Sodium N-(hydroxymethyl)glycinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Rats treated with Sodium hydroxymethyl glycinate at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and which also fell within normal ranges. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article. It is concluded that Sodium hydroxymethyl glycinate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 1200 mg/kg/day (the maximum tolerated dose for this study).

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study according to an OECD guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Han Wistar Crl:WI (Glx/BRL/Han) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River UK Ltd, Margate, UK- Age at study initiation: * Range-finder: Males 6-8 weeks; Females 7-8 weeks * Main study: Males 7-9 weeks- Weight at study initiation: * Range-finder: Males 176-242 g; Females 159-184 g * Main study: Males 187-242 g- Assigned to test groups randomly: * Range-finder: No * Main study: Yes, 51 male rats were randomised to groups of at least six using a system of randomly generated numbers. Cages were then suitably labelled (using a colour coded procedure) to clearly identify the study number, cage number, start date, species and strain, together with a description of the treatment and dose level, route of administration and proposed time of necropsy.- Fasting period before study: Not specified- Housing: in groups of no more than four animals of the same sex in solid-floored cages, cleaned and dried before use with wood shavings for bedding.- Diet: ad libitum- Water: ad libitum- Acclimation period: * Range-finder: at least 2 nights * Main study: at least 5 days- Identification: by either numbered ear tag/tail marking (range-finder) or by numbered ear tag (main study). ENVIRONMENTAL CONDITIONS- Temperature (°C): 19-25°C - Humidity (%): 40-70%- Air changes (per hr):- Photoperiod (hrs dark / hrs light):IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: substance is sufficiently soluble in water- Concentration of test material in vehicle: * Range-finder: 120 to 200 mg/mL * Main study: 30 to 120 mg/mL- Amount of vehicle: 10 mL/kg.

Details on exposure:

Range-finder:groups of three male and three female rats were treated with the test article at suitable doses. Animals were dosed once daily for two consecutive days with the test article and observations made over a two day period following the second administration. Clinical signs of toxicity and body weight over this period were recorded and a maximum acceptable dose (14) determined based on these data. This was used as the highest dose level in the main study. Main study: Animals were dosed once daily for two consecutive days with the test article or vehicle. The positive control was given as a single administration at 20 mg/kg, on the second day of dosing.

Duration of treatment / exposure:

once daily

Frequency of treatment:

two consecutive days

Remarks:

Doses / Concentrations:Range-finder: 1000, 1200, 1400, 2000 mg/kg bwBasis:actual ingested

Remarks:

Doses / Concentrations:Main study: 300, 600, 1200mg/kg bwBasis:actual ingested

No. of animals per sex per dose:

- Range-finder: 3- Main study: 6 (males only); control: 12 (males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide- Justification for choice of positive control(s): induces a statistically significant increase in the frequency of micronucleated PCE - Route of administration: oral (gavage)- Doses / concentrations: single administration at 20 mg/kg, on the second day of dosing

Tissues and cell types examined:

bone marrow from a single femur.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):A single femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shank. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal calf serum into appropriately labelled centrifuge tubes (one per animal). The tubes were centrifuged (1250 x 'g', 2-3 minutes) and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube and from each tube a small volume of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and tag number. The latter served as a code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide. DETAILS OF SLIDE PREPARATION:Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol, followed by rinsing several times in distilled water. One slide from each set of two was taken ( the other kept in reserve) and, after a second fixing/rinsing procedure, were stained for 4 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer and allowed to dry. When dry, slides were mounted with coverslips. SCORING:The slides from all control and dose groups were arranged in numerical order by sampling time and analysed by a person not connected with the dosing phase of the study. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed analysed. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored. OTHER:

Evaluation criteria:

Treatment of data:After completion of microscopic analysis and decoding of the data, the ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequency of micronucleated PCB/1000 cells (± standard deviation) were also determined. PCB/NCB ratios were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity. The group mean frequencies of micronucleated PCB in vehicle control animals were compared with the historical negative control range to determine whether or not the assay was acceptable. Acceptance criteria: The assay is considered valid if the following criteria are met: 1. the incidence of micronucleated PCB in the vehicle control group falls within or close to the historical vehicle control range as given in Appendix 6, and2. at least five animals out of each group are available for analysis, and3. the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCB.Evaluation criteria: A test article is considered as positive in this assay if:1. a statistically significant increase in the frequency of micronucleated PCE occurs at least at one dose, and2. the frequency of micronucleated PCE at such a point exceeds the historical vehicle control range.

Statistics:

The group mean frequencies of micronucleated PCB in vehicle control animals were compared with the historical negative control range to determine whether or not the assay was acceptable. If the heterogeneity x2 test provides evidence of significant (p <= 0.05) variability between animals within at least one group, non-parametric analysis is more appropriate. Provision was made to use the Wilcoxon rank sum test under these circumstances.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

at 1200 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE FINDER:Clinical findings:The test article was administered once daily on two consecutive days to groups of three male and three female rats at doses of 1000, 1400 or 2000 mg/kg/day. Observations were made over a 2 day period following the second administration and signs of toxicity recorded. Severe clinical signs including mortalities were observed at the 1400 and 2000 mg/kg/day dose levels. Piloerection, hunched posture and weight loss were observed at 1000 mg/kg/day in female animals only. No substantial difference in toxicity was observed between males and females therefore, the main study was conducted using male rats only. Due to the number and severity of clinical signs observed at the 1400 and 2000 mg/kg/day dose levels, an intermediate dose level of 1200 mg/kg/day was tested in male animals. Weight loss was observed in all animals and one animal had noisy breathing. Accordingly, 1200 mg/kg/day was considered an acceptable estimation of the maximum tolerated test dose and was selected as the maximum dose level for this study.MAIN STUDY:Validity of study: 1. the incidence of micronucleated PCE in the vehicle control group fell within the historical vehicle control range, and2. at least five animals out of each group were available for analysis, and3. the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE.Clinical findings: Clinical signs including mortalities were observed in two animals tested at 1200 mg/kg/day. Due to the mortalities observed at the maximum dose group, additional work was performed with Sodium hydroxymethyl glycinate at 1200 mg/kg/day and appropriate negative and positive controls. Clinical signs including lethargy, cold to the touch, abnormal gait, abnormal breathing, loose faeces, blue extremities, piloerection and dilated pupils were observed in some animals tested at 1200 mg/kg/day. No other clinical signs were observed in any other main study animal. Groups of at least six male rats were tested and killed 24 hours after the second administration. All results are reported as one experiment. Analysis of data:Groups of rats treated with Sodium hydroxymethyl glycinate exhibited PCE/NCE ratios which were similar to vehicle controls. Group mean frequencies of micronucleated PCB were also similar to that seen in the vehicle control group and were not significantly different by x.2 analysis

Conclusions:

Interpretation of results (migrated information): negativeSodium hydroxymethyl glycinate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 1200 mg/kg/day, a dose at which clinical signs of toxicity were observed.

Executive summary:

Sodium hydroxymethyl glycinate was assayed in vivo m a rat bone marrow micronucleus test at three dose levels. The Sponsor supplied information indicating that the oral LD50 value for Sodium hydroxymethyl glycinate in rats is 2100 mg/kg. In order to establish if there were any substantial inter-sex differences in toxicity, both male and female rats were used. The choice of dose levels was based on an initial toxicity range-finding study in which Sodium hydroxymethyl glycinate, formulated in purified water was administered to rats orally by gavage. The test article was administered once daily on two consecutive days to groups of three male and three female rats at doses of 1000, 1400 or 2000 mg/kg/day. Observations were made over a 2 day period following the second administration and signs of toxicity recorded. Severe clinical signs including mortalities were observed at the 1400 and 2000 mg/kg/day dose levels. Piloerection, hunched posture and weight loss were observed at 1000 mg/kg/day in female animals only. No substantial difference in toxicity was observed between males and females therefore, the main study was conducted using male rats only. Due to the number and severity of clinical signs observed at the 1400 and 2000 mg/kg/day dose levels, an intermediate dose level of 1200 mg/kg/day was tested in male animals. Weight loss was observed in all animals and one animal had noisy breathing. Accordingly, 1200 mg/kg/day was considered an acceptable estimation of the maximum tolerated test dose and was selected as the maximum dose level for this study.

In the main study, Sodium hydroxymethyl glycinate was formulated as described and administered at 300, 600 and 1200 mg/kg/day to groups of six male rats killed 24 hours after the second administration. Severe clinical signs including mortalities were observed in some animals tested at 1200 mg/kg/day. No other clinical signs were observed in any other main study animal. Due to the mortalities observed at the maximum dose group, additional work was performed with Sodium hydroxymethyl glycinate at 1200 mg/kg/day and appropriate negative and positive controls. Groups of at least six male rats were tested and killed 24 hours after the second administration. All results are reported as one experiment.

The negative (vehicle) control in the study was purified water also administered orally by gavage once daily on two consecutive days. Groups of six male rats treated with this were killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered orally by gavage as a single dose of 20 mg/kg to groups of six male rats which were killed after 24 hours. Positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls.

Negative (vehicle) control rats exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies ofmicronucleated PCE within historical negative control (normal) ranges. Rats treated with Sodium hydroxymethyl glycinate at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and which also fell within normal ranges. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article.

It is concluded that Sodium hydroxymethyl glycinate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 1200 mg/kg/day (the maximum tolerated dose for this study).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Genetic toxicity in vitro:

The results of the Ames test indicate that under the conditions of this study, a significant 1.5-fold dose-responsive increase in the number of TA100 revertants per plate was present in the presence of the S9-fraction, but did not meet the criteria for a positive response as defined by the protocol used for this study. As no significant responses were observed in the other strains, the results obtained in this study with sodium hydroxymethyl glycinate (Suttocide A) are not considered to be positive.

The test article, Suttocide A, 50% w /v active, was tested in the chromosome aberration assay using Chinese hamster ovary cells. The initial assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 2, 4, 8, 15, 30, and 60 µg/ml, and 3, 6, 11, 23, 45, and 90 µg/ml, respectively. The test article was soluble in the medium at all concentrations tested. Metaphase cells were collected for microscopic evaluation at 20 hours after the initiation of treatment. Toxicity, as measured by mitotic inhibition, was approximately 71 % at the highest dose level evaluated for chromosome aberrations in the nonactivated study, 60 µg/ml. In the S-9 activated study, no toxicity was observed relative to the solvent control at the highest dose level evaluated for chromosome aberrations, 90 µg/ml. An increase in chromosome aberrations was observed at dose levels of 30 and 60 µg/ml in the nonactivated study (p≤0.01, Fisher's exact test) and at 45 and 90 µg/ml in the S-9 activated study (p≤0.01, Fisher's exact test) at the 20 hour harvest time. The Cochran-Armitage trend test for dose-responsiveness was positive in both the nonactivated and S-9 activated test systems. An independent repeat assay was conducted in the absence of a metabolic activation system at dose levels of 8, 15, 30, 45, and 60 µg/ml and in the presence of an Aroclor-induced S-9 metabolic activation system at dose levels of 30, 45, 60, and 90 µg/ml. Metaphase cells were collected for microscopic evaluation at 20 and 44 hours after the initiation of treatment. Toxicity at 60 µg/ml, measured by mitotic inhibition, was approximately 57% and 59% at the 20 hour and 44 hour harvest times, respectively. In the S-9 activated studies, toxicity at 90 µg/ml was approximately 28% and 20% at the 20 and 44 hour harvests, respectively. Statistically significant increases in structural chromosome aberrations were observed in the nonactivated test system at both harvest times at dose levels of 45 and 60 µg/ml (p≤0.01, Fisher's exact test). The Cochran-Armitage trend test for dose­responsiveness was positive at both harvest times in the nonactivated study. A statistically significant increase in the number of numerical aberrations was found at the 44 hour harvest time in the nonactivated test system at dose levels of 45 and 60 µg/ml, (p≤0.01, Fisher's exact test). In the S-9 activated test system, statistically significant increases in structural chromosome aberrations were found at the dose level of 90 µg/ml at both the 20 and 44 hour harvest times, (p≤0.01 and p≤0.05, respectively). The Cochran-Armitage trend test for dose-responsiveness was positive only at the 20 hour harvest time. No statistically significant increase in numerical aberrations was observed in the S-9 activated test system at the 44 hour harvest time. Based on the findings of this study, Suttocide A, 50% w /v active was concluded to be positive in the in vitro mammalian cytogenetic assay using Chinese hamster ovary cells.

Suttocide A (50%; w/v) was evaluated in the Rat Hepatocyte Primary Culture/DNA Repair Test to evaluate its ability to induce unscheduled DNA synthesis in vitro. The test article initially was prepared and serially diluted in serum-free WME medium to prepare the appropriate dosing solutions to yield concentrations of sodium hydroxymethyl glycinate of 0.75, 2.5, 5, 7.5, 10, 20, 40, 60, 80 and 100 µg/mL. The test article, as well as the untreated and 2AAF positive controls, were evaluated in triplicate cultures containing 10 µCi/mL 3H-thymidine and the appropriate concentration of the test article. Following an 18- to 20- hour exposure, the cultures were washed and autoradiographs were prepared. Cultures treated at treated at concentrations of 60 µg/mL and higher exhibited severe toxicity. In addition, the 40 µg/mL concentration exhibited a high variability in the number of scorable cells. Therefore, 20 µg/mL initially was selected as the highest concentration scored in the assay. Three lower concentrations ( 2. 5, 7. 5 and 15 µg/mL) also were scored, as well as the untreated and positive controls. The average NNG observed for the WME negative control cultures was -3.1 ± 3.2, while those of the cultures treated with Suttocide A at concentrations of 2.5 to 20 µg/mL ranged from -4.2 ± 2.2 to -2.7 ± 2.7, approximating the negative control values. The single remaining culture treated with Suttocide A at a concentration of 40 µg/mL exhibited a slight elevation in NNG (1.1 ± 11.4 NNG), and a more significant elevation in percentage of cells in repair (29.3%). Thus, none of the concentrations of Suttocide Ac induced a mean NNG count >5. Therefore, suttocide Ac is considered to have produced a negative response in the Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions of this test.

Sodium Hydroxymethylglycinate as tested for induction of chromosome aberrations in cultured human peripheral blood lymphocytes. The test article dose levels for chromosome analysis were selected by evaluating the effect of Sodium Hydroxymethylglycinate on mitotic index in the absence and presence of S-9. Chromosome aberrations were analysed at three dose levels (see below). The highest concentrations chosen for analysis, 87.34 µg/mL in the absence of S-9 and 170.6 µg/mL in the presence of S-9, induced approximately 59% and 66% mitotic inhibition (reduction in mitotic index) in the absence and presence ofS-9 respectively.

Treatment of cultures with Sodium Hydroxymethylglycinate in the presence of S-9 resulted in frequencies of cells with structural aberrations that were significantly elevated as compared to the concurrent vehicle control for all concentrations analysed. The aberrant cell frequency of all Sodium Hydroxymethylglycinate treated cultures exceeded historical negative control (normal) ranges. Treatment of cultures with Sodium Hydroxymethylglycinate in the absence of S-9 resulted in significantly increased frequencies of cells with structural aberrations at the intermediate and high doses analysed (44.72 and 87.34 µg/mL). However, it was only at the highest concentration analysed (87.34 µg/mL) where large numbers of aberrant cells ( exceeding normal ranges) were observed in both replicate cultures. It is concluded that Sodium Hydroxymethylglycinate induced chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

Sodium hydroxymethyl glycinate was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by a single mutation experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). In the cytotoxicity range-finding experiment, six doses were tested in the absence and presence of S-9, ranging from 37.5 to 1271 µg/mL (10 mM). Extreme toxicity was observed at the highest five doses tested in the absence and presence of S-9 (75-1271 µg/mL). The highest dose tested that survived treatment (75 µg/mL) yielded 3% relative survival in the absence of S-9 and 11 % relative survival in the presence of S-9. Accordingly, nine doses were chosen for the mutation experiment in the absence and presence of S-9, ranging from 2.5 to 120 µg/mL. Two days after treatment, the highest dose tested in the absence of S-9 (120 µg/mL) was considered to be too toxic for selection to determine viability and 5-trifluorothymidine (TFT) resistance. Furthermore, the lowest dose tested in the absence of S-9 (2.5 µg/mL) and the lowest two doses tested in the presence of S-9 (2.5 and 5 µg/mL) were clearly non-toxic, and were also not selected to determine viability and 5-trifluorothymidine resistance. The highest doses selected were 100 µg/mL in the absence of S-9 and 120 µg/mL in the presence of S-9, which yielded 3% and 8% relative survival, respectively. Negative (solvent) and positive control treatments were included in the mutation experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid. Statistically significant increases in mutant frequency were observed following treatment with Sodium hydroxymethyl glycinate at the highest five doses analysed in the absence of S-9 (20- 100 µg/mL) and in the presence ofS-9 (40- 120 µg/mL). It was concluded that, under the conditions employed in this study, Sodium hydroxymethyl glycinate is highly mutagenic in this test system.

Genetic toxicity in vivo:

Sodium hydroxymethyl glycinate was assayed in vivo m a rat bone marrow micronucleus test at three dose levels. The Sponsor supplied information indicating that the oral LD50 value for Sodium hydroxymethyl glycinate in rats is 2100 mg/kg. In order to establish if there were any substantial inter-sex differences in toxicity, both male and female rats were used. The choice of dose levels was based on an initial toxicity range-finding study in which Sodium hydroxymethyl glycinate, formulated in purified water was administered to rats orally by gavage. The test article was administered once daily on two consecutive days to groups of three male and three female rats at doses of 1000, 1400 or 2000 mg/kg/day. Observations were made over a 2 day period following the second administration and signs of toxicity recorded. Severe clinical signs including mortalities were observed at the 1400 and 2000 mg/kg/day dose levels. Piloerection, hunched posture and weight loss were observed at 1000 mg/kg/day in female animals only. No substantial difference in toxicity was observed between males and females therefore, the main study was conducted using male rats only. Due to the number and severity of clinical signs observed at the 1400 and 2000 mg/kg/day dose levels, an intermediate dose level of 1200 mg/kg/day was tested in male animals. Weight loss was observed in all animals and one animal had noisy breathing. Accordingly, 1200 mg/kg/day was considered an acceptable estimation of the maximum tolerated test dose and was selected as the maximum dose level for this study. In the main study, Sodium hydroxymethyl glycinate was formulated as described and administered at 300, 600 and 1200 mg/kg/day to groups of six male rats killed 24 hours after the second administration. Severe clinical signs including mortalities were observed in some animals tested at 1200 mg/kg/day. No other clinical signs were observed in any other main study animal. Due to the mortalities observed at the maximum dose group, additional work was performed with Sodium hydroxymethyl glycinate at 1200 mg/kg/day and appropriate negative and positive controls. Groups of at least six male rats were tested and killed 24 hours after the second administration. All results are reported as one experiment. The negative (vehicle) control in the study was purified water also administered orally by gavage once daily on two consecutive days. Groups of six male rats treated with this were killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered orally by gavage as a single dose of 20 mg/kg to groups of six male rats which were killed after 24 hours. Positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls. Negative (vehicle) control rats exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies ofmicronucleated PCE within historical negative control (normal) ranges. Rats treated with Sodium hydroxymethyl glycinate at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and which also fell within normal ranges. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article. It is concluded that Sodium hydroxymethyl glycinate did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 1200 mg/kg/day (the maximum tolerated dose for this study).

Suttocide A was tested in the In Vivo - In Vitro Rat Hepatocyte Unscheduled DNA Synthesis Assay. The test article was administered via oral gavage at three dose levels of 2000, 700 and 200 mg/kg rat as well as the negative control (sterile deionized water, 10 ml/kg rat) and the two positive controls (methyl methanesulfonate, 200 mg/kg rat, and 2-acetylaminofluorene, 100 mg/kg rat) . The hepatocytes were harvested 2 to 4 and 12 to 18 hours after test article administration. The results of the In Vwo - In Vuro UDS assay indicate that under the test conditions, the test article did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control) in hepatocytes isolated from treated animals. Only the positive controls, MMS and 2-AAF, induced significant increases in the mean number of net nuclear grain counts over that in the vehicle control. On the basis of the results of this study, Suttocide A is considered to be negative in the In Vivo - In Vitro Rat Hepatocyte Unscheduled DNA Synthesis Assay.

The results of a Micronucleus Test in mice indicated that Suttocide A did not induce a statistically significant increase in micronucleated PCEs at any of the dose levels or time intervals evaluated. Suttocide A also did not cause a statistically significant shift in the PCE/NCE ratio. In contrast, CP produced a positive and significant increase in micronucleated PCEs and decrease in the PCE/NCE ratio. Therefore, Suttocide A is considered negative in the in-vivo micronucleus test.

Justification for selection of genetic toxicity endpoint
In-vivo micronucleus assay.

Justification for classification or non-classification

Summary of available study results:

Type of study	in vitro or in vivo	without metabolic activation	with metabolic activation	Reference
bacterial reverse mutation assay	in vitro	negative	negative	Haworth S.R. (1983)
mammalian cell gene mutation assay	in vitro	positive	positive	Lloyd M. (2002)
in vitro mammalian chromosome aberration test	in vitro	positive	positive	Whitwell J. (2002); Putman D.L. & Schadly E.H. (1992)
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro	in vitro	negative	negative	Stankowski, Jr. L.F. (1995)
micronucleus assay (chromosome aberration)	in vivo	NA	negative	Howe J. (2002); SanSebastian J.R. (1987)
unscheduled DNA synthesis (DNA damage and/or repair)	in vivo	NA	negative	San R.H.C. & Raabe H.A. (1994)

The in-vivo chromosome aberration tests do not confirm the positive responses in the in-vitro tests. Although a posivtive response was observed in the in vitro chromosome aberration tests with the use of an exogenous source of metabolic activation, such a metabolic activation system cannot mimic completely in vivo conditions. Further, results should be interpreted with care when conditions that lead to positive results also show DNA damage associated with cytotoxicity. In conclusion, the potential for mutagenic or genotoxic activity of sodium N-(hydroxymethyl)glycinate is not conclusive to support classification.