Cyclohex-1,4-ylenedimethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A trio of in vitro bacterial and mammalian genetox studies, (Ames, mammalian mutagenicity and chromosomal aberration) did not demonstrate any signs of mutagenic or clastogenic effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 April 2002 to 20 May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted by GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Qualifier:

according to guideline

Guideline:

other: Joint Directives of JEPA, JMHW and JMITI. (31 October 1007) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine auxotrophic

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254-induced male Sprague-Dawley rat liver

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000ug/plate for S. typhimurium TA 98, TA100, TA1535, TA1537, and E. coli WP2 uvr A pKM 101 (range-finding)
50, 150, 500, 1500, 5000ug/plate for S. typhimurium TA 98, TA100, TA1535, TA1537, and E. coli WP2 uvr A pKM 101(with pre-incuvation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of CHDM was assessed at 50mg/ml in water, in which it dissolved. Water (purified in-house by reverse osmosis) was, therefore, used as the solvent for this study.

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix Migrated to IUCLID6: Supplier: Sigma Chemical Company, Purity: min 99.5%

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix Migrated to IUCLID6: Supplier: Sigma Chemical Company, Purity: >97%

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix Migrated to IUCLID6: Supplier: Aldrich Chemical Company, Purity: 98%

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

other: 2-(20Furyl)-3-(5-nitro-2-fuyrl)acrylamide (AF-2)

Remarks:

In the absence of S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix Migrated to IUCLID6: Supplier: Aldrich Chemical Company, Purity: 98%

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:

First test (range-finding test)
CHDM was added to cultures of the five tester strains at seven concentrations separated by ca half-log10 intervals. The highest concentration of CHDM tested was 50 mg/ml in the chosen solvent, which provided a final concentration of 5000 ug/palte. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, water. The appropriate positive controls were also included.

Aliquots of 0.1 ml of the test dilution, positive control or negative control were placed in glass vessel. S9 mix(0.5mg) or 0.1M pH 7.4 phosphate buffer (0.5ml) was added, followed by 0.1ml of a 10 hour bacterial culture and 2 ml of agar containing histidine (0.5mM) and tryptophan (0.5mM). The mixture was thoroughly shaken and overlaid onto previously prepared Pertri dishes containing 25ml minimal agar. Each Petri dish was individually labeled with a unique code corresponding to a sheet, identifying the contents of the dish. Three Petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of CHDM, S9 mix and sodium phosphate buffer. All plates were incubated at 37℃ for ca 72 hours. After this period the appearance of the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

Any toxic effects of CHDM would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed a lower concentration might be chosen, ensuring that sings of bacterial inhibition are present at the top concentration. Ideally a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37℃ for 30 minutes with shaking before the addition of the ager overaly. The top concentration chosen was again 5000ug/plate, but only five concentrations were used.


DURATION
- Preincubation period: 10 hours
- Exposure duration: 72 hours (range-finding test) 30 minutes (second test)

SELECTION AGENT (mutation assays): Aroclor 1254

NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 1E009

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

If exposure to a test substance produces an increase in revertant colony numbers of at least twice th concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain ethier in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system.

Statistics:

No statistical analysis will be performed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to CHDM at any concentration in either the presence or absence of S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current histiorical control range of the laboratory (except strain TA98, range finding test, and strain TA100, second test, where mean control counts in the absence of S9 mix slightly exceeded the upper limit. This was not considered to affect the integrity of the suty). Appropriate positive control chemicals (with S9 mix where required) induced substantial increase in revertant colony numbers with all strains, conforming sensitivity of the cultures and activity of the S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tester strain		Chemical treated	Dose (mg/plate)	Colonies/plate (mean±SD)
				Without S9 mix	With S9 mix
TA 98		Test item	0	35±11	58±5
			50	49±3	67±3
			150	39±5	59±5
			500	45±4	60±6
			1500	44±4	58±8
			5000	28±2	60±12
TA 100		Test item	0	160±11	142±13
			50	138±33	175±4
			150	158±5	114±2
			500	152±12	141±13
			1500	144±19	139±19
			5000	154±14	148±29
TA 1535		Test item	0	21±1	25±3
			50	9±3	20±4
			150	8±6	21±8
			500	16±5	22±1
			1500	16±3	11±2
			5000	22±15	17±0
TA 1537		Test item	0	30±6	37±10
			50	14±3	28±2
			150	20±6	27±12
			500	16±1	27±1
			1500	23±3	29±5
			5000	18±5	33±17
E. coli WP2uvrA		Test item	0	141±15	187±18
			50	184±16	170±21
			150	159±11	226±31
			500	182±20	203±23
			1500	138±19	229±21
			5000	119±36	238±20
Positive control
TA 98	benzo(a)pyrene		5mg/plate	460±51
	2-nitrofluorene		1mg/plate	401±75
TA 100	benzo(a)pyrene		5mg/plate	628±48
	sodium azide		0.5mg/plate	538±59
TA 1535	2-aminoanthracene		2mg/plate	55±3
	sodium azide		0.5mg/plate	378±77
TA 1537	benzo(a)pyrene		5mg/plate	344±26
	9-aminoacridine		50mg/plate	1326±123
WP2uvrA	2-aminoanthracene		10mg/plate	1316±182
	2-(2-Furyl)-3-(5-nitro-2-fuyrl)acrylamide		0.05mg/plate	1061±77

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that, under the test conditions employed, CHDM showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of CHDM, histidine dependent auxotrophic mutants of Salmonella typhimuirium, strains TA 1535, TA 1537, TA 98 and TA 100, and a tryptophan dependent mutant of Escherichia coil, strain WP2uvrA/pKM101 (CM891), were exposed to CHDM diluted in water. Water was also used as a negative control.

Two independent mutation test were performed in the presence and absence of liver preparations from Aroclor 1254 -induced rats (S9 mix). The first (range-finding) was a standard plate incorporation assay; the second involved a pre-incubation stage.

Concentrations of CHDM up to 5,000ug/plate were tested in the mutation tests. This is the standard limit concentration recmmended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the hightest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of CHDM in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, CHDM showed no evidence of mutagenci activity in this bacterial system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2002 - 7 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The method described was also designed to comply with ICH (1995 & 1997).

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 1 I.U./ml sodium heparin, 20 I.U./ml penicillin/20ug/ml streptomycin and 2.0mM glutamine
- Properly maintained: yes
Others: Human blood was collected aseptically from healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum Aliquots (0.4 ml blood : 4.5ml medium : 0.1 ml phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37℃ for approximately 48 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254-induced male Sprague-Dawley derived rat liver

Test concentrations with justification for top dose:

0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 mM for the first test.
0.078, 0.156, 0.313, 0.625, 1.25, 2.6, 5, and 10 mM for the second test with the condition without S9 mix
0.625, 1.25, 2.5, 5, and 10mM with the condition with S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: The solubility of CHDM was assessed at 144.21 mg/ml in water. Water was, therefore, used as the solvent for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

purified water in 50 ul aliquots

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 mix

Migrated to IUCLID6: Supplier: Sigma chemical Co Ltd, Batch number 80K2526(Test 1) and 31K 2502(Test 2) Final Conc.:

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 mix

Migrated to IUCLID6: Supplier: Asta Medica Ltd Final Conc. 10 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Culture of Lymphocytes: Human blood was collected aseptically from healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 1 I.U./ml sodium heparin, 20 I.U./ml penicilline /20 ug/ml streptomycin and 2.0 mN glutamine. Aliquots of the cell suspension were placed in sterile universal containers and incubated at 37 ℃ for approximately 48 hours. The cultures were shaken daily to resuspend the cells.

Test method:
Treatment of cells with test substance-First test: After approximately 48 hours, 50 ul aliquots of CHDM were added to one set of duplicate cultures to give final concentrations. Purified water, the solvent control, in 50 ul aliquots, was added to sultures. Mytomycin C at a final concentration of 0.2 ug/ml, was added to duplicate cultures.
Immediately before treatment of the second set of cultures, 1 ml of medium was removed from each culture and discarded. This was replaced with 1 ml of S9 mix, followed by 50 ul aliquots of the various dilutions of CHDM, giving the same series of final concentrations as above. Cyclophosphamide was added to duplicate cultures at a final concentration of 10 ug/ml. Three hours after dosing, the cultures were centrifuged at 500 g for 5 minutes. The cell pellets were rinsed and resuspended in fresh medium. They were then incubated for a further 17 hours.
Harventing and Fixation : Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at an final concentration. After 2 hours incuvation, each cell suspension was transferred to a centrigue tube and centrifuged for 5 minutes at 500 g. After a 10 minute period of hypotonic incuvation at 37, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methonol: 1 part glacial acetic acid). The fixative was replaced twice.

Anaylsis: The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. Metaphase cells were identified using a low power objective and examined at a magnification of ×1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture.

Second Test: A continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a three hour treatment was used, as in the first test. Duplicate cultures were used for each treatment and two cultures were treated with the solvent control. Mytomycin C and Cyclophosphamide were added to duplicate cultures.
All cultures were treated with colcemid, at a final concentration of 0.1 ug/ml, two hours before the end of the the end of the incubation period.

DURATION
- Preincubation period: 48hours
- Exposure duration: First test- 3hours treatment in both the presence and the absence of S9 mix
Second test- 20 hours continuous treatment without S9 mix, 3hours treatment with S9 mix
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): 2hours
- Fixation time (start of exposure up to fixation or harvest of cells): Fixation time-after 2hours incubation
Harvest tion- 17hours for the first test, 20hours for the second test


SPINDLE INHIBITOR (cytogenetic assays): mitotic inhibitor, Colcemid
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8)


NUMBER OF REPLICATIONS: duplication

NUMBER OF CELLS EVALUATED: 1000 mitotic cells per each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: The incidence of polyploid metaphase cells, out of 500 metaphase cells, was determined quantitatively for negative control cultures and cultures treated with the highest dose level of the test substance used in the analysis for chromosomal aberrations.

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
-Statistically significant increases (P<0.0l) in the frequency of metaphases with aberrant chromosomes (excluding- gaps) are observed at one or more test concentration.
-The increases exceed the negative control range of this laboratory, ta.ken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
-Evidence of a dose-relationship is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.

Statistics:

The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both the absence and presence of S9 mix, CHDM caused no statistically significant increase in the proportion of metaphase figures containing choromosomal aberrations at any dose level, when compared with solvent control in either test.
A quantitative analysis for polyploidy was made in cultures treated with negative control and highest dose level. No increase in the proportion of polyploid cells was seen in either test.

All positive control compounds caused large statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Remarks on result:

other: strain/cell type: Human blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results
The First Test: 3 hours treatment and 17 hours recovery
Exposure period (hours)	S9 mix	Concentration of SKYCHDM(mM)	Cells with aberrations excluding gaps			Cells with aberrations Including gaps			Relative Mitotic Index (%)
			individual values	(%)	Mean(%)	Individual values	(%)	Mean (%)
3	-	0 (Purified water)	1	0	0.5	1	3	2	100
		2.5	1	1	1	3	2	2.5	81
		5	1	5	3	2	7	4.5	84
		10	4	1	2.5	5	1	3	58
		0.2 micro g/ml (Mitomycin C)	30	26	28.0 a ***	32	30	31.0 a ***	-
3	+	0 (Purified water)	3	0	1.5	5	1	3	100
		2.5	2	3	2.5	3	3	3	103
		5	0	5	2.5	2	7	4.5	107
		10	2	2	2	2	3	2.5	111
		10 micro g/ml (Cyclophosphamide)	26	24	25.0 a ***	28	24	26.0 a ***	-
*** P<0.001
Otherwise P>=0.01
a 50 cells were analysed for these cultures due to the high levels of aberrations seen.
The Second Test: Without S9 mix, 20 hours continuous treatment
Exposure period (hours)	S9 mix	Concentration of SKYCHDM(mM)	Cells with aberrations excluding gaps			Cells with aberrations Including gaps			Relative Mitotic Index (%)
			individual values	(%)	Mean(%)	Individual values	(%)	Mean (%)
20	-	0 (Purified water)	0	0	0	0	2	1	100
		2.5	2	3	2.5	4	5	4.5	81
		5	2	2	2	5	4	4.5	80
		10	3	2	2.5	7	3	6	80
		0.1 micro g/ml (Mitomycin C)	20	22	21.0 a ***	22	22	22.0 a ***	-
3	+	0 (Purified water)	2	2	2	3	2	2.5	100
		2.5	1	2	1.5	1	4	2.5	87
		5	4	0	2	4	1	2.5	90
		10	0	0	0	0	0	0	77
		10 micro g/ml (Cyclophosphamide)	16	14	15.0 ***	18	15	16.0 ***	-

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test substance CHDM has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of CHDM to induce chromosomal aberrations in human lymphocytes cultured in in vitro.

In other to assess the toxicity of CHDM to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis these data, the following concentrations were selected for metaphase analysis:

First test

With and without S9 mix: 3hours trestment, 17hours recovery:2.5, 5 and 10mM

Second test

With S9 mix- 20hours continuous treatment: 2.5, 5 and 10mM

With S9 mix- 3hours treatment, 17hours recovery: 2.5, 5 and 10mM

In both the absence and presence of S9 mix, CHDM caused no statistically significant increase in the proportion of metaphase figures containing choromosomal aberrations at any dose level, when compared with solvent controls in either test.

A quantitative analysis for polyploidy was made in cultures treated with negative control and highest dose level. No increase in the proportion of polyploid cells was seen in either test.

All positive control compounds caused large statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Reference 3

Endpoint:

genetic toxicity in vitro, other

Remarks:

Type of genotoxicity: cytotoxicity test

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 April 2002 - 13 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD)

Qualifier:

according to guideline

Guideline:

other: ISO 10993-5, 1999 using MRC-5 cell line

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro cytotoxicity study of leachable endogenous or extraneous substances using elution technique

Species / strain / cell type:

mammalian cell line, other: ATCC CCL 171 MRC-5

Details on mammalian cell type (if applicable):

cell cultures of Human embryonic lung, obtained from ECACC
- Type and identity of media: Stock cells were stored frozen in croytubes and stored at -196°C under liquid nitrogen, as 1 ml volumes of cell suspension in medium containing 5% dimethyl sulphoxide.
Stock cultures grown in Minimum Essential Medium containing Earle's salt, buffered with 2.2 mg/ml sodium bicarbonate and supplemented with 1% non-essential amino acids 100x, anitbiotics, 2mM L-glutamine and 10% v/v FCS.
- Properly maintained: yes

Test concentrations with justification for top dose:

elution ratio of 0.2 g/ml in medium and eluate dilutions of Neat, 1/2, 1/4, 1/8 and 1/16.

Vehicle / solvent:

BME formulation of Eagle's Basal Medium (BME) with Earle's salts, containing 2mM L-glutamine, 0.25 micro g/ml amphotericin B and 50 micro gram/ml gentamicin sulphate, buffered with 1.68 mg/ml sodium bicarbonate, supplemented with foetal calf serum 10% v/v, which is instructed in the guideline

Untreated negative controls:

yes

Remarks:

medical/food grade silicone rubber (ESCO Rubber Ltd.) cut into approximately 1cm x 1cm pieces and eluted in medium at a ratio of 0.2 g/ml of medium

Positive controls:

yes

Remarks:

PVC discs containing 0.57% dibutyl tin dimaleate (Portex Ltd) eluted in medium at a ratio of 1 disc to 5 ml of medium

Positive control substance:

other: 0.57% dibutyl tin dimaleate (Portex Ltd)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Frozen vials of MRC-5 cells were thawed rapidly at 37°C in a water bath and the contents pooled and diluted with medium. The cell suspension was then centrifuged for 5 minutes at approximately 200 g in a bench top centrifuge, the supernatant was removed and the cell pellet resuspended in medium and distributed into tissue culture flasks. The flasks were incubated until near confluent cell monolayers had been obtained.

Preparation of test cell cultures: Cells from 4 flasks of near confluent MRC-5 cells were detached and disaggregated by treatment with trypsin as described above. The number of viable cells in the prepared cell suspension was determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. For the test, the cell suspension was diluted with medium to give a final concentration of 250000 viable cells/ml. A volume of 100ul was pipetted into each of the wells of the required number of sterile 96-well tissue culture plates.

The test and control samples were eluted in sterile containers. Test and control samples were allowed to equilibrate to approximately pH 7.0 in an atmosphere of 5% CO2 in air at 37±1°C for a maximum of two hours. The containers were then transferred to an orbital shaker at 100:5 cpm at 37±1°C for the remainder of me extraction period of 24±0.5 hours.

After 24±0.5 hours the eluates were removed from the test and control samples. The test sample and positive control eluates were serially diluted in two-fold steps with medium to give a dilution range 1/2 - 1/16. The negative control eluate was used undiluted. The eluates and dilutions were allowed to equilibrate in an atmosphere of 5% CO2 prior to testing.

After 24±1 hours incubation, the plates were examined using a microscope to confirm the presence of near confluent monolayers prior to addition of` the eluates. Neat eluates or dilutions of the eluates were tested on quadruplicate cultures of MRC-5 cells. The medium was removed from the wells and replaced with 100 micro l of freshly prepared eluate or eluate dilution. Treated cultures were then incubated for 24±0.5 hours at 37±1°C in a humidified atmosphere of 5% CO2 in air.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 24 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): Treated cultures were then incubated for 24±0.5 hours at 37±1°C in a humidified atmosphere of 5% CO2 in air.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

NUMBER OF REPLICATIONS: quadruplicate

NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

Evaluation criteria:

After 24±0.5 hours incubation each—plate-was fixed using methanol, stained with 20% Giemsa's stain and examined microscopically using an inverted microscope at 100x magnification. The condition of the cells in each was assessed and the results recorded as follows:

0 = no cell damage Non-cytotoxic
1 = up to 25% cell damage Slightly cytotoxic
2 = over 25% and up to 50% cell damage Mildly cytotoxic
3 = over 50% and up to 75% cell damage Moderately cytotoxic
4 = over 75% cell damage Severely cytotoxic

Statistics:

No statistical analysis was performed

Species / strain:

mammalian cell line, other: ATCC CCL 171 MRC-5

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic titre of >= 1/16 (grade 4)

Sample	Eluate dilution	Grade	Cytotoxic titre
CHDM	Neat+	4,4,4,4	>= 1/16
	1/2	4,4,4,4
	1/4	4,4,4,4
	1/8	4,4,4,4
	1/16	4,4,4,4
Positive control	Neat	4,4,4,4	1/8
	1/2	4,4,4,4
	1/4	4,4,4,4
	1/8	2,2,2,2
	1/16	0,0,0,0
Negative control	Neat	0,0,0,0	non-toxic

Conclusions:

Interpretation of results (migrated information):
positive cytotoxicity grade 4

The sample CHDM (1,4-cyclohexanedimetlianol) elicited a cytotoxic titer of >= 1/ 16 (grade 4) and the positive control eluate elicited a cytotoxic titre of 1/8 to MRC-5 cells under the conditions described in this report. The negative control was non-toxic.

Executive summary:

The sample, CHDM (1,4-cyclohexanedimetlianol) was treated for cytotoxicity using an elution technique in accordance with ISO 10993 -5, 1999. The test sample was eluted using a weight: volume ratio of 0.2 g/ml of medium and incubated at 37 +/-°C on an orbital shaker for 24±0.5 hours. The eluate and doubling dilution of 1/2 to 1/6 were applied to monolayer cultures of MRC-5 cells and toxicity was determined by morphological assessment of cell damage following 24±0.5 hours exposure to these eluates. The neat eluate from sample CHDM elicited a cytotoxic grade of 4 at 1/16 dilution to MRC-5 cells, under the conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mouse micronucleus study did not show any clastogenic effects.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 May 2002 - 30 May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: young adult
- Weight at study initiation: Male-between 28 and 32 grams, Female-between 22 and 26 grams (preliminary toxicity test)
- Assigned to test groups randomly: yes, tail marked
- Housing: Each group was kept, with the sexes separated, in cages and miantained in a controlled environment, with thte thermostat
and relative humidity target ranges set at 21°C and 55% respectively.
- Diet (e.g. ad libitum): free access to pelleted expanded rat and mouse No.1 maintenance diet
(QSC grade obtained from Special Diets Serves Ltd, Witham, Essex, UK)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21 °C
- Humidity (%): 37-58 %
- Photoperiod (hrs dark / hrs light): artifial light for 12 hours per day

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: not available
- Concentration of test material in vehicle: Preliminary toxicity test-100mg/ml, Micronucleus test-25, 50, 100 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg bodyweight
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not available

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: freshly prepared on the day of the test in purified water

Duration of treatment / exposure:

treated once

Frequency of treatment:

treated once

Post exposure period:

Seven males from the negative control, each of the test substance groups and five males from the positive control group were sacrificed 24hours after dosing. In addition seven males animals in the positive control and high level treatment groups were sacrificed 48hours after dosing.

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
Preliminary test: 2000mg/kg (100mg/ml), Micronucleus test: 500mg/kg (25mg/ml), 1000mg/kg (50mg/ml), 2000mg/kg (100mg/ml)

No. of animals per sex per dose:

-Preliminary Toxicity test: male 2, female 2
-Micronucleus test:
negative control: male 14
Dosage 500, 1000 mg/kg: male 7
Dosage 2000 mg/kg: male 14
positive control: male 5

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): not available
- Route of administration: orally gavaged
- Doses / concentrations: Doses 12 mg/kg, concentration 0.6 mg/ml

Tissues and cell types examined:

Bone marrow cell

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The preliminary toxicity test had previously shown that a dose of 2000 mg/kg
(the standard limit dose for the micronucleus test) was tolerated.


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Seven males from the negative control, each of the test substance groups and five males from the positive control group were sacrificed 24hours after dosing. In addition seven male animals in the positive control and high level treatment groups were sacrificed 48hours after dosing.


DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml pre-filtered foetal calf serum. The cells were sedimented by centrifugation, the supernatant was discarded suspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. Three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Giemsa with purified water. Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with coverslips using DPX.

METHOD OF ANALYSIS:
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic per animal. One smear per animal was examined and the remaining smears were held temporarily in reserve in case of technical problems with the first smear. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

Evaluation criteria:

Identification of Micronuclei

-Large enough to discern morphological characteristics
-Should possess a generally rounded shape with a clearly defined outline
-Should be deeply stained and similar in colour to the nuclei of other cells - not black
-Should lie in the same focal plane as the cell
-Lack internal structure, ie they are pyknotic
-There should be no micronucleus-like debris in the area surrounding the cell

A Positive response is normally indicated by a statically significant dose-related increase in the incidence of micronucleated immature erythrocytes group compared with the concurrent control group (P<0.001); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal is obtained when the results do not meet the criteria specified for a positive or negative response. Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related in the proportion of immature erythocytes (P< 0.01).

Statistics:

-The results for each treatment group were compared with the results for the concurrent control group using non-parametric stastic:
For incidences of micronuleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Coporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al.1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythocytes, equivalent permutation tests based on rank scores are used, i.e. exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Clinical signs of toxicity in test animals:
Apart from underactivity, no other clinical signs were observed and all animals dosed survived to scheduled termination.


RESULTS OF DEFINITIVE STUDY
- Clinical signs and mortalities:
Animals dosed with CHDM at concentration of 2000 mg/kg showed clinical signs of under activity and partially closed eyelids. All animals dosed survived to scheduled termination. No clinical signs were noted for the remaining groups of animals over the duration of the test.
- Induction of micronuclei (for Micronucleus assay): see the following table
- Ratio of PCE/NCE (for Micronucleus assay): see the following table
- Statistical evaluation:

The test substance did not cause any statically significant increases in the number of micronucleated immature erythrocytes at either samopling time (P>0.001)
Mitomycin C caused a significant increased (P<0.001) in the frequent of micronucleated immature erythrocytes.


The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.


The test substance failed to cause any significant decreases in the proportion of immature erythrocyte (P>0.001).
Mitomycin C did not cause any significant decreases in the proportion of immature erythrocytes (P>0.001).

Summary of results and stastistical ananlysis

Samplin time	Teratment	Dose (mg/kg)	% ie/(ie+me) (mean)	Incidence mie (mean)	Incidence mme (group mean)
24 Hours	Vehicle control	-	35	1.1	0.8
	CHDM	500	32	0.3	1.1
	CHDM	1000	32	0.6	0.8
	CHDM	2000	33	0.4	0.4
	Mitomycin C	12	32	36.0 **	1.1
48 hours	Vehicle control	-	34	0.4	1.5
	SKYCHDM	2000	35	0.6	0.0

Vehicle control Purifed water

% ie/(ie+me) Proportion of immature erythrocytes

mie Number of micronucleated cells observed per 2000 immature erythcytes examined

mme Number of micronucleated cells calculated mature erythrocytes

Results of stastical anaysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

** P < 0.001 (significant)

otherwise P > 0.001 (not significane)

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that CHDM dud not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.

Executive summary:

This study was designed to access the potential induction of micronuclei by CHDM in bone marrow cells of mice. Mice were treated with a single oral administration of the test substance at dose levels of 500, 1000 and 2000mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 2000mg/kg (the standard limit dose for the micronucleus test) was tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance, negative and positive control groups were administered orally by gavage. The negative control group received the vehicle, purified water and the positive control group received mitomycin C at 12mg/kg bodyweight.

No statistically significant increases in the frequency of micronucleated immature erythocytes and no subtantial decreases in the proportion of immature erythrocytes were observed in mice treated with CHDM and killed 24 or 48 hours later, compared to vehicle control vales (P>0.01 in each case).

It is concluded that CHDM dud not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not determined

Additional information

None

Justification for classification or non-classification

As thre were no mutagenic/clastogenic effects observed, no classification is required.