic to TA 98 and TA 100 with of metabolic activation. This conclusion is based the increase in revertants per 1E+8 survivors. However, this effect was observed at cytotoxic concentrations. At lower concentrations no distinct increase in revertants per 1E+8 survivors was observed. Additionally, the total number of revertants per plate did not reach a 2-fold increase, and no dose-response relationship can be observed. Based on this fact, the test item is considered to be not mutagenic to bacteria under the conditions of this test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 1980 to 08 November 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study predates the appropriate OECD test guideline and GLP. Nevertheless, the study was conducted comparable to the OECD TG 476.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in Ham's F12 medium without hypoxanthine, supplemented with 10% dialysed heat-inactivated newborn calf serum. In the experiments, the media also contained 50 U/mL penicilin and 50 µg/mL streptomycin. The pH was kept at 7.2 by adding 25 mM HEPES buffer. In experiments with metabolic activation, the media were further supplemented with 1 mM MgCl2, 4.7 mM glucose-6-phosphate, 1.5 mM NADP, and 1 mM NADH. - Properly maintained: yes (37°C, 5% CO2, and 90+% relative humidity)- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
other: BH4 clone of the CHO-K1 cell line
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of Aroclor 1254 induced male rats
Test concentrations with justification for top dose:
- without metabolic activation: 48.3, 80.6, 112.9, 129.0, and 145.1 µM- with metabolic activation: 4.8, 16.1, 32.2, 40.3, and 48.3 µM (Trial 1), 4.8, 16.1, 32.2, 48.3, and 64.5 µM (Trial 2), 48.3, 64.5, 74.1, 83.8, and 96.7 µM (Trial 3)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Exposure duration: 18-19 h (without metabolic activation), 5 h (with metabolic activation, followed by further 21-25 h in growth medium without test material treatment)- Expression time (cells in growth medium): 7 days- Selection time (if incubation with a selection agent): 7 daysSELECTION AGENT (mutation assays): 6-thioguanineNUMBER OF REPLICATIONS: 2 trials with 5 dishes each (without metabolic activation) and 3 trials with 5 dishes each (with metabolic activation), respectivelyDETERMINATION OF CYTOTOXICITY- Method: relative total growthIn the preliminary cytotoxicity experiments both the test item concentrations, as well as the optimal concentration for the S9 mix were established. The purpose of this experiment was to establish the concentration of S9 that gives the greatest cytotoxicity. In most cases, the concentration of S9 giving the greatest cytotoxicity would also be expected to be the condition, in which the greatest mutagenic activity would be observed. The concentrations of S9 mix tested were 0.2, 0.5, and 1 mg/mL in the treatment medium. The experiments were carried out in duplicate. In mutagenicity assays, cells were splitted after the test item treatment and one portion of cells was plated to assess cytotoxicity. 6 dishes with 200 cells each were incubated for 7 days and the colonies were stained and counted. Cell survival was determined by dividing the total number of colonies by the total number of cells plated and was expressed both as the percent plated and as the percent of the solvent control survival.
Evaluation criteria:
A test sample is classified as a nonmutagen if- the probability is greater than 0.05 that the numbers of revertants at each of the test sample concentrations studied are not greater than the number of revertants in the solvent control and- the probability is greater than 0.05 that there is not a positive correlation between the number of revertants and increasing concentrations of the test item. A test sample is classified as mutagen if - the probability is less than 0.01 that the numbers of revertants at one or more of the test sample concentrations studied are not greater than the number of revertants in the solvent control and- the probability is less than 0.01 that there is not a positive correlation between the number of revertants and increasing concentrations of the test item. Test materials that cannot be classified with the above criteria are dealt with on a case-by-case basis.
Statistics:
Because of the complex nature of the experimental errors in this assay, mutation frequency data were transformed prior to analysis. The formula y=(mutation frequency+1)^0.15 was used because this power transformation provided data which satisfied assumptions required for perfoming parametric statistical analyses. The analysis used a two variable (dose and experiment) Analysis of Variance (ANOVA) Model that allows for unequal numbers of dose points and unequal numbers in test results in each trial. In one analysis, the effect of each chemical dose was compared to the solvent control by a t-test of significance to determine whether a dose caused a significant increase in the mutation frequency. The second analysis was an ANOVA to evaluate a dose-response relationship. Linear, quadratic and higher order effects were tested by an F-test of significance.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a dose-dependent decrease in relative cell survival was observed over all test item concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- All experiments were carried out in glass flasks because the test item reacted with plastic flasks.RANGE-FINDING/SCREENING STUDIES:- After an initial cytotoxicity experiment was performed with 1 mg/mL of S9 mix, a second cytotoxicity experiment was conducted using various amounts of S9 mix. The test chemical was most cytotoxic with 1 mg/mL S9 mix in the treatment medium and therefore, this concentration was used for the mutagenicity experiments. ADDITIONAL INFORMATION ON CYTOTOXICITY: - The test item was most cytotoxic in the presence of the metabolic activation system.

Tab. 1: Toxicity and mutagenicity in CHO without metabolic activation

Trial

Treatment

Conc.

[µM]

Toxicity

Mutagenicity

Cell survival

Cell survival

Mutants

% plated

% control

% plated

Total

Frequency*

1

Acetone

-

77.2

76.3

100+

62.5

64.2

34

22

54.4

34.2

Test item

48.3

76.8

70.3

100.0

91.5

56.6

76.3

18

38

31.8

49.8

80.6

42.8

57.2

55.7

74.5

80.4

66.4

9

8

11.2

12.0

112.9

32.4

32.3

42.2

42.1

61.6

75.1

27

33

43.8

43.9

129.0

29.3

42.3

38.2

55.1

69.7

74.6

40

31

57.4

41.6

145.1

18.3

31.8

23.8

41.4

56.3

65.3

19

61

33.4

93.4

EMS

805

52.6

58.5

68.5

76.2

63.4

66.0

258

187

406.9

283.3

2

Acetone

-

83.8

76.0

100+

80.0

88.3

3

13

3.8

14.7

Test item

48.3

72.8

76.2

91.1

95.4

89.3

91.1

2

2

2.2

2.2

80.6

70.3

27.6

88.0

34.5

76.5

85.5

1

4

1.3

4.7

112.9

2.0

1.1

2.5

1.4

86.3

71.9

0

0

0

0

129.0

4.6

12.7

5.8

15.9

83.3

85.3

0

3

0

3.5

145.1

3.7

1.6

4.6

2.0

85.8

81.8

0

0

0

0

EMS

805

75.9

71.3

95.0

89.2

79.6

73.7

165

128

207.3

173.7

* Mutants per 1E+6 cells

Tab 2.: Toxicity and mutagenicity in CHO with metabolic activation

Trial

Treatment

Conc.

[µM]

Toxicity

Mutagenicity

Cell survival

Cell survival

Mutants

% plated

% control

% plated

Total

Frequency*

1

Acetone

-

82.1

74.8

100+

64.5

63.4

3

3

4.7

4.7

Test item

4.8

86.3

78.5

109.9

100.0

49.2

76.3

10

15

20.3

19.7

16.1

66.3

73.4

84.3

93.5

73.9

63.1

1

1

1.4

1.6

32.2

55.8

44.8

71.1

57.1

68.1

64.4

11

11

16.2

17.1

40.3

42.3

37.9

53.9

48.3

68.7

66.6

7

2

10.2

3.0

48.3

32.6

46.9

41.5

59.7

61.5

65.6

0

10

0

15.2

DMBA

27.3

74.8

58.6

95.3

74.6

61.3

63.3

187

198

305.1

312.8

2

Acetone

-

62.3

49.9

100+

75.3

52.6

3

6

4.0

11.4

Test item

4.8

57.5

53.6

102.5

95.5

69.0

70.1

6

3

8.7

4.3

16.1

41.8

49.1

74.5

87.5

61.2

58.9

10

9

16.3

15.3

32.2

28.5

30.8

50.8

54.9

56.3

66.8

0

7

0

10.5

48.3

28.5

28.3

50.8

50.4

59.7

62.3

0

0

0

0

64.5

18.7

16.5

33.3

29.4

58.3

63.3

2

7

3.4

11.1

DMBA

27.3

48.7

52.1

86.8

92.9

46.4

55.2

109

138

234.9

250.0

3

Acetone

-

81.3

68.3

100+

59.4

52.5

7

5

11.8

9.5

Test item

48.3

37.3

41.3

49.9

55.2

44.7

60.4

2

2

4.5

3.3

 

64.5

30.8

28.8

41.2

38.5

58.2

59.9

0

0

0

0

 

74.1

19.7

11.8

26.3

15.8

54.1

42.6

5

0

9.2

0

 

83.8

6.2

5.2

8.3

7.0

48.7

45.6

12

1

24.6

2.2

 

96.7

3.8

0.5

5.1

0.7

45.7

38.7

14

1**

52.5

6.5

DMBA

27.3

65.7

62.1

87.8

83.0

62.3

57.1

183

204

293.7

357.6

* Mutants per 1E+6 cells

** Only 4E+5 cells evaluated due to sparse growth

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mammalian cytogenicity: (Rodent Dominant Lethal Test): negative (similar to OECD 478)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / 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
Remarks:
The study was conducted similar to an appropriate OECD TG, but without GLP
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Version / remarks:
(1984)
Deviations:
no
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
other: Sprague-Dawley (Spartan substrain)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Spartan Research Animals, Haslett, Michigan- Age at study initiation: 10-12 weeks- Housing: 2 per cage in wire-bottom cages- Diet: Purina laboratory chow, ad libitum (during the non-exposure period)- Water: ad libitum (during the non-exposure period)
Route of administration:
inhalation: vapour
Vehicle:
- Vehicle used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: no dataGENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: 4.3 m³ Rochester-type chamber at dynamic airflow conditions- System of generating vopour: Vapour was generated by counter-current exchange between air and liquid test material in a 15-plate perforated plate distillation column. To increase the stability of the system, the distillation was water jacketed and thermostated to a few degrees below ambient room temperature. The generating system was arranged so that air was continuously pumped through a closed loop containing the distillation column and a 5 liter plenum flask. The apparatus was designed to hold the concentration of the test material in the plenum flask at a level of approximately 100 ppm. The chambers were supplied by pumping air from the plenum flask via positive displacement piston pumps, and diluting the stream 400 - 4000 fold to achieve the target concentrations. - Temperature and humidity in air chamber: approximately 72 °F (22.2 °C) and 50% humidity - Air flow rate: 700 l/minTEST ATMOSPHERE- Brief description of analytical method used: To ensure stable operation, the test material concentration in the plenum flask was monitored periodically by a semiautomated gas chromatographic method. Primary analysis of chamber air was made via a liquid chromatographic procedure. 20 l of the chamber air was bubbleed through an impinger at a rate of 2 l/min into a solution containing p-nitrobenzyl propyl amine. The test item reacts quantitatively with this material to form the urea derivate of the isocyanato group. An aliquot of this solution containing the urea derivate of the test material and the unreacted p-nitrobenzyl propyl amine, was analysed by HPLC with an UV detector at 250 nm. This method was used to precisely measure test material concentrations in the chamber, whereas a second on-line analytical method was employed to detect fluctuations in chamber concentrations. Therefore an MDA paper tape detector (MDA Scientific Inc., Park Ridge, Illinois), specific for isocyanates, was connected to each chamber to provide a continous record of the test material concentration. - Samples taken from breathing zone: yes
Duration of treatment / exposure:
6 h
Frequency of treatment:
10 weeks: 4 days for the first week, and 5 days/week for the subsequent 9 consecutive weeks
Post exposure period:
week 1 post exposure: each male rat was mated with 2 adult (at least 10-12 weeks old) untreated virgin femalesweek 2 post exposure: each male rat was mated with 2 other females
Dose / conc.:
25 other: ppb (nominal)
Remarks:
24.1±2.3 ppb (analytical conc.)
Dose / conc.:
80 other: ppb (nominal)
Remarks:
79.1±3.6 ppb (analytical conc.)
Dose / conc.:
250 other: ppb (nominal)
Remarks:
245±12 ppb (analytical conc.)
No. of animals per sex per dose:
20 male rats
Control animals:
yes, concurrent no treatment
Tissues and cell types examined:
Number of corpora lutea and live and dead implantations were counted.
Statistics:
Body weight, number of implantations, and number of corpora lutea were evaluated by a one-way analysis of variance; differences between experimental groups and the controls were analysed using Dunnett's test. The fertility index was analysed by the Fisher's exact propability test. The pre-implantation loss and resorption rate data were analysed using the Wilcoxon test as modified by Haseman and Hoel.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined

During the third week of exposure, all animals at every dose level and the control group exhibited signs of consistent sialodacryoadentitis (swelling in the neck area and the appearance of a red crusty material around the eyes and nasal area). After approx. ten days, these signs subsided. Beginning with the seventh week of exposure and lasting throughout the rest of the treatment period, five animals at the 250 ppb level exhibited swelling and ulcerated abscesses in the throat region. Control animals appeared normal in appearance and behaviour, and did not show these signs.

Two rats at 250 ppb (high dose group) died during the study. One animal showed signs of a distended and hard abdomen after the first 6 - hour exposure period and died after eleven exposures to 250 ppb of the test material. The second animal died after ten weeks of exposure to 250 ppb of the test item and upon gross examination this animal exhibited a distended and hard abdomen. Gross pathology on both high dose males revealed that death was caused by a large abscessed mass and an infection in the abdominal cavity. Hence, the deaths are considered to be not related to the test material treatment, but attributed to an incidental infection.

Throughout the study, mean body weights of male rats at all dose levels were comparable to those in the control group. Weight losses occurred in some rats during 3rd and 4th weeks of exposure when the signs of sialodacryoadentitis were evident; however, no statistical differences were observed between the rats exposed to the test item and the control animals.

Vaginal plugs were observed with equal frequency beneath the cages of control and of treated rats suggesting that mating occurred in all groups. The fertility indices of male rats exposed to the test item were comparable to the control group. All groups had 95% or higher fertility rate for both weeks of mating. The average number of implantations, corpora lutea, and resorptions in the unexposed female rats bred to the male rats exposed to the test material are given in Tab.1. All exposure levels had values for these parameters which were comparable to the control group. For the first week of breeding the average pre- implantation loss for all dose groups was not significantly different from the control data. For the second week of breeding the average pre- implantation loss at the 80 ppb level was significantly lower than the control mean (Tab. 2). This was not a detrimental effect and was not considered to be of toxicological importance. The average resorption rates in the treatment levels for the first and second week of breeding were comparable to the respective control group resorption rate (Tab. 2).

Tab. 1: Reproductive parameters – average number of implantations, corpora lutea, and resorptions in unexposed female rats bred to male rats exposed to the test item by inhalation

 

Post exposure breeding

Exposure level [ppb]

control

25

80

250

Week 1

Average corpora lutea

13 ± 1

14 ± 1

13 ± 1

14 ± 1

Average implantations

13 ± 2

13 ± 1

13 ± 2

13 ± 1

Average resorptions

1 ± 1

1 ± 1

1 ± 1

1 ± 1

Week 2

Average corpora lutea

14 ± 2

14 ± 2

14 ± 2

14 ± 2

Average implantations

13 ± 2

13 ± 1

13 ± 1

13 ± 2

Average resorptions

1 ± 1

1 ± 1

1 ± 1

1 ± 1

Mean ± SD. The average number of implantations, corpora lutea, or resorptions for the two females bred to each male was calculated. Group means were then calculated from the averaged values.

No values were significantly different from control values by an analysis of variance and Dunnett’s Test or by a modified Wilcoxon Test, p < 0.05.

 

Tab. 2: Reproductive parameters – average pre- implantations loss and average resorption rate in unexposed female rats bred to male rats exposed to the test item by inhalation Average pre- implantation loss [%]

 

Post exposure breeding

Exposure level [ppb]

control

25

80

250

Average pre- implantation loss [%]

Week 1

6± 7

8 ± 8

6 ± 8

5 ± 5

Week 2

9 ± 10

7 ± 5

5 ± 6*

9 ± 9

Average resorption rate [%]

Week 1

5 ± 6

3 ± 5

6 ± 7

5 ± 4

Week 2

6 ± 4

4 ± 5

5 ± 6

6 ± 7

* Significantly different from the control mean by a modified Wilcoxon Test, p < 0.05

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genotoxicity in vitro

In the available key study (DuPont, 1980) the test item was investigated for mutagenicity to bacteria in a study, which predates the appropriate OECD test guideline and GLP. However, the study was conducted according to a protocol that is similar to the OECD test guideline 471. S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 were tested both with and without metabolic activation at doses up to cytotoxic concentrations in medium. No additional strain for the detection of crosslinking agents was included into this test. The authors concluded the test item to be not mutagenic to S. typhimurium strains TA 1535 and TA 1537 with and without metabolic activation system, and in TA 98 and TA100 in the absence of a metabolic activation system. However, the authors also concluded the test item to be mutagenic to TA 98 and TA 100 with of metabolic activation. This conclusion is based the increase in revertants per 1E+8 survivors. However, this effect was observed at cytotoxic concentrations. At lower concentrations no distinct increase in revertants per 1E+8 survivors was observed. Additionally, the total number of revertants per plate did not reach a 2-fold increase, and no dose-response relationship can be observed. Based on this fact, the test item is considered to be not mutagenic to bacteria under the conditions of this test.

 

In the available key study (Dow Chemical USA, 1980), the submission substance was investigated for DNA damage and subsequent repair (indicative for cytogenicity) in the unscheduled DNA synthesis (UDS) assay in vitro. The study predates the appropriate OECD test guideline, but was conducted according to a protocol that is similar to the OECD test guideline 482 and in compliance with GLP. Primary rat hepatocytes were cultured and treated with the test item up to cytotoxic concentrations. No metabolic activation system was added, since primary hepatocyte cultures have sufficient metabolic capacity. Following treatment, the cells were cultured with 3H-Thymidine at 10 µCi/mL to radiolabel replicating DNA. After fixation, the cells were submitted to autoradiography and UDS was quantified by counting the number of grains in 3 nuclear seized areas adjacent to the nucleus in the cytoplasm. The test material failed to elicit UDS at any concentration. Appropriate positive, solvent, and negative controls were included into the test and gave the expected results. Hence, the test material was concluded not to cause DNA damage inducible repair under the conditions of this test.

 

In the available key study (DuPont, 1980) the test substance was investigated for mammalian mutagenicity (HPRT Test) in a study predating the appropriate OECD test guideline and GLP. Nevertheless, the study was conducted according to a protocol that is comparable to the OECD test guideline 476. Chinese Hamster Ovary cells were treated with the test item up to cytotoxic concentrations in both the presence and absence of a metabolic activation system. No increase in mutant frequency was observed at any concentration up to cytotoxicity with metabolic activation at 5 h treatment and without metabolic activation at 18 -19 h exposure. Expected results were obtained with positive and solvent controls. It is concluded that test item is negative for the induction of mutations in Chinese hamster Ovary cells (CHO) under the conditions of the test.

 

Genotoxicity in vivo

In the available key study (Murray et al., 1980) the submission substance was tested for chromosome aberration in vivo in the rodent dominant lethal test. The study predates the appropriate OECD test guideline and GLP, but was conducted according to a protocol that is comparable to the OECD test guideline 478. Male Sprague-Dawley rats (Spartan substrain, 10 -12 weeks of age) received the test material via inhalation at vapour concentrations of 25, 80, and 250 ppb for 6 h/day, 5 days/week, and 10 weeks.

All rats were observed at the end of each daily exposure for changes in appearance or behaviour. The body weight of each male rat on the study was recorded at weekly intervals throughout the exposure period. Animals which appeared moribund or died spontaneously during the course of the study were submitted for gross pathologic evaluation. At the end of the exposure period, each male was bred with two adult (at least 12 weeks old) virgin, unexposed females during week 1, and with two other females during week 2. The area beneath each cage was observed each morning for copulatory plugs as evidence for mating. The bred females were sacrificed 12 days after the last day of cohabitation. The number of corpora lutea and live and dead implantations were counted. The male fertility index (%) and for each litter the pre-implantation loss (%) and the resorption rate (%) were calculated. All experimental males were sacrificed after reviewing the second week breeding data.

All treated animals exhibited signs of toxicity, and two rats at 250 ppb (high dose group) died during the study. However, gross pathology revealed that the deaths were not related to the test material treatment. Vaginal plugs were observed with equal frequency beneath the cages of control and of treated rats suggesting that mating occurred in all groups. The fertility indices of male rats exposed to the test item were comparable to the control group. All groups had 95% or higher fertility rate for both weeks of mating. All exposure levels had average number of implantations, corpora lutea, and resorptions comparable to the control group. For the first week of breeding the average pre- implantation loss for all dose groups was not significantly different from the control data. For the second week of breeding the average pre- implantation loss at the 80 ppb level was significantly lower than the control mean. The authors concluded that this was not a detrimental effect and was not considered to be of toxicological importance. It can be concluded from the study that repeated exposure to the test item via inhalation does not produce dominant lethal mutations under the conditions of this test.

 

In conclusion, the submission substance was not mutagenic to bacteria and mammalian cells in vitro both with metabolic activation, neither was cytogenicity observed in mammalian cells in vitro with and without metabolic activation. Moreover, in vivo data confirm that the submission substance does not cause germ cell mutagenicity. It can thus be concluded that the submission substance does not exhibit a risk of genotoxicity in mammals.


Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on this data, classification for genetic toxicity according to 67/584/EEC and EC/1272/2008 is not warranted.