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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purity 99.852%, analytical certificate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Age/Weight: at the beginning of the treatment period, the males were approximately 9 weeks old and had a mean body weight of 331 g, the females were 6 weeks old and had a mean body weight of 184 g. The animals were sexually mature at the time of mating and the females were virgin.
Acclimation: an 11-day acclimation period to the conditions of the study preceded the beginning of the treatment period. A larger number of animals than necessary was acclimated to permit selection and/or replacement of individuals.
Allocation to study: before the beginning of the treatment period, the required number of animals (40 males and 60 females) was selected according to body weight and clinical condition and allocated to the groups, according to a stratification procedure, so that the average body weight of each group was similar. Identification: each animal was individually identified by an ear tattoo and received a unique CIT identity number.
From arrival at CIT, the animals were housed in a barriered rodent unit, under specific pathogen free (SPF) standard laboratory conditions.
The animal room conditions are set as follows: temperature: 22 +/- 2°C, relative humidity : 50 +/- 20%, light/dark cycle: 12h/12h (7:00 - 19:00), ventilation: about 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are checked regularly and records filed. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.
The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
The F0 females were housed individually in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Autoclaved wood shavings (SICSA, Alfortville, France) were provided as nesting material, a few days before delivery and during the lactation period. The cages were placed in numerical order on the racks. Every 6 weeks, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
The animals had free access to pelleted maintenance diet distributed weekly. The animals had free access to bottles containing tap water (filtered with a 0.22 um filter).
Bacterial and chemical analyses of sawdust, diet and water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (sawdust: pesticides and heavy metals; diet and water: pesticides, heavy metals and nitrosamines). No contaminants were present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on oral exposure:
The test item was administered as a solution in the vehicle. The test item dosage forms were prepared at weekly intervals and stored at +4°C and protected from light prior to use. Due to physical and chemical properties of the test item, special care was taken in order to ensure minimal dispersion into the environment and exposure of the technicians.
Before the start of treatment, the suitability of the proposed dosage form preparation procedure was determined. During the treatment period, the concentration of dosage forms prepared for use in the study was checked.
Before the start of treatment, two dosage forms containing 3 and 15 mg/mL of test item were prepared for stability analysis. Each dosage form was sampled in duplicate after 0 (just after preparation), 4 and 9 days storage at +4°C and protected from light. The aliquot sampled on day 4 was stored frozen at -20°C pending analysis on the last sampling occasion (day 9) when all samples were assayed.
The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8 and 12 was determined.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
ca. 7 weeks
Frequency of treatment:
once daily; 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
0.192 mmol/kg bw/d
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
0.640 mmol/kg bw/d
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Remarks:
0.960 mmol/kg bw/d
No. of animals per sex per dose:
10 (main groups); 5 (satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were determined in agreement with the Sponsor, following the results of a preliminary 2-week toxicity study by oral route in rats (CIT/Study No. 24603 TSR). The test item, 2-MERCAPTOETHANOL, when administered daily for 2 weeks was well tolerated at 10 and 30 mg/kg/day. At 100 mg/kg/day, treatment-related changes were noted resulting in:
. the death of a few animals,
. decrease in body weight and food consumption in females,
. increase of coagulation factors and transaminase activity in males and females,
. increase urea and decrease sodium blood level in females,
. increase in the weight and the size of the liver in males and females.
Consequently, the dose-levels of 15, 50 and 75 mg/kg/day were selected.
The animals were allocated in 4 main groups (10 males and 10 females) and 4 satellite groups (5 females). The reprotoxicity end-points were evaluated in the male and female animals from the main groups. The repeated dose toxicity end-points were evaluated in the five first males from the main groups and the five females of the satellite groups.

Examinations

Observations and examinations performed and frequency:
Each animal was observed at least once a day, at approximately the same time for the recording of clinical signs.
All animals of each group were observed in the cage, in the hand and in the standard arena, by observers unaware of the animal’s treatment, before the first day of treatment and then once a week thereafter.
The animals were randomized in order to ensure "blind" evaluation, except for examination performed before the first day of treatment.
The following parameters were assessed:
. "touch escape" or ease of removal from the cage,
. in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
. in the standard arena (two-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions, gait, arousal (hypo- and hyperactivity),
. posture, stereotypic behavior and breathing, ataxia, hypotonia.
In the first five males of the main groups and in the five females of the satellite groups, the examinations listed below were conducted during the week before mating (before laboratory investigations). The observer performing the evaluation was not aware of the treatment group of the animal.
The animals were randomized in order to ensure "blind" evaluation. The following measurements, reflexes and responses will be recorded:
. touch response,
. forelimb grip strength,
. pupil reflex,
. visual stimulus,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot play,
. rectal temperature.
Motor activity was measured in the first five males of the main groups and in the five females of the satellite groups, during the week before mating (before laboratory investigations), using an automated infra-red sensor equipment recording individual animal activity over a 10-minute period.
The body weight of each male of the main groups and female of the satellite groups was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female of the main groups was recorded once a week during the premating and mating periods, then on days 0, 7, 14, 20 post-coitum and on days 1, 7, 14 and 21 post-partum.
The quantity of food consumed by male of the main groups and female of the satellite groups was recorded once a week, over a 7-day period, from the first day of treatment until sacrifice.
main groups.
Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper the next day.
Sacrifice and pathology:
On completion of the treatment period, all surviving animals were asphyxiated by carbon dioxide and killed by exsanguination. Any moribund animals were killed in the same way.
Statistics:
Number of corpora lutea (implantations and pups) or as proportions (pre-implantation loss, post-implantation loss and pup findings). Whenever necessary, the experimental unit of comparison was the litter. Mean quantitative values are compared by one-way analysis of variance and Dunnett test (mean
values being considered as normally distributed and variances being considered as homogeneous). Proportions are compared by Fisher exact probability test.

Results and discussion

Results of examinations

Details on results:
CLINICAL EXAMINATIONS
- no mortality except 1 female in the high dose group; death considered to be not treatment related (no clinical signs, slight perilobular vacuolated hepatocytes and congestion of the lung)
- ptyalism noted from day 15 or 17 in 5 females of the mid dose group and in 4 females at 75 mg/kg/day; ptyalism from day 15 also in all males of the mid and high dose group and in 1 control male; no further clinical signs
- reactivity to manipulations or to different stimuli not altered as well as the motor activity
- body weight decreased in males but slightly increased in TS treated females
- food consumption was not altered in males; in females there was a slightly higher mean food consumption (days 1-50; correlated with body weight gain); the effect was not significant; however, it was considered to be treatment related
- the estrous cycle was not affected
HEMATOLOGY
- parameters not affected in males or females
BIOCHEMISTRY
- no relevant changes were observed in females
The altered urea level was considered to be without toxicological relevance (no effect on creatinine, no histopathological effect); effects on triglycerides (Trig) and cholesterol (Chol) were considered to be treatment related; other alterations were contributions of a few individuals and within historical range.
URINE ANALYSIS
- parameters were unaffected by the treatment
SEMINOLOGY
- no effect was observed on sperm motility and morphology
- the number of testicular sperm heads was slightly (not significantly) reduced in high dose males (113.3E6 versus 121.9E6 per g of testis in controls)
- further more epididymal sperm count was slightly decreased in all treatment groups; however, the effect was not dose dependent and/or within historical control range
NECROPSY
- liver paleness: in 1/10 males and 1/5 females of the control group, at 15 mg/kg in 1/5 females and 0/10 males, at 50 mg/kg in 4/10 males and 3/5 females, at 75 mg/kg in 6/10 males and 3/5 females;
- accentuated lobular pattern of the liver: at 50 mg/kg in 3/10 males and in 0/5 females, at 75 mg/kg in 7/10 males and 1/5 females;
- enlargement of the liver in 1/10 males given 75 mg/kg
HISTOPATHOLOGY
- minimal to slight hypertrophy of liver cells recorded in 2/5 females at 50 mg/kg and in 3/10 males and 4/5 females given 75 mg/kg

No treatment related effects were observed in other organs including reproductive organs.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weight change of surviving rats (g):

 Males (n = 10)    control  15 mg/kg  50 mg/kg  75 mg/kg
   days 1 - 15  +49  +43  +40  +38
   days 1 - 29  +96  +90  +73  +78
   days 1 - 50  +140  +140  +107  +124
 Females (n = 5)  days 1 - 15  +47  +47  +54  +45
   days 1 - 29  +71  +82  +82  +72
   days 1 - 50  +96  +116  +113  +109

Changes in males (n = 5 per group) suggested an effect on the liver (correlated with histopathology):

   control  15 mg/kg  50 mg/kg  75 mg/kg
 K+ (mmol/l)  3.97  4.02  4.36  4.67
 Cl- (mmol/l)  104.1  105.5  106.9  108.6
 urea (mmol/l)  3.7  4.2  7.0  7.1
 creatinine (umol/l)  36  33  35  39
 bile acids (umol/l)  49.8  49.4  69.8  116.7
 Chol (mmol/l)  1.6  1.3  0.9  0.6
 Trig (mmol/l)  0.85  0.67  0.39  0.23
 ALAT (IU/l)  2  24  37  37
 ASAT (IU/l)  59  61  69  134
 Glucose (mmol/l)  6.72  7.29  7.16  8.25

Peri-/medio-lobular vacuolated hepatocytes:

 males    0 mg/kg  15 mg/kg  50 mg/kg  75 mg/kg
   incidence  2/5  3/5  5/7  9/10
   mean severity*  1.0  1.0  2.8  2.8
 females  incidence  3/5  5/5  5/5  5/5
   mean severity*  1.0  1.2  2.8  3.4

(* severity not specified by the authors)

The marginally higher incidence and/or severity of vacuolated hepatocytes in the low dose group was considered to be not treatment-related (can be recorded in untreated rats) in contrast to effects at the mid enad high dose.

Degenerative cardiomyopathy:

 males    0 mg/kg  15 mg/kg  50 mg/kg  75 mg/kg
   incidence  3/5  1/5  3/5  5/5
   mean severity*  1.7  1.0  1.3  2.4
 females  incidence  0/5  0/5  3/5  3/5
   mean severity*  -  -  1.3  1.3

(* severity not specified)

Treatment-related effects on the heart in females at > 50 mg/kg and in males at 75 mg/kg.

Organ weights (differences in organ weights (in % of controls; n = 5):

     15 mg/kg  50 mg/kg  75 mg/kg
 Males  kidney absolute  -4  -1  +9
   kidney relative  -3  +7  +21
   liver absolute  0  -4  +22
   liver relative  +2  +5  +36
 Females  kidney absolute  +7  +10  +16
   kidney relative  -2  +6  +17
   liver absolute  +6  +32  +36
   liver relative  -3  +28  +37

These effects in the liver of males and females correlated with hepatocellular hypertrophy and/or vacuolated hepatocytes.

Applicant's summary and conclusion

Conclusions:
No toxic effects were recorded at 15 mg/kg in males and females; a slightly higher body weight gain and food consumption was seen in females (also at higher dose levels). In males at >= 50 mg/kg ptyalism, a lower body weight gain, minimal to marked vacuolated hepatocytes accompanied by lower cholesterol and triglyceride levels, paleness and accentuated lobular pattern of the liver were observed.