Reaction mass of isopropyl dihydrogen phosphate and phosphoric acid and propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The structural analogues and single components of Hordaphos CC MIN (Hordaphos CC MIS, phosphoric acid, diphosphorus pentaoxide and isopropanol) showed negative results in several studies for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The studies were performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A. Test concentrations up to the limit concentration of 5000 µg/plate were tested in the experiments. The test compounds proved to be not mutagenic to the bacterial strains.

The structural analogues/mixture components of Hordaphos CC MIN, also yielded negative results in two in vitro gene mutation study in mammalian cells with and without metabolic activation and provedto be non-mutagenic/clastogenic in two in vitro chromosome aberration studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March - May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimurium
Tryptophan for E. Coli

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 mix

Test concentrations with justification for top dose:

0.312, 0.625, 1.25, 2.5 and 5 mg/plate (according to guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:distilled water
- Justification for choice of solvent/vehicle: solubility

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 4-nitroquinoline 1-oxide

Details on test system and experimental conditions:

1. Initial Cytotoxicity Test for Dose Selection

Based on the results of solubility and precipitation test, an initial cytotoxicity test was conducted for the selection of test doses for the mutation assay. Salmonella typhimurium TA100 strain was exposed to vehicle control, 1, 2, 3, 4 and 5 mg/plate of test item. An initial cytotoxicity test was conducted using 15 hrs old culture of Salmonella typhimurium TA100 tester strain, both in the presence and the absence of metabolic activation, in triplicate, along with concurrent vehicle control (distilled water). Each concentration of test item was mixed with soft agar containing histidine and biotin, either S9 mix (for presence of metabolic activation) or phosphate buffer saline (for absence of metabolic activation), Salmonella typhimurium TA100 of cell density approximately 18×108 cells/mL and overlaid on to pre-labeled minimal glucose agar plates. The plates were incubated at 37°C for 48 hrs.

2. Plate Incorporation Method

In the first trial, which is a plate incorporation method, the bacterial suspensions are exposed to the test item, vehicle and the positive controls in the presence and absence of an exogenous metabolic activation system. These bacterial suspensions are then mixed with soft agar and plated immediately onto minimal glucose agar medium viz., his- for Salmonella typhimurium and try- for Escherichia coli.
Plate Incorporation method was carried out with concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate; where the test item, vehicle, positive control and the tester strains along with S9/phosphate buffer saline were mixed with 2 mL soft agar and poured on to minimal glucose agar plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37°C for 48 hrs.

The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.

3. Preincubation Method

The confirmatory trial or second experiment is a preincubation method. The test constituents are mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with soft agar and plated immediately onto minimal glucose agar medium his- for Salmonella typhimurium and try- for Escherichia coli.
After 48 to 72 hrs of incubation at 37±1ºC, revertant colonies are counted and compared to the number of spontaneous revertants on vehicle control plates.
Preincubation method was carried out with concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate; where the test item, vehicle, positive control and the tester strains along with S9/Phosphate Buffer Saline were mixed with 2 mL soft agar and poured on to minimal glucose agar plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37°C for 48 hrs.

The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.

Rationale for test conditions:

Based on solubility and cytotoxicity pretest; according to guideline

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

The data (in the form of appendix) were verified with the raw data. The data of number of revertants were subjected to computer statistical processing using Graphpad prism software version 5 wherever possible. All individual data were summarized and presented as tables. All findings were presented in the report as per the standard reporting procedure. Data was analyzed for differences among vehicle control, treatment and positive control group using ANOVA. Differences between the vehicle control, treatment with different concentrations of test item and positive control groups were tested by one-way ANOVA with Dunnett test at a 5% level (p<0.05) of significance. The statistical significances are designated by superscripts as given below:

Key result

Species / strain:

other: S. thyphimurium TA 98, TA 100, TA 1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

From the results obtained, the test item HORDAPHOS CC MIS is found to be non-mutagenic at and up to the highest concentration of 5 mg/plate in Bacterial Reverse Mutation Test in the tester strain Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) in the presence and absence of metabolic activation in plate incorporation and preincubation methods under laboratory conditions tested.

Executive summary:

The test item HORDAPHOS CC MIS was assessed for its mutagenic effects using Salmonella typhimurium strains: TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101). The test item was tested at the concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/plate using distilled water as vehicle based on the precipitation and initial cytotoxicity test. The study was conducted, with and without the metabolic activation(S9 fraction). The S9 fraction was prepared from sodium phenobarbitone and β-naphthoflavone induced ratliver. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine,4-nitroquinoline 1-oxidefor trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicates with plate incorporation method (Trial I) and preincubation method (Trial II). Data were statistically analyzed and expressed as mean ±SD.

From the experimental results obtained, the mean numbers of revertant colonies at the above mentioned concentrations were comparable to those of the dimethyl sulphoxide, in both the trials - plate incorporation method and preincubation method, in the presence and absence of metabolic activation. There was no significant increase in number of revertant colonies at any of the concentrations tested in both plate incorporation and preincubation method. The number of revertant colonies in the positive controls has shown 2.3 to 20.1 fold increase with statistical significance at a 5% level (p<0.05) underidenticalconditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug - Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: in vitro mammalian chromosome test

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.25, 0.5 and 1.0 mg/mL due to cytotoxicity

Vehicle / solvent:

distilled water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Chromosome aberration Test Procedure
• Whole blood of volume 0.5 mL was added to each tube containing culture media of volume 4.5 mL supplemented with final concentration of 40 µg/mL of phytohaemagglutinin (PHA) and incubated for 44 to 48 hours at 37ºC and 5% CO2.
• Post 44 to 48 hours of incubation with PHA, cells from tubes were centrifuged at 1500 rpm for 10 minutes, supernatant was discarded.
• Cell pellet was resuspended with fresh media.
• The treatment details for the initial cytotoxicity test are as mentioned below:
Set No. Metabolic
Activation Treatment Duration
1 +S9 Vehicle control/Test item/positive control (Cyclophosphamide) 3 Hours
2 - S9 Vehicle control/Test item/positive control (Mitomycin C) Hours
3 - S9 Vehicle control/Test item positive control (Mitomycin C) 20 Hours
• For tubes with metabolic activation (+S9) - set 1, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle and 0.5 mL of S9 mix was added, volume was made up to 5 mL with culture media.
• For tubes without metabolic activation (-S9) - set 2 and 3, cell suspension was mixed with 50 µL each of the respective test concentrations/vehicle with respective concentration of test item was mixed. The volume was made up to 5 mL with culture media.
• All the test item concentrations and controls were maintained in duplicates.
• Cells were incubated both with metabolic activation (+S9) and without metabolic activation (-S9) for 3 to 6 hours (Set 1 and 2) and without metabolic activation (-S9) for 20 to 24 hours (Set 3) at 37ºC and 5% CO2 as mentioned in the table.
• The treatment for Set 1 and 2 tubes were terminated post 3 to 6 hours of incubation, by centrifugation at 1500 rpm for 10 minutes.
• Supernatant was discarded and cell pellet was mixed with 5 mL of culture medium and incubated further to complete 20 to 24 hours starting from the start of treatment.
• The treatment for Set 3 tubes was terminated post 20 to 24 hours of incubation, by centrifugation at 1500 rpm for 10 minutes. Supernatant were discarded and cell pellet was mixed and made up to 5 mL with culture medium.
• Before 1 to 3 hours of harvesting, Colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 2 hours with colchicine, cell suspension was collected to pre labeled tubes and centrifuged for 10 minutes at 1500 rpm.
Three slides were prepared for each treatment replicate.


Slide Preparation
Pellet was mixed with 4 mL of freshly prepared 0.56% warm Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later centrifuged at 2000 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 2 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 2 mL of cold acetic acid:methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator before use.
The cell suspension was mixed using a pipette and few drops of the suspension was aspirated and dropped onto the chilled slides pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment and slide number. The slides were air dried.
Three slides were prepared for each treatment. Slides were stained using 5% Giemsa stain for 20 minutes.


Slide Evaluation
• All slides including vehicle control, treatment and positive controls of chromosome aberration test were coded before evaluation.
• Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
• Concurrent measures of mitotic index for all treated and vehicle control cultures were determined.
• The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data and presented in study report.
• Gaps were recorded separately and reported but generally not included in the total aberration frequency.

Rationale for test conditions:

based on pretests and according to guideline

Evaluation criteria:

• Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
b) The increase is dose-related when evaluated with an appropriate trend test
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
• Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control
b) There is no concentration-related increase when evaluated with an appropriate trend test
The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.
• An increase in the number of polyploid cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations.
• An increase in the number of cells with endoreduplicated chromosomes may indicate that the test item has the potential to inhibit cell cycle progression.

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p < 0.05) and the statistical significance was designated by the superscripts though out the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results obtained, the test item, HORDAPHOS CC MIS is considered as non-clastogenic at and up to the dose of 1.0 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

Test item was soluble in distilled water at 200 mg/mL. No precipitation was observed at and up to 2 mg/mL. No change in pH was observed at any of the concentrations tested up to 2 mg/mL. As there was no precipitation and no change in pH at 2 mg/mL, the same was selected as the highest concentration for testing in the initial cytotoxicity test.

In the initial cytotoxicity test, the test itemresulted in severe reduction of Mitotic Index (MI) both with and without metabolic activation for both short term (3 to 6 hours) and long term treatments (20 to 24 hours) at 2.0 mg/mL which was in the range of 72.26 to 75.50%. Whereas at 1.0 mg/mL of test item treatment, the percentage reduction of Mitotic Index was not greater than 36.08 which was inside the acceptable range of 45±5% reduction in Mitotic Index. Hence, 1.0 mg/mL was selected as highest dose and the other doses selected as low and mid doses were 0.25 and 0.5 mg/mL for the chromosomal aberration test.

In the chromosomal aberration test, the cells were treated withthe test itemat the doses of0.25, 0.5 and 1.0mg/mL as low, mid and high doses respectivelyusing distilled water as vehicle. The treatment was carried out in duplicates for short term (3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation.

Concentration of 10 µg/mL ofCyclophosphamide Monohydrate (+S9) and           0.05µg/mLof Mitomycin-C (-S9 both for short term and long term) were used as positive controls. Cells were arrested at metaphase using 0.3 µg/mL of colchicine. The cells were harvested between 2 hours of cell cycle arrest and slides were prepared and stained using 5% Giemsa stain.

The results indicatedno statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentrations tested.The mean percentage reduction in mitotic index observed at 1.0 mg/mL, the highest concentration tested was 32.63 in the presence of metabolic activation and 32.49 and 36.95 in the absence of metabolic activation for short and long term treatments, respectively.

The respective positive controls tested induced 8.0% to 9.0% of mean aberrated cells which was statistically significant. The mean percentage reduction in mitotic index was in the range of 17.10 to 22.28 when compared with the respective vehicle controls.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March - Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro gene mutation in mammalian cells

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.0125, 0.025, 0.05 and 0.1 mg/mL based on solubility and cytotoxicity tests

Vehicle / solvent:

distilled water (based on solubility)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Test Procedure for Gene Mutation Test
Gene Mutation test was carried out as described in section 7.4 and 7.5. Cells were pooled from replicate treatments and cell count was performed. The treatment schedule for the experiment was maintained as indicted in the following table.
Set No. Metabolic
Activation Treatment Duration
1 +S9 Vehicle control/Test item/
Positive control (3 µg/mL of Benzo(a)pyrene) 3 to 6 Hours
2 - S9 Vehicle control/Test item/
Positive control (1 µg/mL of
4 nitro quinolone 1-oxide) 3 to 6 Hours
3 - S9 Vehicle control/Test item/
Positive control (1 µg/mL of 4 nitro quinolone 1-oxide) 18-24 Hours


Expression of Mutant Phenotype
Duplicate cultures pooled were subcultured in duplicates at a density of 0.5×106 cells/culture flask. Cells were incubated at 37oC with 5% CO2, followed by subculturing with an interval of 2 and 3 days for the remaining 7 days of expression period (2 sub cultures).


Selection of Mutant Phenotypes and Plating for Cloning Efficiency
Post expression period of 7 days of Mutant phenotype, each pooled treatment cultures were subcultured in quintuplicates at a density of 2×105 cells per culture flask with culture media containing 10-20 µM of 6 Thioguanine and 200 cells /flask in triplicates without 6-Thioguanine for determination of cloning efficiency. Flasks were incubated at 37°C with 5% CO2 for 7 days. Post incubation period, medium from each flask was aspirated and stained with 5% Giemsa for 10 minutes. Numbers of colonies formed were counted manually.

Rationale for test conditions:

based on pretests and according to guideline

Evaluation criteria:

• There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
• A test item, for which the results do not meet the above criteria is considered non-mutagenic in this system.
• Positive results for an in vitro mammalian cell gene mutation test indicate that the test item induces gene mutations in the cultured mammalian cells used. A positive concentration-response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test item does not induce gene mutations in the cultured mammalian cells used. A test item for which the results do not meet the above criteria is considered as non-mutagenic in this test system.

Statistics:

Data was analyzed for differences among vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at 95% level of confidence (p<0.05). The statistical significance was designated by the superscripts though out the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results obtained, the test items HORDAPHOS CC MIS is considered as non-mutagenic at and up to the concentration of 0.1 mg/mL both in the presence and absence of metabolic activation under the laboratory conditions tested.

Executive summary:

The test item resulted in no precipitation up to theconcentration of 5 mg/mL.However, there was significant change in pH from 0.5 to 5 mg/mL and no significant change in pH was observed at and up to 0.1 mg/mL. Hence, the test item concentrations greater than of 0.1 mg/mL were not tested in the initial cytotoxicity test.

In the initial cytotoxicity test, there was no significant reduction in the number of colonies formed in any of the concentrations treated in all the treatment sets, when compared to the vehicle control. The Relative Cloning Efficiency (RCE) of all the concentrations treated with test item was not lesser than 96.5% both in the presence (3 to 6 hours) and absence of metabolic activation (for 3 to 6 hours and 18 to 24 hours). Hence, concentrations of 0.0125, 0.025, 0.05 and 0.1 mg/mL were selected for the gene mutation test.

In the gene mutation test, the cells were treated with test item, at the concentrations of 0.0125,0.25, 0.5 and 0.1 mg/mLusing distilled water as the vehicle in duplicates for a period of 3 hours (short term treatment) both in the presence and absence of metabolic activation, and for 18 to 24 hours (long term treatment) in the absence of metabolic activation.

The test item,HORDAPHOS CC MIS resulted in mutant frequencies of 8.8 to 9.8 mutant colonies/10⁶cells in the presence of metabolic activation with 13.2 mutant colonies/10⁶cells in the vehicle control. In the absence of metabolic activation(3 to 6 hours), mutant frequenciesof 11.6 to 13.5 mutant colonies/10⁶cells were observed with 13.7 mutant colonies/10⁶cells in the vehicle control. In the absence of metabolic activation(18 to 24 hours), mutant frequenciesof 11.8 to 13.2 mutant colonies/10⁶cells were observed with 12.4 mutant colonies/10⁶cells in the vehicle control. There was no statistically significant increase in the number of mutant colonies observed when compared with vehicle control at any of the dose levels of the test item tested.

Positive control, 3 µg/mL of Benzo (a) pyrene, in the presence of metabolic activation, resulted in 139.3mutant colonies/10⁶cellswhich were statistically significant when compared with the vehicle control for short term treated flasks (3 to 6 hours).

Positive control, 1 µg/mL of Nitro quinoline 1- oxide, in the absence of metabolic activation, resulted in 145.3mutant colonies/10⁶cellsand 171.6mutant colonies/10⁶cellswhich were statistically significant when compared with that of vehicle control for    3 to 6 hours and 18 to 24 hours respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

not mandatory

Justification for classification or non-classification

Hordaphos CC MIN does not have to be not classified for mutagenicity since the structural analogues as well single components of this mixture did not reveal any mutagenic effect in the bacterial reverse mutation assays in the presence or absence of metabolic activation in concentrations up to 5000 µg/plate, in the in vitro gene mutation assays or in the in vitro chromosome aberration studies.