Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item, TM 11-0078, was assessed according to internationally recognized guidelines in two in vitro tests. Firstly, an in vitro gene mutation study in bacteria (Ames test) and secondly an in vitro mammalian chromosome aberration study.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation in the Ames test.

In the chromosome aberration test, the test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system, and was therefore considered non-clastogenic to human lymphocytes in vitro. Based on two negative results the test item, TM 11-0078 is considered non-mutagenic.

 

Link to relevant study records

Reference

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:

The study was conducted between 06 December 2013 and 27 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 473 using an in vitro mammalian chromosome aberration method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 182.13, therefore, the maximum dose level was 1821 μg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. The purity of the test item was > 97% and was, therefore, accounted for in the test item formulations.

Information from the sponsor indicted that the test item was immiscible in aqueous solution. However, the test item was miscible in dimethyl sulphoxide at 182.1 mg/mL in a solubility check performed in-house. Prior to each experiment, the test item was accurately weighed, formulated in dimethyl sulphoxide and serial dilutions prepared.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

In the absence of S9, mitomycin C was used at 0.4 and 0.2 µg/mL for cultures in expts 1 & 2 respectively. It was dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used at 5 µg/mL in both expts. It was dissolved in DMSO.

Details on test system and experimental conditions:

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Culture Conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 – 9.10 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.70 - 0.75 mL heparinized whole blood

With Metabolic Activation (S9) Treatment:
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the
Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment:
In Experiment 1, after approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium.After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.


Preliminary Toxicity Test:
Three exposure groups were used:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 0, 7.11, 14.23, 28.45, 56.91, 113.81, 227.63, 455.25, 910.5 and 1821 μg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.


Experiment 1:
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 30, 60, 90, 120, 180 and 240 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 30, 60, 120, 240, 360 and 480 μg/mL.

Experiment 2:
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 60, 90 and 180 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 30, 60, 120, 240, 300 and 360 μg/mL.


Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawnoff and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Premiminary Toxicity Test:
The dose range for the Preliminary Toxicity Test was 0 to 1821 μg/mL. The maximum dose was the 10 mM concentration.

A greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 227.63 μg/mL, in the 4(20)-hour exposure group (without S9), at 1821 μg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 455.25 μg/mL in the continuous exposure group.

Haemolysis was observed following exposure to the test item at and above 28.45 μg/mL in the 4(20)-hour exposure groups and at and above 113.81 μg/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 113.81 μg/mL in the absence of metabolic activation (S9) and up to 227.63 μg/mL in the presence of metabolic activation (S9). The test item induced evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level was based on toxicity for both Experiment 1 and Experiment 2.

Chromosome Aberration Test - Experiment 1:
The dose levels of the controls and the test item are given in the table below:

4(20)-hour without S9 at a final concentration of 0*, 30, 60*, 90*, 120*, 180, 240, MMC 0.4*
4(20)-hour with S9 (2%) at a final concentration of 0*, 30, 60*, 120*, 240*, 360, 480, CP 5*

Where:
* = Dose levels selected for the metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 240 μg/mL in the presence of metabolic activation (S9). In the absence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 120 μg/mL.

No precipitate was observed at the end of exposure, however, haemolysis was observed following exposure to the test item at and above 60 μg/mL in both exposure groups. The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 76% mitotic inhibition was achieved at 180 μg/mL in the absence of S9. In the presence of S9, 25% and 34% inhibition of mitotic index was observed at 120 and 240 μg/mL, respectively. The toxicity of the test item was steep in that there were no or very few scorable metaphases at and above 180 μg/mL but no decrease in mitotic index at 120μg/mL in the absence of S9. In the presence of S9, there was a substantial decrease in mitotic index at 240 μg/mL, but it did not achieve the optimum level of 50±10%. There were no metaphases present for scoring above 240 μg/mL. The maximum dose level selected for metaphase analysis was 120 μg/mL and 240 μg/mL in the absence and presence of S9, respectively.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2:

The dose levels of the controls and the test item are given in the table below:

24-hour without S9 at a final concentration of 0*, 3.75, 7.5, 15, 30*, 60*, 90*, 180*, MMC 0.2*
4(20)-hour with S9 at a final concentration of 0*, 30, 60, 120*, 240*, 300*, 360*, CP5*

Where:
* = Dose levels selected for the metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide


The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 180 μg/mL in the absence of S9 and 360 μg/mL in presence of S9. No precipitate was observed at the end of exposure, however, haemolysis was observed following exposure to the test item at and above 60 μg/mL in the 24-hour exposure group and 30 μg/mL in the 4(20)-hour exposure group.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in the absence of S9. However, there were reduced numbers of cells due to test item induced toxicity but there were sufficient numbers of metaphases present at the maximum concentration of 180 μg/mL to analyze this dose level for chromosome damage. The dose range for this exposure was determined by the preliminary toxicity test which suggested a plateau of toxicity between 14.23 and 113.81 μg/mL. Unfortunately, this plateau of toxicity did not repeat in Experiment 2. In the presence of S9, there was clear toxicity to the cells at 360 μg/mL but again enough metaphases were present to allow analysis for chromosomal damage. Therefore, only 360 μg/mL exhibited an inhibition of mitotic index (43%). It was considered that the test item had been adequately tested because of the reduced cell pellet observations that were taken to indicate a physical toxic response was occurring that did not affect the growth rate of the lymphocytes. In both exposures, haemolysis was observed confirming that there had been adequate exposure to the test item.

Therefore, due to the reasons stated above, the maximum dose level selected for metaphase analysis was 180 μg/mL in the absence of S9 and 360 μg/mL in the presence of S9. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mitotic Index - Preliminary Toxicity Test

4-Hour Treatment, 20-Hour Recovery -S9

4-Hour Treatment, 20 -Hour Recovery +S9

24-Hour Treatment -S9

Mitotic Index- Preliminary Toxicity Test

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	7.60	100	4.50	100	8.10	100
7.11	8.80	116	-	-	6.30	78
14.23	8.75	115	5.90	131	4.40	54
28.45	8.00 H	105	6.95 H	154	5.25	65
56.91	7.35 H	97	6.70 H	149	4.45	55
113.81	3.65 H	48	5.75 H	128	3.65 H	45
227.63	-NM H GP	-	3.30 H	73	-NM H	-
455.25	-NM H GP	-	-NM H	-	-NM H GP	-
910.5	-NM H GP	-	-NM H	-	-NM H GP	-
1821	-NM H GP	-	-NM H GP	-	-NM H GP	-

 

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

GP = Greasy/oily precipitate observed at end of exposure period in blood-free cultures

H = Haemolysis observed at the end of exposure in blood cultures

Mitotic Index - Experiment 1

Dose Level (µg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of control	A	B	Mean	% of Control
0	6.40	8.50	7.45	100	8.40	7.00	7.70	100
30	8.95	6.05	7.50	101	7.10	6.45	6.78	88
60	9.30 H	7.15 H	8.23	110	6.40 H	5.00 H	5.70	74
90	8.40 H	8.40 H	8.40	113	NA	NA	NA	NA
120	8.45 H	5.70 H	7.08	95	5.90 H	5.60 H	5.75	75
180	0.75 H	2.80 H	1.78	24	NA	NA	NA	NA
240	-H NM	-H NM	-	-	4.45 H	5.70 H	5.08	66
360	NA	NA	NA	NA	-H NM	-H NM	-	-
480	NA	NA	NA	NA	-H NM	-H NM	-	-
MMC 0.4	2.60	2.05	2.33	31	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	2.50	1.60	2.05	27

 

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

NM = No or too few metaphases suitable for scoring

H = Haemolysis

- = Not assessed for mitotic index

Mitotic Index – Experiment 2, Without S9

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	3.25	5.45	4.35	100
3.75	-	-	-	-
7.5	-	-	-	-
15	-	-	-	-
30	5.65	4.75	5.20	120
60	4.35 H	6.20 H	5.28	121
90	4.35 H	5.10 H	4.73	109
180	2.75 H R	3.85 H R	3.30	76
MMC 0.2	2.05	1.90	1.98	45

 

Mitotic Index – Experiment 2, With S9

Dose Level (µg/mL)	24-Hour With S9
	A	B	Mean	% of Control
0	4.65	4.95	4.80	100
30	- H	-H	-	-
60	5.65 H	4.20 H	4.93	103
120	4.55 H	4.15 H	4.35	91
240	3.05 H	5.60 H	4.33	90
300	2.20 H	5.80 H	4.00	83
360	2.00 H R	3.50 H R	2.75	57
CP 5	2.25	3.70	2.98	62

 

MMC = Mitomycin C

CP = Cyclophosphamide

H = Haemolysis

R = Reduced numbers of cells but metaphases present

- = Not assessed for mitotic index 

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro.

Executive summary:

The chromosome aberration potential of the test item, TM 11-0078, was negative according to OECD Test Guideline 473 using an in vitro mammalian chromosome aberration method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.

The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

The in vitro gene mutation study (Ames) was designed to assess the mutagenic potential of the test material using a bacterial test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al. In this study mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. This method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

The Salmonella strains used in the test are incapable of synthesising histidine and are therefore dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent, these bacteria may undergo a reverse mutation to histidine independent form which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional strains have been introduced. These include a deletion though the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of E. coli (WP2uvrA-) which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the salmonella strains. This strain also has a deletion in the excision repair gene (uvrA-). Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.

From the results of the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested, with any dose of the test item, either with or without metabolic activation. The test item, TM 11-0078 was therefore considered to be non-mutagenic in vitro.

The purpose of the chromosome aberration study was to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocytes. The detection of structural chromosomal aberrations in cultured mammalian cells supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). Human peripheral blood lymphocytes are recognized in the OECD 473 guideline as being a suitable cell line for the Mammalian Chromosome Aberration Test. Numerical and structural chromosome aberrations are implicated in the pathology of neoplasia (Radmanet al., 1982; Cairns, 1981) and also occur in a high proportion of spontaneous abortions and abnormal live births (Chandley, 1981). Furthermore, most carcinogens are capable of inducing such changes in chromosome fidelity. Metaphase analysis in vitro involves recording such structural and numerical aberrations in the chromosomes of exposed cells. Many of these changes are lethal to the cells in which they occur and are therefore not of heritable significance. However, it is assumed that agents capable of inducing gross chromosomal changes also induce more subtle changes (translocations, inversions and small deletions) which are not cell lethal, and therefore represent a hazard. The ability to induce chromosome aberrations also correlates well with the induction of gene mutations (Hollsteinet al., 1979). This study was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test".

It was established in the chromosome aberration test that the test item was toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two expeiments, using a dose range that included a dose level which induced approximately 50% mitotic inhibition or greater. The test item, IFF TM 11-0078 was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint

The study was conducted on the target substance using an appropriate in vitro method according to internationally recognised test guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity testsin vitroand in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in two in vitro tests. Firstly, an in vitro gene mutation study in bacteria (Ames test) and secondly an in vitro mammalian chromosome aberration study.

In the in vitro gene mutation study in bacteria (Ames) no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

In the in vitro mammalian chromosome aberration study, the test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either with or without metabolic activation and was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Based on two negative results in vitro, the test item is considered non-mutagenic.