Methyl 3-mercaptopropionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-06-06 to 2007-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted with methods similar to OECD Guideline 407. This study was part of a reproductive/developmental toxicity screening study. Males were the only sex of animals included for the repeat dose endpoint. Females were included in the reproduction endpoint study listed below in the "cross-reference to same study record" section. Guideline 407 states to have at least 10 animals (5 from each sex). However, even though females were not included in this study record (based on the fact that they were pregnant and this can alter toxic effects), there were 10 males included in the study record. Therefore, enough animals were included.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Only males were included for this endpoint.

Principles of method if other than guideline:

N/A

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-mercaptopropionate

EC Number:

220-912-4

EC Name:

Methyl 3-mercaptopropionate

Cas Number:

2935-90-2

Molecular formula:

C4H8O2S

IUPAC Name:

methyl 3-sulfanylpropanoate

Details on test material:

- Name of test material (as cited in study report): Methyl 3-Mercaptopropionate
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: active
- Physical state: liquid

Test animals

Species:

rat

Strain:

other: HanRcc:WIST (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Laboratory Animal Services; Wolferstrasse 4; CH-4414 Fullinsdorf/Switzerland
- Age at study initiation:10 wks at delivery
- Weight at study initiation: Males: 286-331 g
- Fasting period before study: N/A
- Housing: Animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding. This repeat dose study was part of a 1-generation reproduction study and during the pairing period, the rats were housed one male/one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males were housed individually again.
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 rat/mouse maintenance diet was available ad libitum.
- Water (e.g. ad libitum): Tap water from Fullinsdorf in bottles was available ad libitum.
- Acclimation period: 7 days (minimum) under test conditions with an evaluation of health status


ENVIRONMENTAL CONDITIONS
- Temperature (deg. C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (background music played at a centrally defined low volume for at least 8 hours during the light period.)


IN-LIFE DATES: From: 2006-06-13 To: The males were sacrificed on Day 29 of the study (~2006-07-11).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous mixture was prepared. Having obtained a homogeneous mixture, vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration. During the daily administration period, homogeneity of the test substance in the vehicle was maintained using a magnetic stirrer. Doses were prepared at least weekly (7-day stability was determined). All dose formulations were stored at room temperature (20 +/- degrees C), away from direct sunlight. All animals received a dose volume of 4 mL/kg bw in vehicle with a daily adjustment of the individual volume to the actual body weight.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): 17678674
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneity and stability of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the administration period.
On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. The samples were frozen (-25 to -15 degrees C) pending analysis. Samples were sent on dry ice to RCC Ltd., Environmental Chemistry & Pharmanalytics, CH-4452 Itingen/Switzerland. Analysis was performed using a method developed by RCC Ltd. The test substance concentrations were determined by GC couple to a FID detector and quantified with the area under the peak. After analysis, the analytical results were communicated to the Study Director. Upon receipt and evaluation of these results, the Study Director decided about discarding the samples.
The identity of the test substance was confirmed by its retention time, which was similar to that measured in the working standard. The test substance content in all samples was found to be within the accepted range of +/-20% of the nominal content. In addition, the homogeneous distribution of the test substance in corn oil was demonstrated. The application formulations were considered to be stable for at least 7 days when kept under storage conditions.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 50 and 100 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study (RCC Study No. A57802) and following discussions with the sponsor.
- Rationale for animal assignment (if not random): Prior to start of treatment, animals were assigned to the different groups using a computer-generated random algorithm. In addition, body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice weekly
- Cage side observations included: All animals were checked for any mortalities and signs of reaction to treatment and/or symptoms of ill health. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first test substance administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed daily during the entire study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined weekly during the study (except during the pairing period with the female rats used for the reproductive toxicity portion of the study) and was reported as g/animal/day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of scheduled necropsy. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes; Blood samples were collected sublingually with the animal under light isoflurane anaesthesia.
- Animals fasted: Yes
- How many animals: 5 males (randomly selected) from each group
- Parameters examined.:erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, platelet count, total leukocyte count, differential leukocyte count, coagulation (thromboplastin time and activation partial thromboplastin time)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of scheduled necropsy. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: Yes
- How many animals: 5 males (randomly selected) from each group
- Parameters examined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), aspartate aminotransferase, alanine aminotransferase, bile acids, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus inorganic, protein (total), albumin, globulin, albumin/globulin ratio


URINALYSIS: No
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: N/A
- Animals fasted: N/A
- Parameters examined: N/A


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The examinations were performed shortly before scheduled sacrifice, following the daily dose administration.
- Dose groups that were examined: all (5 rats per group)
- Battery of functions tested: Animals were observed for the following:
1. cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.
2. hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reactivity to handling.
3. open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
4. categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer's reflex), urine or faeces, soiling, general abnormalities, posture.
5. measurements/counts: hind limb/fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity of the animals (basing on beam count) was recorded for 6-minute intervals over a period of 30 minutes.



OTHER: N/A

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; All surviving animals were sacrificed on day 29. Animals were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. All animals were subject to a gross necropsy examination. The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The testes* and epididymides* were weighed. In addition for five males randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: liver, adrenals*, thymus, kidneys*, spleen, brain and heart (*paired weights).
HISTOPATHOLOGY: Yes; Of all males the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: prostate, seminal vesicles with coagulation gland, testes (in Bouin's fixative) and epididymides (in Bouin's fixative).
In addition, of the five males selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: gross lesions, brain, spinal cord, small and large intestines (incl. Peyer's patches), stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative), urinary bladder, lymph nodes, peripheral nerve and bone marrow.
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Examinations were extended to the animals of the other dosage groups, if treatment-related changes were seen in the highest dose group.
All gross lesions were examined.
Microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Other examinations:

N/A

Statistics:

The following statistical methods were used to analyze body weights, food consumption, and skeletal examination data:
-Means and standard deviations of various data were calculated and included in the report.
-If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e., single treatment groups against the control group).
-The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
-Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: All animals survived until scheduled necropsy. In the high dose group, all animals pushed their heads through the bedding after administration of the test substance starting on day 5 and continuing until the end of the treatment period. One high dose male had salivation on two days during the study. No further observations were noted in any group. No test substance-related findings were noted during detailed weekly clinical observations at any dosage. Common findings such as spontaneous vocalization when the rat was removed from the cage observed in all groups was considered to be incidental.


BODY WEIGHT AND WEIGHT GAIN: Mean absolute body weights and body weight development were similar in all groups and gave no indication of a test substance-related effect. In the low dose group, mean body weight gain was statistically significantly increased in the first half of the study, which was considered to be incidental. Mean body weight loss was noted in all groups during a two-day period and was attributable to the overnight fasting before taking blood samples.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Mean food consumption was not affected by treatment with the test substance and was generally similar in all groups.


FOOD EFFICIENCY: N/A


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A


OPHTHALMOSCOPIC EXAMINATION: N/A


HAEMATOLOGY: The assessment of the hematology data did not reveal any test substance-related effects in the males.


CLINICAL CHEMISTRY: The assessment of clinical biochemistry data did not reveal any test substance-related effects in the males.

URINALYSIS: N/A


NEUROBEHAVIOUR: 1. FOB-None of the parameters under investigation during the functional observational battery were considered to be affected by treatment with the test substance. Common findings such as spontaneous vocalization when the rat was removed from the cage observed in the vehicle, mid and high dose groups was considered to be incidental.
-Grip strength, landing foot splay and body temperature-mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of test substance-related effects. Body temperature was statistically lower in the mid and high dose groups compared to the control group (37.5 degrees C each compared to 38.0 degrees C in the control group). This finding was considered to be incidental.
-Locomotor activity-Locomotor activity was assessed quantitatively in terms of low beam counts in activity monitor. The level of locomotor activity was similar in all groups and gave no indication of a test substance-related effect.


ORGAN WEIGHTS: For the mid and high dose groups, liver weights relative to body weights and brain weights were dose-dependently increased. Liver weights relative to the brain weights did not reach statistical significance in the mid dose. In the absence of a histopathological correlation these higher weights were considered to be of no adverse character.


GROSS PATHOLOGY: During necropsy no test substance-related findings were noted. The gross necropsy findings noted were within the range of spontaneous findings which may be recorded in rats of this strain and age. Their inter-group distribution did not suggest an effect of treatment at any dose level. The findings consisted of testes and epididymides reduced in size, and seminal vesicles or thymus with focal discolorations.


HISTOPATHOLOGY: NON-NEOPLASTIC: A minimal to slight hyperplasia of the forestomach squamous epithelium partly associated with a minimal to slight hyperkeratosis and minimal inflammatory cell infiltrations was recorded in four males in the high dose group. Proliferative lesions of the rodent non-glandular stomach region are relatively common in gavage and feeding studies ranging from mild hyperplasia of the keratinized stratified squamous epithelium to extensive papillomatous hyperplasia. As no similar findings were noted in the forestomach epithelium of the control group, this finding was considered to be test substance-related.
All other microscopic findings recorded in various organs of all groups treated with the test substance did not differ significantly from the control group. All findings were considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of this strain and age.


HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A


HISTORICAL CONTROL DATA (if applicable): N/A


OTHER FINDINGS: N/A

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: 50 mg/kg bw/day was determined to be the NOAEL based on a minimal to slight hyperplasia of the forestomach squamous epithelium noted in males at 100 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

N/A

Applicant's summary and conclusion

Conclusions:

Male rats were treated with the test substance for 28 days to evaluate toxicological effects. Based on the results of the study, the NOAEL was determined to be 50 mg/kg bw/day.

Executive summary:

N/A