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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06-JUN-2006 to 13-JUN-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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 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

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
LOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: organ weights; increased relative liver weight. In the absence of a histopathological correlation these higher weights were considered to be of no adverse character.
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.
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:
not specified

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:
The findings made in this study were either not adverse (liver weight increase in males) or not relevant for humans (slight forestomach hyperplasia).
The general NOAEL in this study was therefore greater than or equal to 100 mg/kg bw/day.