Butyl 3-mercaptopropionate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-JUN-2006 to 13-JUN-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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): C4H8O2S
- Molecular weight (if other than submission substance): 120.17
- Smiles notation (if other than submission substance): COC(=O)CCS
- InChl (if other than submission substance): InChI=1/C4H8O2S/c1-6-4(5)2-3-7/h7H,2-3H2,1H3
- Physical state: Liquid
- Analytical purity: 99.81%
- Purity test date: 2006-01-25
- Lot/batch No.: 6ATMMP09
- Expiration date of the lot/batch: 2007-01-01
- Stability under test conditions: stability and homogeneity were analytically verified
- Storage condition of test material: At room temperature (20 ± 5°C)/under nitrogen

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 286 - 331 grams, Females: 180 - 207 grams
- Fasting period before study: only before blood sampling
- Housing: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding. During the pre-pairing period, males and females were housed individually. Cages of males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, rats were housed one male/one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males and the females were housed individually again. During the lactation period (until day 4 of lactation), dams were housed together with their litters.
- Diet: Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) available ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 13-JUN-2006 To: 30-JUL-2006

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item 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 homogenous mixture was prepared. Having obtained a homogenous 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 item in the vehicle was maintained using a magnetic stirrer.


VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is the vehicle of choice for substances with low water solubility
- Concentration in vehicle: 6.25, 12.50, 25.00 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneity and stability (7 days) 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°C to -15°C) pending analysis. Samples were sent on dry ice to Dr. D. Flade, RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen / Switzerland. Analysis was performed using a method developed by RCC Ltd. 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.

Duration of treatment / exposure:

males: at least 28 days; females for 14 days prior to pairing, through pairing and gestation until day 4 post partum

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

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none
- 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.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical signs. Additionally, the females were observed for signs of difficult or prolonged parturition.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first test item administration and weekly thereafter.


BODY WEIGHT: Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day before or on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters:
Erythrocyte count, Hemoglobin concentration distribution width, Haemoglobin Platelet count, Haematocrit Total leukocyte count, Mean corpuscular volume, Differential leukocyte count, Red cell volume distribution width, Mean corpuscular hemoglobin concentration, Mean corpuscular haemoglobin
Coagulation: Thromboplastin time, Activated partial thromboplastin time



CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on the day before or on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5 per sex per group
- Parameters:
Glucose, Sodium, Urea, Potassium, Creatinine, Chloride, Bilirubin, total Calcium, total Cholesterol, inorganic Phosphorus, Aspartate aminotransferase, total Protein, Alanine aminotransferase, Albumin, Bile acids, Globulin, Alkaline phosphatase, Albumin/Globulin ratio, Gamma-glutamyl-transferase


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were evaluated for five P generation males and five P generation females randomly selected from each group.
- Dose groups that were examined: all
- Battery of functions tested: Cage side observations, Hand-held observations, Open field observations, Categorical observations, Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The testes and epididymides of all parental males were weighed.
In addition for five adult males and females, randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
liver, spleen, adrenals, brain, thymus, heart, kidneys

HISTOPATHOLOGY: Yes
prostate, testes, seminal vesicles with coagulation gland, epididymides, ovaries, gross lesions, heart, brain, thymus, spinal cord, thyroid, small and large intestines (incl. Peyer's patches), trachea and lungs, stomach, uterus (with vagina), liver, urinary bladder, kidneys, lymph nodes, adrenals, peripheral nerve, spleen, bone marrow

Statistics:

The following statistical methods were used to analyze body weights, food consumption, reproduction 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:

no effects observed

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy. In group 4, all animals pushed their heads through the bedding after administration of the test item starting on day 5 of the prepairing period and continuing until the end of the treatment period.

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 item-related effect.

HAEMATOLOGY
The assessment of the hematology data did not reveal any test item-related effects in males and females.

CLINICAL CHEMISTRY
The assessment of clinical biochemistry data did not reveal any test item-related effects in males and females.

NEUROBEHAVIOUR
None of the parameters under investigation during the functional observational battery was considered to be affected by treatment with the test item.
Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of test item-related effects.
Body temperature in males was statistically significantly lower in groups 3 and 4 compared to the control group (37.5°C each compared to 38.0°C in the control group). Since the difference is very small and the body temperature was similar in all groups in females this finding was considered to be incidental.
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 itemrelated effects.

ORGAN WEIGHTS (see Table 1)
For groups 3 and 4 males, 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 group 3.
In the absence of a histopathological correlation these higher weights were considered to be of no adverse character.
In females, mean absolute organ weights as well as organ/body weight ratios and organ/brain weight ratios were not affected by exposure to the test item.

GROSS PATHOLOGY
During necropsy of parent animals no test item-related findings were noted.

HISTOPATHOLOGY (see Table 2):
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 and four females in group 4. 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 item-related.
All other microscopic findings recorded in various organs of all groups treated with the test item 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.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

LOEL

Effect level:

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

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

3

Dose descriptor:

LOEL

Effect level:

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

Sex:

female

Basis for effect level:

other: A minimal to slight hyperplasia of the forestomach squamous epithelium was noted in males and females. This effect is not relevant since humans have no forestomach.

4

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Neither the slight forestomach hyperplasia not the liver weight increase are adverse or relevant effects.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Organ weights P-generation males

MALES	Group 1 0 mg/kg/d	Group 2 25 mg/kg/d	Group 3 50 mg/kg/d	Group 4 100 mg/kg/d
BODY W. [g]	342.3	360.3	352.1	355.9
ST.DEV.	17.8	17.2	23.9	23.4
N	10	10	10	10
BRAIN [g]	2.02	2.01	2.08	1.97
% BW	0.60	0.57	0.59	0.57
N	5	5	5	5
HEART [g]	0.95	1.02	1.04	1.01
% BW	0.28	0.29	0.29	0.29
N	5	5	5	5
LIVER [g]	8.30	8.62	9.40	9.38
% BW	2.46	2.43	2.65*	2.70*
N	5	5	5	5
THYMUS [g]	0.306	0.364	0.287	0.348
% BW	0.091	0.103	0.082	0.099
N	5	5	5	5
KIDNEYS [g]	2.26	2.23	2.34	2.40
% BW	0.67	0.63	0.66	0.69
N	5	5	5	5
ADRENALS [g]	0.074	0.081	0.081	0.088
% BW	0.022	0.023	0.023	0.025
N	5	5	5	5
SPLEEN [g]	0.69	0.72	0.76	0.68
% BW	0.21	0.21	0.22	0.20
N	5	5	5	5
TESTES [g]	3.63	3.69	3.76	3.83
% BW	1.06	1.02	1.07	1.08
N	10	10	10	10
EPIDIDYMIDES [g]	1.225	1.231	1.240	1.275
% BW	0.358	0.342	0.352	0.359
N	10	10	10	10

*/**: Dunnett-test based on pooled variance sig. at 5% or 1% level.

Table 2: Histopathology

Dose group	1	2	3	4	1	2	3	4
Sex	Males				Females
Stomach No. examined	5	5	5	5	5	5	5	5
epithelial hyperplasia	–	–	–	4	–	–	–	4
grade 1	–	–	–	1	–	–	–	1
grade 2	–	–	–	3	–	–	–	3
hyperkeratosis	–	–	–	3	–	–	–	1
grade 1	–	–	–	3	–	–	–	–
grade 2	–	–	–	–	–	–	–	1
mononuclear infiltrate	–	–	–	1	–	–	1	2
grade 1	–	–	–	1	–	–	1	2
inflammation	–	–	–	2	–	–	–	3
grade 1	–	–	–	2	–	–	–	3

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavageadministration of the test item MMP to rats over approximately 28 days. The test item was administered in corn oil as vehicle at dosages of 25, 50, and 100 mg/kg body weight/day, and controls received the vehicle only.The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Administration of 50 and 100 mg/kg/day resulted in dose-dependently increased liver weights relative to body weights and brain weights in males. In the absence of a histopathological correlation these higher weights were considered to be of no adverse character. Furthermore at 100 mg/kg/day, a minimal to slight hyperplasia of the forestomach squamous epithelium was noted in males and females. Based on these results a general NOAEL (No Observed Adverse Effect Level) was established at 50 mg/kg body weight/day. The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 100 mg/kg/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 was to generate preliminary information concerning the effects of MMP on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. Methyl 3-Mercaptopropionate was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. The following dose levels were applied: Group 1: 0 mg/kg body weight/day (vehicle control) Group 2: 25 mg/kg body weight/day Group 3: 50 mg/kg body weight/day Group 4: 100 mg/kg body weight/day A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

 

PARENT ANIMALS

GENERAL TOLERABILITY

Males All animals survived until scheduled necropsy and no clinical signs were noted. At 100 mg/kg/day, animals pushed their heads through the bedding after administration of the test item starting on day 5 of the prepairing period and continuing until the end of the treatment period. This was considered to be a sign of reaction to the treatment with the test item. Neither food consumption nor body weight development were affected by exposure to the test item at any concentration. None of the parameters under investigation during the functional observational battery was considered to be affected by treatment with the test item.

 

REPRODUCTION DATA

The fertility rate was high resulting in 9, 8, 9 and 10 litters in order of ascending dose levels for evaluation of reproduction data. At all concentrations, there were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to scheduled sacrifice on day 4 post partum.

 

CLINICAL LABORATORY INVESTIGATIONS

The assessment of clinical biochemistry and hematology data did not reveal any test item-related effects at any concentration.

 

ORGAN WEIGHTS

At 50 and 100 mg/kg/day, liver weights relative to body weights and brain weights were dose-dependently increased in males. In the absence of a histopathological correlation these higher weights were considered to be of no adverse character.

 

MACROSCOPIC FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS

During necropsy of parent animals no macroscopical test item-related findings were noted. During histopathological examinations 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 and four females at 100 mg/kg/day. The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups and gave no indication of a test item-related effect.

 

LITTER DATA

No abnormal findings were noted for pups at first litter check or during the first 4 days post partum. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item. Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. Mean pup weight development during the first 4 days post partum lactation was unaffected by treatment with the test item.

 

CONCLUSION

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavageadministration of the test item MMP to rats over approximately 28 days. The test item was administered in corn oil as vehicle at dosages of 25, 50, and 100 mg/kg body weight/day, and controls received the vehicle only.The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Administration of 50 and 100 mg/kg/day resulted in dose-dependently increased liver weights relative to body weights and brain weights in males. In the absence of a histopathological correlation these higher weights were considered to be of no adverse character. Furthermore at 100 mg/kg/day, a minimal to slight hyperplasia of the forestomach squamous epithelium was noted in males and females. Based on these results a general NOAEL (No Observed Adverse Effect Level) was established at 50 mg/kg body weight/day. The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 100 mg/kg/day.