Semicarbazide hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ICN-K and K Laboratories, Plainview, N. Y.
- Expiration date of the lot/batch:
- Purity test date: 95-99%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

8,3, 17, 34, 67 and 133µmol
The test item is tested at subtoxic concentrations.

Vehicle / solvent:

the test item was dissolved and diluted in distilled water.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Bacterial Mutagenesis Assays:
Mutagenicity was checked by means both of the plate incorporation test and of the spot test, basically as described in detail by Ames e/ al. (1 ).
Briefly, the plate incorporation test was performed by mixing 50 p.\ of overnight broth cultures (2.5% Oxoid Nutrient Broth No. 2) of each bacterial tester strain, 100 ¡uolf test hydrazine, 0.5 ml of S-9 mix (when needed), and 2.5 ml of molten top agar (0.6% Difco agar:0.5% NaCI solution) supplemented with 10% of a solution of 0.5 mM i_-histidine-HCI:0.5 mM biotin. The mixture was immediately poured on a minimal-glucose agar medium (1.5% Difco agar in Vogel-Bonner Medium E with 2% glucose) solidified layer in Pétriplates (25 ml of medium in 90-mmdiameter plastic plates). A number of assays was also carried out by means of a liquid preincubation test, as described in "Results."
The spot test was performed by placing 10 jul of hydrazine solution in sterile 7-mm filter paper discs at the center of plates over the top agar layer incorporating bacteria and when needed S-9 mix.
All mutagenicity assays were per formed in triplicate, except controls (100 /J of distilled water or dimethyl sulfoxide), which were run on 5 test plates.

Revertant colonies were scored after 48 hr at 37° in the dark.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other:

Remarks:

Weak positive

Conclusions:

SCH was found to be weak without S-9 mix and almost inactive in the presence of S-9 mix in terms of mutagenic potency.

Executive summary:

Sixteen hydrazine derivatives including Semicarbazide hydrochloride were tested for mutagenic activity in the Salmonellamicrosome (Ames) test.

Thirteen of the first 14 compounds listed (81% of the total) including SCH were positive in the Ames test, with a broad range of activity towards the five bacterial strains of Salmonella typhimurium used (TA1535, TA100, TA1537, TA1538, and TA98) and of metabolic behavior in the presence of S-9 mix containing rat liver, mouse liver, or mouse lung postmitochondrial preparations from Aroclor-treated animals.

Under these test conditions, SCH was found to be weak without S-9 mix and almost inactive in the presence of S-9 mix in terms of mutagenic potency.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo studies are available : a guideline study, Klimisch score 1, a COMET Assay in the rat and a publication, Klimisch score 4, a Micronucleus assay in the mouse.

As they are both in vivo studies and negative, it is strongh enough to conclude on the genotoxicity of the test item without performing in vitro tests.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55507861
- Expiration date of the lot/batch: 28/04/2019
- Purity test date: 99,3%
- Physical state/Appearance : white solid

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was freshly prepared as required as a solution at the appropriate concentration in distilled water.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HsdRCCHan™WIST

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo (UK)
- Age at study initiation: eight to ten weeks old
- Weight at study initiation: 184.2 to 209.9 g,
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in solid-floor polypropylene cages with woodflake bedding
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

OTHER:

The diet and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The animals were provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). These enrichment items were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

Route of administration:

oral: gavage

Vehicle:

- Vehicle: distilled water
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): 3012486
- Purity:
- Supplier : Aguettant
- Expiry date : 01/11/2018
-Storage conditions : Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:

24 hours

Frequency of treatment:

twice with a 24 hour interval

Post exposure period:

4 hours following the second administration

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 males rats per dose
7 males rats for vehicule group
3 males rats for positive control group

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

N-Nitriso-N-methylurea (MNU)
- Justification for choice of positive control(s): MNU is a positive control item that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.
- Route of administration: oral gavage
- Doses / concentrations: 25mg/kg bw 2,5mg/mL
- Supplier: AstaTech Inc
- Supplier’s Lot Number: 4486-027
- Physical state/ Appearance: Pale orange solid
- Purity: 90% (corrected for in the formulation)
- Expiry: 05 December 2018
- Storage Conditions: Approximately -20 ºC
- Solvent: Distilled water

Tissues and cell types examined:

Humane euthanasia was performed on the animals at the end of the exposure period using a method that did not affect the integrity of the required tissues (carbon dioxide asphyxiation). Samples of liver and glandular stomach were obtained from each animal for comet processing.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: See table in template any other information on materials and methods
A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex.
The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:
Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered through gauze to provide a single cell suspension.
Glandular Stomach– The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.

DETAILS OF SLIDE PREPARATION:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.


METHOD OF ANALYSIS:
The slides were coded prior to scoring to allow “blind” scoring. The slides were stained just prior to analysis for comets. To each dry slide, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, Comet IV.

Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparisons between the vehicle control group response and that of the test item dose groups was made.

The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity.
Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

OTHER:

Evaluation criteria:

The following criteria will be used to determine a valid assay:
• The concurrent negative control is comparable with the laboratory historical negative control range.
• The positive controls induce responses that are comparable with those in the laboratory historical positive control range.
• Adequate numbers of cells and doses have been analyzed.
• The highest dose level selected meets the requirements of the guideline and the study plan.

Statistics:

A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were also made when it was considered that there was a marked increase over the vehicle control value.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mortality Data and Clinical Observations :
One premature death occurred (animal 12) in the 700 mg/kg dose group prior to termination. The clinical signs observed in the 700 mg/kg dose group were unexpectedly more severe than those seen in the range-finding test and included hunched posture, lethargy, ataxia, increased salivation, ptosis, pilo-erection and elevated-tail. No clinical signs were observed in animals dosed with the test item at 350 and 175 mg/kg.

Evaluation of Comet Assay Slides :
A summary of the results for each of the tissues of the Comet Assay, liver and glandular stomach, is given in Table 2. Individual slide data and animal data for each tissue are presented in Tables 3 to 12 with the means, medians and standard deviations calculated from the individual animal data and the group data.

The vehicle control group demonstrated percentage tail intensities which were consistent with the current laboratory historical control range. The positive control item (MNU) produced a statistically significant marked increase in the percentage tail intensity and median percentage tail intensity in the liver and glandular stomach, with values which were comparable with the laboratory historical control range for these tissues. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.

There were no statistically significant increases in percentage tail intensity for any of the test item dose levels in the glandular stomach or liver tissues which exceeded the current laboratory historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver or glandular stomach. The premature death of one animal in the 700 mg/kg dose group was considered to have no impact on the results of the study as there were adequate numbers of animals remaining in this group and there was no indication of a response in the remaining animals.
There was no marked increase in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. The hedgehog frequency data for each tissue is included in Tables 3 to 12.

Conclusions:

The test item Semicarbazide hydrochloride, did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity in the liver or glandular stomach and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.

Executive summary:

Before performing the Comet Assay, a range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex.

In a Comet assay, performed according to the OECD 489 Guideline and to the GLP, 7 male rats (Wistar) administered by oral gavage with 700, 350 and 175 mg/kg of Semicarbazide hydrochloride at time 0 and 24 hours after the initial dosing. A further group of 7 rats were treated as the vehicle control group and a group of 3 rats were treated with as positive control, the N-Nitroso-N-methylurea. Animals were killed 4 hours after the second administration, at a sampling time of 28 hours. The glandular stomach and liver tissues were sampled and processed, the slides were then prepared prior to scoring for the presence of Comets.

The presence of clinical signs indicated that systemic absorption had occurred.

There was no evidence of an increase in the liver or glandular stomach in the percentage tail intensity or median percentage tail intensity in the test item dose groups when compared to the concurrent vehicle control groups.

The positive control item produced a marked increase in the percentage tail intensity value in the liver and glandular stomach, indicating that the test method was working as expected. The vehicle control group for the liver and the glandular stomach had percentage tail intensity values which were consistent with the current laboratory historical range for a vehicle.

The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity values in any of the tissues investigated when compared to the concurrent vehicle control group. The test item was considered to be unable to induce DNA strand breakage to the liver and glandular stomach in vivo, under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

As they are both in vivo studies and negative, it is strongh enough to conclude on the genotoxicity and the classification of the test item without performing in vitro tests.

Semicarbazide is not genotoxic and not classified for mutagenicity.