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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: 50% solution in water
Details on test material:
- Name of test material (as cited in study report): Sodium Hydroxymethylglycinate- Received on 11 April 2000- Physical state: clear colourless liquid (solution)- Analytical purity: 51.06%- Lot/batch No.: 04000023467- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
other: Han Wistar Crl:WI (Glx/BRL/Han) BR
Sex:
male/female
Details on test animals 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:

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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

Applicant's summary and conclusion

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).