Methanethiol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

other information

Study period:

First day of treatment: 13 April 2004. Experimental completion date: 16 June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

yes

Remarks:

4-week premating treatment instead of 2 weeks

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium methanethiolate

EC Number:

225-969-9

EC Name:

Sodium methanethiolate

Cas Number:

5188-07-8

IUPAC Name:

sodium methanethiolate

Details on test material:

Test substance: SODIUM METHYLMERCAPTIDE
CAS Number : 5188-07-8
Supplier: Arkema SA
Batch number : SMM33 0050-7
Purity : 32.9% solution in water

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
- Breeder: Charles River Laboratories France, L'Arbresle, France.
- Age/Weight: at the beginning of the treatment period, the males were 6 weeks old (body weight range: 183 g to 221 g), the females were 8 weeks old (body weight range: 183 g to 232 g). 
- Acclimation: 7 days before the beginning of the treatment period.

Environmental conditions: 
- temperature : 22 ± 2°C
- relative humidity : 50 ± 20%
- light/dark cycle : 12h/12h (7:00 - 19:00)  
- ventilation : approximately 12 cycles/hour of filtered, non-recycled air.

Housing:  Individually in suspended wire-mesh cages. Prior to delivery and during lactation, the animals were housed individually in polycarbonate cages.

Food and water:
- food: A04 C pelleted maintenance diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- water: tap water (filtered with a 0.22 µm filter) ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Control of the stability: before the start of treatment
- Control of the concentrations: on weeks 1, 4 and 8

Duration of treatment / exposure:

. for the males, throughout the pre-mating period (28 days), during the mating and post-mating periods until final sacrifice (approximately 8 weeks in total),
. for the females, throughout pre-mating (28 days) and mating periods, during pregnancy and lactation, until day 4 post-partum inclusive (between 8 and 9 weeks).

Frequency of treatment:

once a day, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 15, 45 mg/kg/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

Examinations

Observations and examinations performed and frequency:

Mortality and clinical signs were checked daily in all animals. Body weight and food consumption were recorded at designated intervals. 
- morbidity and mortality checked twice daily during treatment period and once daily on other days; morbid rats sacrificed (necropsy) 
- general clinical observations at least once daily
- body weight recorded on day 1 of treatment period and then once weekly 
- food consumption determined once weekly (not in males during mating period)

Detailed clinical observations and neurobehavioral tests were conducted at designated intervals to assess behavior, main functions and reflexes and to measure motor activity over a 60-minute period. 
- detailed clinical observations once weekly on a blind  basis; rats observed in the cage, in the hand and in a standard arena 
- shortly before terminal sacrifice, the reactivity to manipulations or different stimuli was checked in 5 males and 5 females of each group (randomization) as well as the motor activity over a 10 minute period (automatically recorded)

Blood and urine samples were collected from five males and five females at the end of the pre-mating period (ca. 4.5 weeks of treatment)  for hematology, blood biochemistry and urinalysis investigations.
- Hematology: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Mean cell Hemoglobin (MCH), Thrombocytes (PLAT), Leucocytes (WBC), Differential white cell count with cell morphology, Prothrombin time, Activated partial thromboplastin time (APTT), Fibrinogen (FIB).
- Blood biochemistry: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Bile acids (BILAC).
- Urinalysis: Volume (VOLUME), pH, Specific gravity (SP.GRAV), Proteins (PROT), Glucose (GLUC), Ketones (CETO), Bilirubin (BILI), Nitrites (NITR), Blood (BLOOD), Urobilinogen (UROB), Cytology of sediment.

Sacrifice and pathology:

At final sacrifice, specified organs were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on a range of sampled tissue and organs for five males and five females of the control and high-dose groups. Additionally, a microscopic examination of the spleen was performed for animals of the low and mid-dose groups. Furthermore, the spleen of animals of the control and the high-dose group was stained using Perls staining to confirm the nature of the observed pigment (hemosiderin) and for a more accurate evaluation of the severity of hemosiderin deposition.

- Macroscopic post-mortem examination: at termination (or moribund rats during treatment period).
- Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus.
- Microscopic examination:             
. as tabulated in OECD guideline 422 for five males and five females of the control and high-dose groups killed at the end of the treatment period,
. macroscopic lesions of all the animals of the low- and intermediate-dose groups killed on completion of the treatment period.           
. spleen of five males and five females of the low and intermediate dose groups            
. spleen, stained with Perls staining, of five males and five females of the control and high-dose groups.

Statistics:

- mean quantitative values compared by one-way analysis of variance and Dunnett test; proportions are compared by the Fisher exact probability test  
- hematology, biochemistry, urine analysis and organ weight: test for normality of distribution, Bartlett test (homogeneity of variances between groups), Dunn test (not homogeneous) or Dunnett test - *: p<0.05; **: p<0.01

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

45 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Hematological and spleen changes

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSAGE FORMS
- Stability: Satisfactory stability over a 9-day period at
+4°C.
- Concentration: satisfactory agreement between the nominal
and actual concentrations 

CLINICAL EXAMINATION 
- Mortality: there were no deaths in any group.

- Clinical signs:
        . 45 mg/kg/d: hypotonia was transiently observed in all
males and females during the whole dosing period, ataxia was
observed at detailed clinical observations in 4 males and in
one female during week 5 or 6 of dosing, ptyalism was
observed in most males and females a few days after the
beginning of the study until the end of dosing.
        . 5 and 15 mg/kg/d: no clinical sign was noted.

- Body-weight :
        . 45 mg/kg/d: in the males, the group mean body weight gain
was statistically significantly lower during
the first week of dosing (days 1 to 8: 45 g vs. 55 g, -18%,
p<0.01) and remained marginally lower during the dosing
period. In the females, the group mean body weight gain was
marginally lower during the premating period.
        . 5 and 15 mg/kg/d: no effect was noted.

- Food consumption: 
        . 45 mg/kg/d: in the males, the food consumption was
marginally lower during the whole study.
        . 5 and 15 mg/kg/d: no effect was noted.

- Haematology: 
          45 mg/kg/d: lower hemoglobin concentration was
recorded in both males and females. This was associated, in
the males with lower mean cell volume, and in the females
with lower mean cell hemoglobin concentration, lower red
blood cell count, and lower packed cell volume.

- Blood biochemistry: no relevant changes were noted in
blood biochemical parameters at any dose-levels.

- Urinalysis: the urinalysis parameters were unaffected by
the treatment.

PATHOLOGY
- Necropsy: no substance related effect at any dose-levels.

- Organ-weights: 
        . 45 mg/kg/d: higher absolute and relative spleen weights
were noted in the males and females 
        . 15 and 5 mg/kg/d: no substance related effect.

- Microscopic examination: 
        . 45 mg/kg/d: increased incidence and/or severity of
extramedullary hematopoiesis and hemosiderosis in the spleen
of both males and females and sinusoidal ectasia in most of
the males. Extramedullary hematopoiesis was observed with a
higher incidence (but a marginally higher severity) in the
liver of females given 45 mg/kg/day; this was associated
with greenish
pigment in a few Kuppfer's cells in 1/5 males and 2/5
females.
        . 15 and 5 mg/kg/d: no substance related effect.

Applicant's summary and conclusion

Conclusions:

5 males and 5 females in he control group

Executive summary:

In a combined repeated-dose/reproductive/developmental toxicity screening study (OECD TG 422), Sprague-Dawley rats (10/sex/dose) were administered 0, 5, 15 or 45 mg/kg bw/day of sodium methanethiolate in water by gavage daily for 8-9 weeks at a volume of 5 ml/kg. There was no mortality or changes in blood biochemistry values or urinalysis parameters. There were no clinical signs of toxicity or body weight or food consumption effects observed in the 5 or 15 mg/kg bw/day groups. At 45 mg/kg bw/day, clinical signs observed included transient low muscle tone in males and females, incoordination of muscles in four males and one female at weeks 5 or 6 and excessive salivation in most males and females beginning a few days into the study and continuing though the end of dosing. The group mean body weight gain of the males in the 45 mg/kg bw/day group was significantly lower (19%) during the first week of dosing and remained marginally reduced during the dosing period relative to the control group. In the females exposed to 45 mg/kg bw/day, the mean group body weights were marginally reduced during premating and significantly reduced during the first week of gestation in comparison to the control group. The food consumption values were marginally reduced throughout the study for the 45 mg/kg bw/day males but not the females. Decreased hemoglobin concentration was observed in the males and females of the 45 mg/kg/day dose group. This was associated with reduced mean cell volume in the males and with decreased mean cell hemoglobin concentration, red blood cell count and packed cell volume in the females. At necropsy, there were no macroscopic treatment-related effects observed in any dose group. There were no organ weight effects or microscopic effects observed in the 5 or 15 mg/kg bw/day dose groups. Higher absolute and relative spleen weights were observed for the male (22 and 33% increased, respectively) and female (49 and 57% increased, respectively) rats exposed to 45 mg/kg bw/day. Increased incidence and/or severity of extramedullary hematopoiesis and hemosiderosis in the spleen of both male and female rats and sinusoidal ectasia in the male rats was observed at 45 mg/kg bw/day (p<0.01). In the 45  mg/kg bw/day females, a higher incidence of extramedullary hematopoiesis in the liver was observed that was associated with greenish pigment in a few Kupffer’s cells. The NOAEL and the LOAEL of sodium methanethiolate in rats were considered to be 15 and 45  mg/kg bw/day, respectively.