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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Guideline:
other: according to the OECD guideline No. 407
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No. 15/10/02, purity 99.85%

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, batch Nos. 21017 and 21206 (UAR, Villemoisson, Epinay-sur-Orge, France) 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:
other: sterile isotonic saline solution
Details on exposure:
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.
Details on mating procedure:
Within the main groups, females were paired with males from the same dose-level group: one female was placed with one male, in the latter’s cage, during the night. Confirmation of mating was made in the morning by checking the presence of a vaginal plug or of sperm in a vaginal lavage. The day of confirmed mating was designated day 0 post-coitum (p.c.). Each female was placed with the same male until mating occurred or 14 days had elapsed.The pre-coital time was calculated for each female.
Duration of treatment / exposure:
Exposure period: females - throughout the premating period (5 weeks), during mating, pregnancy and lactaiton period until day 21 post natal; males - throught premating period (5 weeks), mating andp ostmating periods.Premating exposure period (males and females): 5 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:
Total: 110 rats (44 males and 66 females)
Control animals:
yes, concurrent vehicle
Details on study design:
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. Handling and preparation procedures are documented in a Specific Operating Procedure.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.During the mating period, the food consumption was noted for neither males nor females of the 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.

Examinations

Parental animals: Observations and examinations:
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 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, theexaminations listed below were conducted during the week before mating (before laboratoryinvestigations). The observer performing the evaluation was not aware of the treatment group ofthe 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.
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:. for females from the satellite groups, during the last 3 weeks of treatment,. for females from the main groups, during the last 3 weeks of the premating period, during the mating period, until the females were mated.
Sperm parameters (parental animals):
In the first five males of the control and high dose-groups, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell.Results are expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).
Litter observations:
The total litter size and number of pups of each sex were recorded as soon as possible after birth. The litters were observed daily in order to note the number of live, dead and cannibalized pups. Any gross malformation in pups was noted.On day 4 post-partum, the size of each litter was adjusted by randomly culling extra pups to obtain, as nearly as possible, four males and four females per litter. Whenever necessary, partial adjustment (for example five males and three females) was permitted. Standardization of litter size is considered to reduce litter size-induced variability in growth and development of the pups and thus increase the sensitivity of statistical analysis. This also ensuresthat any adverse effects on pup growth and development are not masked by a treatment-related reduction in litter size.The pups were observed daily for clinical signs or abnormal behavior. Dead pups and pups killed on day 4 post-partum (not selected) were discarded after external examination for gross abnormalities.The anogenital distance (AGD) was measured on day 1 post-partum.
Postmortem examinations (parental animals):
Each animal was checked for mortality or signs of morbidity:. at least twice a day during treatment period,. at least once a day on other days.Any animal showing signs of poor clinical condition, was humanely killed. Animals found dead or killed prematurely were subjected to a macroscopic examination.
Postmortem examinations (offspring):
The pups were observed daily for clinical signs or abnormal behavior.Dead pups and pups killed on day 4 post-partum (not selected) were discarded after external examination for gross abnormalities.The weight of each pup was recorded on days 1, 4, 7, 14 and 21 post-partum.
Statistics:
Data are expressed as group mean values +/- standard deviation (body weight, food consumption, 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: P0 (first parental animals)

Details on results (P0)

Mortality: no deaths in females of control and low dose group; at 50 mg/kg 2 females found dead at day 21 post coitum and 1 at day 2 post partum; at 75 mg/kg 3 females found dead at days 20 and 21 post coitum and 1 sacrificed at day 19 post coitum, 1 female found dead at day 22 post coitum and 1sacrificed at day 23 post coitum; all females found dead or sacrificed were pregnant; symptoms seen in sacrificed females: piloerection, dyspnea, round back, pallor of extremities, emaciated appearance, oiled urogenital area, chromorrhinorea, chromodacryorrhea; no mortality in males in any treatment group. Ptyalism noted in females of the mid (9/10 during premating period) and high dose (10/10 during premating period); this effect was partly also observed during pregnancy and lactation; ptyalism from day 15 also in all males of the mid and high dose group and in 1 control male; no further clinicalsigns also in detailed clinical observations. Body weight decreased in males; slightly increased bw in TS treated females during premating period butreduced at the end of gestation. Food consumption was not altered in all males or females of the low dose group; in females of the mid dose group there was a slightly higher mean food consumption (except during the 3rd week of pregnancy); the effect was not significant; at 75 mg/kg significant (+14%, p<0.05) increase was seen the 1st 2 weeks of pregnancy and lower from day 14-20 post coitum; however, this result was considered to be treatment related. The estrous cycle was not affected as well as the mating index, fertility index and pre-coital time. Changes in clinical chemistry of males suggested an effect on the liver (correlated with histopathology. The implantation site per litter were within the historical range.Delivery data: females with no delivery: 0 in control and low dose group, but 2 females in the mid dose group and 5 at 75 mg/kg.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Sex:
male
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: male fertility and reproductive performance (migrated information)
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Sex:
female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: maternal toxicity, female fertility, and parturition (migrated information)
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: repeated dose toxicity parameters (migrated information)

Results: F1 generation

Details on results (F1)

No gross external abnormalities in any pup. The number of liveborn pups per litter was not affected in low and mid dose group; significant effects at 75 mg/kg due to one dam with only 1 liveborn pup. Number of pups that died during the 1st 4 days of lactation was increased at >= 50 mg/kg (7.5% versus 3% in controls, no data about significance). Viability index decreased at 50 mg/kg due to a sacrified litter whose mother died. The lactation index was lower in the high dose group compared with controls; at 75 mg/kg the percentage of pups per litter on day 21 post partum was significantly decreased (7.0 vs 7.8 in control). No clinical signs were observed in pups at any dose level. No effect was seen on the anogental distance on day 1 post partum. Pups weight was slightly increased at >= 50 mg/kg (10 and 9%; probably due to increase in duration of gestation). Body weight gain during lactation was slightly increased at 50 mg/kg (10%), but significantly lower in the 1st week of lactation at 75 mg/kg (-20%) (both considered to be treatment related). The sex ratio was not altered.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Due to the high number of dead and prematurely sacrificed females (see below, mortality) in the mid (n=2) and high (n=4) dose group, it was decided to interrupt the treatment at these 2 dose levels from days 19 and 20 post coitum until delivery.

Seminology: No significant effects observed on sperm count, motility and morphology.

Organ weight: Increased weight of the liver of males and females correlated with hepatocellular hypertrophy and/or vacuolated hepatocytes.

Necropsy and histopathology: Effects on the liver (minimal to slight hypertrophy of liver cells, peri/medio-lobular vacuolated hepatocytes) and heart (degenerative cardiomyopathy) in the mid and high dose group are decribed in chapter 5.4 - no treatment related effects were observed in other organs including reproductive organs.

Body weight change of surviving rats (g):

 Males    control  15 mg/kg  50 mg/kg  75 mg/kg
   days 1 - 15  +15  +43  +40  +38
   days 1 - 29  +96  +90  +73  +78
   days 1 - 50  +140  +140  +107  +124
 Females  premating        
   days 1 - 15  +42  +44  +50  +50
   days 1 - 36  +88  +91  +94  +91
   pregnancy        
   days 0 - 20  +145  +146  +146  +107 (-26%)
   days 14 - 20  +84  +79  +78  +44 (-47%)
   lactation        
   days 1 - 21  +21  +23  +32  +35

Gestation data:

   control  15 mg/kg  50 mg/kg  75 mg/kg
 paired females  10  10  10  10
 mated females  9/10  10/10  10/10  10/10
 pregnant females  8/9  10/10  9/10  10/10
 non-pregnant females  1  0  1  0
 females with liveborn pups  8  10  7  4
 gestation index (%)  10  10  78  40
 duration of gestation (d)  21.5  21.2  21.9  22.3
 females delivered on day 21 p.c.  3  8  1  0
 females delivered on day 22 p.c.  4  2  6  3
 females delivered on day 23 p.c.  0  0  0  1
 implantation sites per litter  15.9  16.4  15.4  13.8
 post-implantation loss (%)  6.9  9.1  1.8  27

Applicant's summary and conclusion

Conclusions:
Parameters on gestation and delivery were not affected at 15 mg/kg. No effect was noted on mating and fertility at any dose level.At 50 mg/kg bw/day the duration of gestation and the delivery were affected, the maternal toxicity was substantiated by death of pregnant females; ptyalism was observed in both gender; females showed slightly higher body weight gain and food consumption during exposure period and lactation; pup weight also slightly increased; in males a decrease in body weight gain and effects on the liver were recorded.Additionally to the effects seen at the mid dose 75 mg/kg bw/day resulted in a significant decrease in body weight gain of pregnant dams in the last week of pregnancy accompanied by a decrease in food consumption (not significant); the decreased number of liveborn, the high post-implantation loss and the reduced pups survival and pups body weight gain the 1st week of lactation (significant) were probably related to the treatment; however, evaluation is difficult due to the low number of litters in this dose group.