Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Tests: non mutagenic (OECD 471, WoE using γ-Undecalactone and γ-Caprolactone data)

- MLA/TK Forward Mutation Assay with a Confirmatory Assay (OECD 476, GLP, K, rel. 2): ɣ-Nonalactone non mutagenic up to cytotoxicity limit

-Chromosomal aberation tests: non-clastogenic and non-aneugenic (OECD 490, using γ-Undecalactone and γ-Caprolactone data)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2002-05-14 to 2002-06-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted according to JAPAN Ministry of Labor notice, which is similar to the OECD test guideline No. 471. The only deviation is that duplicate plates per dose level were only used instead of triplicates plates. γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: Notice No. 77, 1988-9-1 and No.67, 1997-6-2 of the Ministry of Labour (JAPAN)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
duplicate plates per dose level instead of triplicate plates
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for Salmonella and Tryptophan gene for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 (fraction of Sprague-Dawley male rat liver with phenobarbital and 5,6-benzoflavone) is obtained from Kikkoman Corporation in Japan. (Lot no. RAA-455, RAA-460 prepared on 2001-11-22 and 2002-03-15.) Cofactor was purchased by Roche Diagnostics (Lot 730).
Test concentrations with justification for top dose:
First pre-incubation test (range finding test): with metabolic activation (10 % S9 liver homogenate): 5, 20, 78, 313, 1250, 5000 µg/plate; without metabolic activation: 5, 20, 78, 313, 1250, 5000 µg/plate.
Second pre-incubation test (main test) : with metabolic activation (10 % S9 rat liver homogenate): 313, 625, 1250, 2500, 5000 µg/plate, without metabolic activation: 313, 625, 1250, 2500, 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection (The Japanese Pharmacopoeia)
- Justification for choice of solvent/vehicle: dissolved in water more than 5% and stable in water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
without metabolic activation Migrated to IUCLID6: dissolved in DMSO, for TA98 (0.1 ug/plate), TA100 and WP2uvrA (0.01 ug/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: dissolved in deionized water, for TA1535 (0.5 ug/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation Migrated to IUCLID6: dissolved in DMSO, for TA1537 (80 ug/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation Migrated to IUCLID6: dissolved in DMSO, for TA98, TA100 and TA1537 (5.0 ug/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene dissolved in DMSO, for TA1535 (2.0 ug/plate) and WP2uvrA (10.0 ug/plate)
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation with (10% S9) and without metabolic activation

DURATION
- Preincubation period: 20 min (37 degrees)
- Expression time (cells in growth medium): 48h

NUMBER OF PLATES EVALUATED: two per dose

NUMBER OF REPLICATIONS: two independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: toxicity to bacteria was assessed by thinning of the bacterial lawns and/or reduction in the number of colonies.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- no contamination of different bacteria
- the solvent control data are within the laboratory's normal control range
- the positive controls induce increases in the mutation frequency which are more than twice compared to the solvent control and within the laboratory's normal range
Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate.
b) it induces a dose-related increase in the mean number of revertants per plate.
c) there is a reproducibility of the result in two independent studies
Statistics:
No
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: more than 5%
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
In the first pre-incubation test with (10% S9) and without metabolic activation, bacteria were treated with doses of 5, 20, 78, 313, 1250, 5000 µg/plate. No bacterial toxicity and no increase of revertant colonies were observed in any bacterial strains. Therefore maximum dose was set 5000 µg/plate for the main study.

COMPARISON WITH HISTORICAL CONTROL DATA:
Values for solvent and positive controls were within the laboratory´s historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no bacterial toxicity observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Sterility control: The plates for sterility control of test item and S9-mix showed no growth.

Table 7.6.1/1 Experiment 1 (dose-range finding study)

Study period

2002-05-14 to 2002-5-17

Metabolic

activation

Dose

(ug/plate)

Revertants colony (colony number / plate)

Base substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9Mix

(-)

control

123, 132

(128)

8, 11

(10)

26, 28

(27)

27, 26

(27)

9, 6

(8)

5

149, 132

(141)

9, 12

(11)

21, 25

(23)

24, 21

(23)

9, 7

(8)

20

124, 133

(129)

11, 11

(11)

29, 23

(26)

25, 22

(24)

9, 11

(10)

78

133, 134

(134)

10, 11

(11)

27, 21

(24)

36, 32

(34)

7, 6

(7)

313

131, 133

(132)

12, 15

(14)

33, 27

(30)

34, 27

(31)

8, 8

(8)

1250

130, 132

(131)

8, 12

(10)

32, 27

(30)

35, 24

(30)

6, 7

(7)

5000

121, 140

(131)

10, 11

(11)

33, 27

(30)

43, 27

(35)

7, 6

(7)

S9Mix

(+)

control

128, 143

(136)

11, 11

(11)

30, 31

(31)

37, 29

(33)

7, 8

(8)

5

144, 134

(139)

13, 11

(12)

23, 31

(27)

33, 33

(33)

5, 6

(6)

20

138, 142

(140)

15, 8

(12)

29, 32

(31)

37, 39

(38)

6, 6

(6)

78

137, 143

(140)

8, 13

(11)

27, 34

(31)

28, 37

(33)

7, 7

(7)

313

136, 125

(131)

13, 12

(13)

30, 27

(29)

37, 32

(35)

6, 10

(8)

1250

131, 122

(127)

13, 12

(13)

33, 22

(28)

32, 28

(30)

8, 6

(7)

5000

139, 143

(141)

13, 8

(11)

24, 21

(23)

35, 34

(35)

8, 8

(8)

Positive

control

S9Mix

(-)

Name

AF-21)

NaN32)

AF-2

AF-2

9AA3)

Dose

(ug/plate)

0.01

0.5

0.01

0.1

80.0

Revertants

/plate

713, 706

(710)

444, 454

(449)

177, 173

(175)

621, 547

(584)

870, 710

(790)

S9Mix

(+)

Name

B[a]P4)

2AA5)

2AA

B[a]P

B[a]P

Dose

(ug/plate)

5.0

2.0

10.0

5.0

5.0

Revertants

/plate

1239, 1264

(1252)

325, 280

(303)

995, 1019

(1007)

344, 352

(348)

60, 79

(70)

1) 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2) sodium azide, 3) 9-aminoacridine,

4) benzo[a]pyrene, 5) 2-aminoanthracene

Table 7.6.1/2 Experiment 2 (Main study)

Study period

2002-05-30 to 2002-6-3

Metabolic

activation

Dose

(ug/plate)

Revertants colony (colony number / plate)

Base substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

S9Mix

(-)

control

134, 138

(136)

9, 12

(11)

22, 23

(23)

22, 23

(23)

8, 8

(8)

313

124, 139

(132)

13, 11

(12)

25, 19

(22)

25, 19

(22)

6, 6

(6)

625

123, 121

(122)

12, 13

(13)

27, 22

(25)

21, 24

(23)

6, 6

(6)

1250

130, 139

(135)

12, 9

(11)

19, 22

(21)

24, 23

(24)

6, 6

(6)

2500

138, 134

(136)

12, 7

(10)

22, 23

(23)

21, 28

(25)

10, 5

(8)

5000

140, 133

(137)

11, 12

(12)

19, 20

(20)

20, 24

(22)

7, 6

(7)

S9Mix

(+)

control

139, 125

(132)

11, 11

(11)

29, 25

(27)

24, 27

(26)

6, 8

(7)

313

136, 141

(139)

12, 7

(10)

24, 32

(28)

20, 28

(24)

6, 5

(6)

625

136, 138

(137)

8, 9

(9)

20, 23

(22)

27, 28

(28)

6, 7

(7)

1250

136, 144

(140)

9, 11

(10)

27, 27

(27)

28, 22

(25)

7, 8

(8)

2500

132, 131

(132)

14, 14

(14)

31, 33

(32)

24, 26

(25)

6, 10

(8)

5000

134, 130

(132)

12, 14

(13)

29, 21

(25)

23, 27

(25)

7, 7

(7)

Positive

control

S9Mix

(-)

Name

AF-21)

NaN32)

AF-2

AF-2

9AA3)

Dose

(ug/plate)

0.01

0.5

0.01

0.1

80.0

Revertants

/plate

748, 722

(735)

395, 355

(375)

170, 161

(166)

589, 589

(589)

844, 802

(823)

S9Mix

(+)

Name

B[a]P4)

2AA5)

2AA

B[a]P

B[a]P

Dose

(ug/plate)

5.0

2.0

10.0

5.0

5.0

Revertants

/plate

1153, 1201

(1177)

231, 221

(226)

611, 620

(616)

328, 286

(307)

68, 76

(72)

1) 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 2) sodium azide, 3) 9-aminoacridine,

4) benzo[a]pyrene, 5) 2-aminoanthracene

Conclusions:
Under the test condition, γ-caprolactone is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA- according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay in bacteria performed according to Japanese ministry of Lavor guideline (which is similar to OECD test guideline No. 471) and in compliance with GLP, strains TA1535, TA 1537, TA 98, TA 100 of S. typhimurium and WP2uvrA of E.coli were exposed to γ-caprolactone diluted in water. Two independent experiments were performed up to the limit concentration of 5000 µg/plate with and without mammalian metabolic activation (rat liver S9-mix 10%). No bacterial toxicity was observed in any of the strain tested. Precipitation was not observed at any dose tested. There was no evidence of increased revertant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic activation. The solvent and positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.

Under the test condition, γ-caprolactone is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA-according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1984
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted similarly to the old version of the OECD test guideline No. 471 (1981) therefore an E. coli or S. typhimurium TA102 strain is missing. Individual/mean plate counts, positive controls counts and lower doses applied were not reported. Only duplicate cultures were used per dose level.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli or S. typhimurium TA102 strain is missing; individual/mean plate count, positive controls and lower doses were not reported; only duplicate cultures used/dose; no data on number of bacterial cells per culture and test strain characteristics
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
30 %v/v S9 fraction of Fischer rats liver, pretreated 5 days before with polychlorinated biphenyls (500 mg/kg bw of Kanechlor KC-400 in olive oil, intraperitoneal)
Test concentrations with justification for top dose:
Maximum concentration: 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
- Source of test strains: Dr B. N. Ames (University of California, Berkeley, USA)

METHOD OF APPLICATION: Preincubation method

DURATION
- Preincubation period: 20 minutes at 37 °C
- Incubation period for treated plates: 2 days at 37 °C

NUMBER OF REPLICATIONS: Duplicate
Evaluation criteria:
Result was considered positive if the number of colonies found twice the number in the control.
Statistics:
None
Species / strain:
other: TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98
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
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
γ-Undecalactone is not mutagenic with and without metabolic activation in S. typhimurium strains TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98 according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD Guideline 471, strains of Salmonella typhimurium (TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98) were exposed to γ-Undecalactone up to 5000 µg/plate, with and without metabolic activation (30 %v/v S9 fraction of Fischer rats liver induced with polychlorinated biphenyls) according to the pre-incubation method. Vehicle control (DMSO) was included in this experiment.

γ-Undecalactone showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains up to 5000 µg/plate in either presence or absence of S9 mix.

γ-Undecalactone is not mutagenic with and without metabolic activation in S. typhimurium strains TA 92, TA 1535, TA 100, TA 1537, TA 94 and TA 98 according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for an in vitro bacterial reverse gene mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 February 2012 to 2 April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 476 without any deviations. γ-Nonalactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
The L5178Y mouse lymphoma (3.7.2c) cells are heterozygous at the thymidine kinase locus, TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: R10p medium was used for cell culture unless otherwise specified. R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6 benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C. Lot No.: 2882 (Date of preparation
Test concentrations with justification for top dose:
Preliminary toxicity test: -S9 mix and +S9 mix
(3 hours and 24 hours) 3.1, 6.1, 12.2, 24.4, 48.8, 97.6, 195.3, 390.5, 781 and 1562 µg/mL

Mutation tests:
-S9 mix (3 hours) 200, 300, 400, 450, 500, 550, 600, 650, 700, 750 and 800 µg/mL
+S9 mix (3 hours) 200, 300, 400, 600, 800, 1000, 1200, 1400 and 1562 µg/mL

+S9 mix additional (3 hours) 200, 400, 800, 1000, 1050, 1100, 1150 and 1200 µg/mL

-S9 mix (24 hours) 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700 and 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Gamma-Nonalactone was dissolved and diluted in acetone (analytical grade), shortly before dosing. The final concentration of acetone added to the cultures was 1% v/v.
- Justification for choice of solvent/vehicle: Prior to commencing testing, Gamma-Nonalactone was confirmed to be soluble at 156.2 mg/mL in acetone. A solution of 1562 mg/mL, dosed at 1% in medium, showed no precipitate in the culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
In the absence of S9 mix
Positive control substance:
methylmethanesulfonate
Remarks:
Solvent: DMSO / Exposure concentration:10 µg/mL (3 hour exposure) and 5 µg/mL (24 hour exposure)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
In the presence of S9 mix
Positive control substance:
benzo(a)pyrene
Remarks:
Solvent: DMSO / Exposure concentration:10 µg/mL (3 hour exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): the mutant 96-well plates had been incubated 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls.

NUMBER OF CELLS EVALUATED: Cloning efficiency was assessed by plating 1.6 cells/well in R20p medium, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in selective medium, two plates being prepared per culture.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (CE); relative suspention growth (RSG); other: mutant frequency (MF)

OTHER EXAMINATIONS:
- Other: The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants.
Evaluation criteria:
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
The criteria used to assess whether an assay was valid are based on the recommendations of the Plymouth and Aberdeen papers (Robinson et al., 1989; Moore et al., 2000; Moore et al., 2002; Moore et al., 2003; Moore et al., 2006 and Moore et al., 2007).

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment.
Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
up to Gamma-Nonalactone cytotoxic concentrations following 3-hour and 24-hour exposure.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No precipitate (observed by eye at the end of treatment) was observed at any concentration tested in the absence and presence of S9 mix following any exposure.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality:no
- Evaporation from medium:no
- Water solubility:no
- Precipitation:no
- Other confounding effects:no

RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No precipitate (observed by eye at the end of treatment) was observed at any concentration tested in the absence and presence of S9 mix following a 3 hour exposure. Exposure to Gamma-Nonalactone at concentrations from 3.1 to 1562 µg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values between 132 and 0% and between 109 and 2% respectively. (see Table 1 in attached document)
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration tested. Exposure to concentrations from 3.1 to 1562 µg/mL resulted in RSG values between 104 and 0%. (see Table 1 in attached document)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

1.1 Main mutation test - 3 hour treatment in the absence of S9 mix (see Table 2 and Table 3 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 800 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 200 to 550 mg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 125 to 23% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (213 x 10-6), of the mean concurrent vehicle control mutant frequency (87 x 10-6) and the Global Evaluation Factor (GEF) of 126 x 10-6, within acceptable levels of toxicity.

Although this assay did not reach the required 10-20% RTG it is considered valid in accordance withMoore et al. (2002)which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

1.2  Main mutation test - 3 hour treatment in the presence of S9 mix (see Table 4 in attached document)

The results are shown inTable4.

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 1562 mg/mL. No precipitate was observed by eye at the end of treatment. RSG values from 90 to 2% were obtained relative to the vehicle control. As an appropriate toxicity profile was not achieved an additional test was performed.

1.3 Additional main mutation test - 3 hour treatment in the presence of S9 mix (see Table 5 and Table 6 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 1200 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 200 to 1050 mg/mL were assessed for determination of mutation frequency. RTG values from 110 to 11% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (214 x 10-6) of the mean concurrent vehicle control mutant frequency (88 x 10-6) and the GEF (126 x 10-6), within acceptable levels of toxicity.

The positive control, benzo[a]pyrene,induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

The results obtained in response to the exposure of cultures to Gamma-Nonalactone in the presence of S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the acceptable vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

1.4    Main mutation test - 24 hour treatment in the absence of S9 mix (see Table 7 and Table 8 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 100 to 800 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 100 to 550 mg/mL were assessed for determination of mutation frequency. RTG values from 131 to 17% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (214 x 10-6) of the mean concurrent vehicle control mutant frequency (88 x 10-6) and the GEF (126 x 10-6), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Conclusions:
Under the test conditions, Gamma-Nonalactone did not demonstrate mutagenic potential in this in vitro cell mutation assay in the absence and presence of metabolic activation.
Executive summary:

Gamma-Nonalactone was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD 476 guideline and in compliance with GLP. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

Gamma-Nonalactone was found to be soluble at 156.2 mg/mL in acetone. A final concentration of 1562 mg/mL (10 mM), dosed at 1% v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the standard limit concentration within this test system as recommended in the regulatory guidelines. 

Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Gamma-Nonalactone at concentrations from 3.1 to 1562 mg/mL, relative suspension growth (RSG) values were between 132 and 0% and between 109 and 2% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG vales were between 104 and 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency (93 x 10-6and 88 x 10-6, respectively) and the Global Evaluation Factor (GEF) of 126 x 10-6, within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and in the presence of S9 mix were 550 and 1050 mg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 23 and 11% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 550 mg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control (88 x 10-6) mutant frequency and the GEF of 126 x 10-6, within acceptable levels of toxicity. The RTG was reduced to 17%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that Gamma-Nonalactone did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2002-06-17 to 2002-11-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to Japanese guideline for medicine and for new chemicals. γ-Caprolactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across-purpose (see §"Toxicokinetics").
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: Japanese guideline for medicine (iyakushin no.1604, 1999-11-1) and Japanese guidline for new chemicals (kanpoann no.287, eisei no.127, kikyoku no.2 in 1997-10-31)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
other: chinese hamster lung cell (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle MEM (Eagle Minimal Essential Medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no information in the report
- Periodically checked for karyotype stability: no information in the report
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 (fraction of Sprague-Dawley male rat liver with phenobarbital and 5,6-benzoflavone)-mix is obtained from Kikkoman Corporation, in Japan. (Lot no. CAM-463 and CAM-465)
Test concentrations with justification for top dose:
Evaluated test concentrations:
Dose range finding test (Experiment I): Growth rate determination
with S9 mix/ 6 hrs exposure + 18 hrs incubation: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
without S9 mix/ 6 hrs exposure + 18 hrs incubation: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
without S9 mix/ 24 hrs exposure: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
without S9 mix/ 48 hrs exposure: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL

Main test (Experiment II): Growth rate determination+ chromosomal aberration
with S9 mix/ 6 hrs exposure + 18 hrs incubation: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
without S9 mix/ 6 hrs exposure + 18 hrs incubation: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
without S9 mix/ 24 hrs exposure: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
without S9 mix/ 48 hrs exposure: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL

Additional test (Experiment III): Growth rate determination+ chromosomal aberration
without S9 mix/ 6 hrs exposure + 18 hrs incubation: 2566, 2822, 3105, 3415, 3757, 3945, 4132, 4545, 5000 µg/mL

Confirmatory test (Experiment IV):
with S9 mix/ 6 hrs exposure + 18 hrs incubation: 143, 285, 570, 1140 µg/mL
without S9 mix/ 6 hrs exposure + 18 hrs incubation: 143, 285, 570, 1140 µg/mL
without S9 mix/ 24 hrs exposure: 143, 285, 570, 1140 µg/mL
without S9 mix/ 48 hrs exposure: 143, 285, 570, 1140 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline (The Japanese Pharmacopoeia)
- Justification for choice of solvent/vehicle: dissolved in water more than 5% and stable in water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
physiological saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
physiological saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation (0.1 µg/mL for 6hr treatment, 0.05 µg/mL for 24 and 48 hr treatment)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (Eagle MEM with 10% FCS)
DURATION
- Exposure duration and Expression time (cells in growth medium):
1. with metabolic activation for 6hr + 18hr incubation for expression
2. without metabolic activation for 6hr + 18hr incubation for expression
3. without metabolic activation for 24hr + no exression time
4. without metabolic activation for 48hr + no exression time
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr respectively

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.4 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2 independent experiments, per group two parallel cultures were set up.
NUMBER OF CELLS EVALUATED: at least 100 methaphase plates were scored per culture. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Numerical aberration was also recorded. Gaps were recorded as well, but not included in the calculation of the aberration rates.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth measured by monocellater

OTHER EXAMINATIONS: recorded as numerical aberration
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
The result is negative if:
- the total rate of structural chromosome aberrations and/or numerical aberrations is less than 5%
The result is equivoval if:
- the total rate of structural chromosome aberrations and/or numerical aberrations is between 5% and 10%
The result is positive if:
- the total rate of structural chromosome aberrations and/or numerical aberration exceeds 10%
Statistics:
Statistical evaluation was not performed in this test.
Species / strain:
other: chinese hamster lung cell (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY 1:

1. Dose range finding test (Experiment I): Growth rate determination
test system 1: with S9 mix/ 6 hrs exposure + 18 hrs incubation: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
test system 2: without S9 mix/ 6 hrs exposure + 18 hrs incubation: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
test system 3: without S9 mix/ 24 hrs exposure: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL
test system 4: without S9 mix/ 48 hrs exposure: 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/mL

More than 50% growth inhibition was observed at 5000 µg/mL in the test system 2, 3 and 4, then IC50 was calculated 3320, 2273 and 2583, respectively. IC50 of test system 1 was speculated between 2500 and 5000 µg/mL. No precipitation of the test substance was observed in any test systems. A pH decrease was observed over 625 µg/mL in the test systme 1, 2 and 4, and over 1250 µg/mL in the test system 3 at the end of the exposure time period.

2. Main test (Experiment II): Growth rate determination+ chromosomal aberration
test system 1: with S9 mix/ 6 hrs exposure + 18 hrs incubation: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
test system 2: without S9 mix/ 6 hrs exposure + 18 hrs incubation: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
test system 3: without S9 mix/ 24 hrs exposure: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL
test system 4: without S9 mix/ 48 hrs exposure: 1635, 1880, 2162, 2486, 2859, 3288, 3781, 4348, 5000 µg/mL

A growth inhibition was observed over 2486 µg/mL in the test system 2, and reached below 50% at the concentration of 4348 µg/mL. A growth inhibition was observed between 3288 and 4348 µg/mL in the test system 1, but did not obtain 50% inhibition. A growth inhibition was observed over 2162 µg/mL in the test system 3, and reached below 50% over 3288 µg/mL. A growth inhibition was observed over 1880 µg/mL in the test system 4, and reached below 50% over 2486 µg/mL.
No precipitation of the test substance was observed in any test systems. A pH decrease was observed in all concentrations tested in all test systems.

3. Additional test (Experiment III): Growth rate determination+ chromosomal aberration
test system 2: without S9 mix/ 6 hrs exposure + 18 hrs incubation: 2566, 2822, 3105, 3415, 3757, 3945, 4132, 4545, 5000 µg/mL

A growth inhibition was observed over 3105 µg/mL in the test system 2, and reached below 50% over 3945 µg/mL.
No precipitation of the test substance was observed in any test systems. A pH decrease was observed in all concentrations tested in all test systems.


CHROMOSOMAL ABERRATION 1 (EXPERIMENT II AND III)

Structual aberration rate at 3105 and 3415 µg/mL in the test system 2 was below 5%, but 39.0% (positive criteria) was obtained at 3757 µg/mL. Numerical aberration rate was below 5%. Structual aberration rate at 2486 and 2859 µg/mL in the test system 1 was below 5%, but 18.5% (positive criteria) was obtained at 3288 µg/mL. Numerical aberration rate was below 5%. Structual aberration rate at 2486 and 2859 µg/mL in the test system 3 was below 5%, but can not be determined at 3288 µg/mL due to the strong cytotoxicity. Structual aberration rate and numerical aberration rate were below 5% at 1880, 2162 and 2486 µg/mL. Structual aberration rates of positive control were 29.0, 65.0, 65.0 and 89.5% in the test system 1, 2, 3 and 4, respectively.


CYTOTOXICITY 2 and CHROMOSOMAL ABERRATION 2:

4. Confirmatory test (Experiment IV):
test system 1: with S9 mix/ 6 hrs exposure + 18 hrs incubation: 143, 285, 570, 1140 µg/mL
test system 2: without S9 mix/ 6 hrs exposure + 18 hrs incubation: 143, 285, 570, 1140 µg/mL
test system 3: without S9 mix/ 24 hrs exposure: 143, 285, 570, 1140 µg/mL
test system 4: without S9 mix/ 48 hrs exposure: 143, 285, 570, 1140 µg/mL

A growth inhibition was not observed at alll concentration tested in all test systems. No precipitation of the test substance was observed in any test systems. A pH decrease was observed over 1140 µg/mL in all test systems.
Structual aberration rate and numerical aberration rate were below 5% in all concentraions tested in all test systems.
Structual aberration rates of positive control were 51.0, 76.0, 79.5 and 91.0% in the test system 1, 2, 3 and 4, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See "Tables for gamma-CL (chromosomal aberration test)" in "Attached background material".

Conclusions:
Under the test conditions, γ-Caprolactone did not show any cytogenic activity in the chromosomal aberration test using hamster cells. Therefore, γ-Caprolactone was not considered as clastogenic in hamster cells in vitro according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and to the GHS. 
Executive summary:

In a chromosomal aberration assay in mammalian cells, performed according to the Japanese guideline of medicine, and in compliance with GLP, γ-Caprolactone (purity 97.4%) diluted in physiological saline was tested in female Chinese Hamster lung cells (CHL/IU) in the presence and the absence of mammalian metabolic activation (S9) at concentrations up to 5000 µg/mL.

γ-caprolactone was incubated with the cells for 6, 24 or 48 hours and the cells were analysed for the presence of chromosomal aberration 18 hours (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period).

In dose range finding test (Experiment I) for growth rate determination, more than 50% growth inhibition was observed at 5000 µg/mL in the test system 2, 3 and 4, then IC50 was calculated 3320, 2273 and 2583, respectively. IC50 of test system 1 was speculated between 2500 and 5000 µg/mL. No precipitation of the test substance was observed in any test systems. A pH decrease was observed over 625 µg/mL in the test systme 1, 2 and 4, and over 1250 µg/mL in the test system 3 at the end of the exposure time period.

In the first main test (Experiment II) for growth rate determination and chromosomal aberration, a growth inhibition was observed over 2486 µg/mL in the test system 2, and reached below 50% at the concentraion of 4348 µg/mL. A growth inhibition was observed between 3288 and 4348 µg/mL in the test system 1, but did not obtain 50% inhibition. A growth inhibition was observed over 2162 µg/mL in the test system 3, and reached below 50% over 3288 µg/mL. A growth inhibition was observed over 1880 µg/mL in the test system 4, and reached below 50% over 2486 µg/mL.

No precipitation of the test substance was observed in any test systems. A pH decrease was observed in all concentrations tested in all test systems.

In the additional test (Experiment III) for growth rate determination and chromosomal aberration in the test system 2, a growth inhibition was observed over 3105 µg/mL in the test system 2, and reached below 50% over 3945 µg/mL. No precipitation of the test substance was observed in any test systems. A pH decrease was observed in all concentrations tested in all test systems.

In the chromosomal aberration study for experiment II and III, structural aberration rate at 3105 and 3415 µg/mL in the test system 2 was below 5%, but 39.0% (positive criteria) was obtained at 3757 µg/mL. Numerical aberration rate was below 5%. Structural aberration rate at 2486 and 2859 µg/mL in the test system 1 was below 5%, but 18.5% (positive criteria) was obtained at 3288 µg/mL. Numerical aberration rate was below 5%. Structural aberration rate at 2486 and 2859 µg/mL in the test system 3 was below 5%, but can not be determined at 3288 µg/mL due to the strong cytotoxicity. Structural aberration rate and numerical aberration rate were below 5% at 1880, 2162 and 2486 µg/mL. Structural aberration rates of positive control were 29.0, 65.0, 65.0 and 89.5% in the test system 1, 2, 3 and 4, respectively.

Structural aberration observed in this study was only at the concentrations about 30mM (over 10mM), therefore this result is considered to be obtained due to unphysiological conditions applied in this test.

In the confirmatory study (Experiment IV), a growth inhibition was not observed at alll concentration tested in all test systems. No precipitation of the test substance was observed in any test systems. A pH decrease was observed over 1140 µg/mL in all test systems.

Structual aberration rate and numerical aberration rate were below 5% in all concentraions tested in all test systems.

Structual aberration rates of positive control were 51.0, 76.0, 79.5 and 91.0% in the test system 1, 2, 3 and 4, respectively.

Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses.

Under the test conditions, γ-Caprolactone did not show any cytogenic activity in the chromosomal aberration test using hamster cells. γ-Caprolactone was not considered as clastogenic in hamster cells in vitro according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and to the GHS. 

This study is considered as acceptable as it satisfied the criteria of the Japanese guideline for medicine.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1984
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Significant methodological deficiencies: only tested without metabolic activation; incubation temperature, signs of toxicity, positive controls and lower doses applied were not reported; no data on no. of replicates and no. of chromosomal aberrations per culture
Reason / purpose:
reference to same study
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo, Japan (Koyama, Utakoji & Ono, 1970)
- CHL was maintained by 4-day passages in Minimum Essential Medium supplemented by 10 % calf serum.
- The modal chromosome number is 25 and the doubling time was approximately 15 h.
- Source: Cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo, Japan (Koyama, Utakoji & Ono, 1970).
- Type and identity of media: Cell line was maintained by 4-day passages in Minimum Essential Medium supplemented by 10 % calf serum.
- The modal chromosome number is 25 and the doubling time was approximately 15 h.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
Maximum concentration: 0.5 mg/mL (highest non-cytotoxic dose)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Species / strain:
other: Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Chromosomal aberrations (48 h exposure):

- Polyploid (%): 0

- Structural aberrations (%): 3

Conclusions:
Under the test conditions, γ-Undecalactone is not clastogenic without metabolic activation in Chinese hamster fibroblast cell line.
Executive summary:

In an in vitro mammalian chromosome aberration test, Chinese hamster fibroblast cell lines were exposed to γ-Undecalactone at concentrations up to 0.5 mg/mL in Minimum Essential Medium without metabolic activation system for 24 and 48 h. Colcemid (0.2 µg/mL) was added to the culture 2 h before cell harvesting. The cells were then treated with a hypotonic solution, fixed and spread on glass slides. The slides were stained with Giemsa and 100 well-spread metaphases were observed for polyploid and chromosomal aberrations.

γ-Undecalactone showed no substantial increases in either the proportion of polyploid cells or the proportion of metaphase figures containing chromosomal aberrations up to 0.5 mg/mL (highest non-cytotoxic dose), without metabolic activation.

Under the test conditions, γ-Undecalactone is not clastogenic without metabolic activation in Chinese hamster fibroblast cell line.

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

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test: ɣ-Undecalactone not clastogenic and not aneugenic in mice bone marrow cells up to limit dose (OECD 474, GLP, K, rel. 2) + in vitro CAT, ɣ-Caprolactone not clastogenic up to limit concentration (S, rel.2).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted similarly to OECD Guideline 474 with minor deviations: no data on the housing conditions and individual body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported. 1000 polychromatic erythrocytes (PCE) were analysed per animal, which was a requirement of the old version of the OECD 474 (1983). The new version requires the analysis of at least 2000 PCE.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
no data on the housing conditions and body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: ddY mice
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan
- Age at study initiation: 8 weeks
- Diet: Food pellets CE-2 (Japan Clea, Tokyo, Japan), ad libitum
- Water: ad libitum
Route of administration:
intraperitoneal
Vehicle:
Olive oil
Details on exposure:
None
Duration of treatment / exposure:
- 250, 500, 1000 and 2000 mg/kg bw: One day
- 500 mg/kg bw: 4 days
Frequency of treatment:
- 250, 500, 1000 and 2000 mg/kg bw: Single injection
- 500 mg/kg bw: 4 injections with 24 h intervals between the injections
Post exposure period:
24 h
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin-C
- Source: Kyowa Hakko Ltd, Tokyo, Japan
- Route of administration: Intraperitoneal
- Doses: 2 mg/kg bw
Tissues and cell types examined:
- Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs) and total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum doses of the test material were set at the supposed maximum tolerated dose referring to LD50.

DETAILS OF SLIDE PREPARATION: Mice were killed by cervical dislocation at 24 h after exposure. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain.

METHOD OF ANALYSIS:
- 1000 polychromatic erythrocytes (PCEs) per mouse were scored using a light microscope with a high power objective (X 100) and the no. of micronucleated polychromatic erythrocytes (MNPCEs) were recorded.
- Proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was evaluated by observing 1000 erythrocytes on the same slide.
Evaluation criteria:
Result was considered as positive if one or more treatment group(s) showed a statistically significant difference (P < 0.01) from the spontaneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05).
Statistics:
Two-stage statistical procedure:
- In the first step of the procedure, the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data (Hayashi et al. 1985).
- In the second step, the dose-response relationship was tested by the Cochran- Armitage trend test (Armitage, 1955; Cochran, 1954; Margolin and Risko, 1988).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- See table 7.6.2/1 for MNPCEs (%) and PCEs (%)

Table 7.6.2/1: Results of micronucleus test

Groups

Dose (mg/kg bw)

No. of doses

Time between doses (24 h)

Sampling time (h)

MNPCEs (%)

PCEs (%)

Mortality

Vehicle (Olive oil)

0

1

-

24

0.10 ± 0.06

54.6 ± 6.4

0/6

Undecalactone

250

0.25 ± 0.12

51.6 ± 7.7

500

0.08 ± 0.08

43.9 ± 7.4

1000

0.18 ± 0.08

43.7 ± 9.2

2000

0.25 ± 0.07

52.3 ± 3.0

500

4

24

0.08 ± 0.10

48.7 ± 8.1

Mitomycin-C

2

1

-

4.78 ± 1.40*

43.5 ± 4.6

 

MNPCEs - Micronucleated polychromatic erythrocytes; PCEs - Polychromatic erythrocytes;

* value of MNPCE differ significantly from the historical control (p < 0.01)

Conclusions:
Under the test conditions, Undecalactone is not considered as clastogenic in the mouse bone marrow micronucleus assay according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vivo mouse bone marrow micronucleus assay performed similarly to OECD Guideline 474, groups of ddY male mice (6/dose) were injected with Undecalactone in olive oil at 250, 500, 1000 and 2000 mg/kg bw (single treatment) and 500 mg/kg bw (4 injections with 24 h intervals between the injections) by intraperitoneal route. The positive control group was injected with mitomycin C at 2 mg/kg bw. Mice were killed by cervical dislocation 24 h after an administration. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain. 1000 polychromatic erythrocytes (PCEs) per mouse were scored for recording the incidence of micronucleated polychromatic erythrocytes (MNPCEs). The proportion of PCEs among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Mice treated with Undecalactone did not show any significant increase in the frequency of MNPCEs. Positive control (mitomycin C) induced a statistically significant increase in MNPCEs indicating the validity of the study.

Under the test conditions, Undecalactone is not considered as clastogenic in the mouse bone marrow micronucleus assay according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

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

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains / Cells tested

Metabolic activation

Test concentration

Statement

1

 

Ishidate, 1984

Ames Test

(eq. OECD 471)

WoE, rel. 4

γ-Undecalactone

 

Gene mutation

TA 1535,

TA 1537,

TA92,

TA94,

TA 98,

TA 100

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Masayuki Kato, 2002

Ames Test

(eq. OECD 471)

WoE, rel. 2

γ-Caprolactone

 

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2uvrA

-S9

+S9

Up to 5000 µg/plate

-S9 : non mutagenic

+S9 : non mutagenic

3

Woods, 2012

 

ML/TK test (OECD 476)

K, rel. 2

γ-Nonalactone

 

Gene mutation

mouse lymphoma L5178Y cells

-S9

+S9

Up to 800 µg/mL –S9

Up to 1562 µg/mL (10mM) +S9

-S9 : non mutagenic

+S9 : non mutagenic

4

 

Hayashi, 1988

In vivo micronucleus (eq. OECD 474)

K, rel. 2

γ-Undecalactone

 

Chromosomal aberration

Bone marrow cells (femur)

NA

Up to 2000 mg/kg bw

Not clastogenic

 5

 

Kawamura, 2002

CHL/CAT

S, rel.2

γ-Caprolactone

 

Chromosomal aberration

Chinese Hamster lung fibroblasts

-S9

+S9

Up to 5000 µg/mL (cytotoxic)

-S9 : non clastogenic

+S9 : non clastogenic

 6

 

Ishidate, 1984

CHL/CAT

S, rel. 3

γ-Undecalactone

 

Chromosomal aberration

Chinese Hamster lung fibroblasts

 

-S9

Up to 500 µg/mL

-S9 : non clastogenic

 

 

Gene mutation Assays (Tests n° 1-3):

Two Bacterial Reverse mutation Assays(Ames test)were performed similarly to the OECD 471 test guideline with γ-Undecalactone, and the read-across substance, γ-Caprolactone (See §”Toxicokinetics” for read-across justification). Both tests were used in a weight-of evidence approach as only four strains of bacteria were used in the Test n°1 whereas all required strains were used in Test n°2. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test materials up to limit concentration, either in the presence or absence of metabolic activation. The tests indicate that γ-Undecalactone and γ-Caprolactone did not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. Based on this weight-of evidence, γ-Undecalactone is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using anin vitroforward mutation assay in mouse lymphoma TK L5178Y cells(ML/TK test)(Test n°3). None of the dose levels up to the cytotoxicity limit with the read-across substance, ɣ-Nonalactone, either with or without metabolic activation, induced significant mutant frequency increases in the initial, or repeat tests. ɣ-Nonalactone does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. ɣ-Nonalactoneis therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of both Ames tests and extends the non-mutagenic effect of aliphatic ɣ-lactones to mammalian cells.

 

Chromosomal aberration (Tests n°4-6)

The clastogenic and aneugenic potential of γ-Undecalactone was determined using an in vivo mammalian erythrocytes micronucleus assay (Test n°4), which measured the potential of a substance to increase the incidence of the structural and numerical chromosome aberrations in mice bone marrow erythrocytes. None of the dose level, up to the limit dose of 2000 mg/kg bw, induced increase in the frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow, whereas positive control chemical induced significant increase in the fMPCE. γ-Undecalactone was therefore considered as negative for inducing chromosomal aberrations in mice bone marrow erythrocytes under the conditions used in this study. This result is supported by two in vitro chromosome aberration tests in Chinese hamster lung fibroblast, conducted on γ-Undecalactone (Test n°5) and on the read-across substance, γ-Caprolactone (Test n°6). In both tests, none of the dose levels up to the cytotoxicity limit, either in the presence of absence of metabolic activation, induced significant increases in the frequency of cells with aberrations.

γ-Undecalactone is therefore considered as non-clastogenic and non-aneugenic.

Justification for classification or non-classification

Harmonized classification:

γ-Undecalactone has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex I of Regulation (EC) No. 1272/2008 (CLP) and to te GHS.