Sodium 2-mercaptoethanolate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

July 27, 1995

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ATOFINA
- Lot/batch No. of test material: 15/10/02
- Appearance: Colourless liquid
- Expiration date of the lot/batch: December 2003
- Purity: 99.852%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and under nitrogen atmosphere.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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.

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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, 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.

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 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.
- The estrous cycle was not affected.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight decreased in males but slightly increased in TS treated females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Parameters not affected in males or females

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

no relevant changes were observed in females.
An 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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Parameters were unaffected by the treatment

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

- 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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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.

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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)

Version / remarks:

March 22, 1996

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-mercaptoethanol

EC Number:

200-464-6

EC Name:

2-mercaptoethanol

Cas Number:

60-24-2

Molecular formula:

C2H6OS

IUPAC Name:

2-sulfanylethanol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ATOFINA
- Lot/batch No. of test material: 15/10/02
- Appearance: Colourless liquid
- Expiration date of the lot/batch: December 2003
- Purity: 99.852%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and under nitrogen atmosphere.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: males ca. 9 weeks, females: ca. 6 weeks
- Weight at study initiation: males: 331 g, females: 184 g.
- 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.
- Housing: From arrival at CIT, the animals were housed in a barriered rodent unit, under specific pathogen free (SPF) standard laboratory conditions. 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.
- Diet: The animals had free access to pelleted maintenance diet, batch Nos. 21017 and 21206 (UAR, Villemoisson, Epinay-sur-Orge, France) distributed weekly.
- Water: The animals had free access to bottles containing tap water (filtered with a 0.22 um filter).
- Acclimation period: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

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 substance, 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated and considered to be suitable for the analysis of the samples of the study.

SPECIFICITY
The specificity of the analytical method was demonstrated as follows:
- analysis of a standard solution of the test substance in methanol
- analysis of a solution of NaCl at 0,9 % (vehicle) diluted ten-fold in methanol.

No relevant interference between the test item peak and vehicle was observed on chromatograms.

LIMIT OF QUANTIFICATION
The limit of quantification of the analytical method was established as 2 µg/mL for a standard solution of the test substance. This limit corresponds to a limit of quantification of 0.02 mg/mL for the test item in the dosage form, the minimum dilution factor being ten-fold to avoid interference.

RELATIONSHIP CONCENTRATION/RESPONSE
The relationship concentration/detector response was checked by analysis of three different sets of six standard solutions containing 2, 5, 10, 20, 50, and 100 µg/mL of the test substance in methanol.
Satisfactory proportionality was demonstrated in the range of 2 to 100 µg/mL using a quadratic regression model since the coefficients of determination obtained were higher than 0.999.

REPEATABILITY OF INJECTIONS
Replicate analysis (n = 10) of a solution containing 108 µg/mL of the test item gave satisfactory results since the coefficients of variation values obtained were as follows:
- 4% based on peak height
- 5% based on peak area.
Considering these similar results, the repeatability of injections of the analytical method was validated taking into account peak area.

ACCURACY AND PRECISION
Six analyses of dosages forms containing 0,2 and 20 mg/mL of the test item in the vehicle were carried out. Samples were diluted appropriately with methanol before analysis. The accuracy and the precision (CV%) obtained is presented in 'Any other information materials and methods incl. tables'.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

The nominal concentration deviated in an acceptable range of ± 10% from the actual concentration

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

The nominal concentration deviated in an acceptable range of ± 10% from the actual concentration

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

The nominal concentration deviated in an acceptable range of ± 10% from the actual concentration

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, 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.

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 ensures
that 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 generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 clinical
signs also in detailed clinical observations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

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 1 sacrificed 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight decreased in males; slightly increased bw in TS treated females during premating period but reduced at the end of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in clinical chemistry of males suggested an effect on the liver (correlated with histopathology.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrous cycle was not affected.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The mating index, fertility index and pre-coital time were not affected.

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 1
sacrificed 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 clinical
signs also in detailed clinical observations. Body weight decreased in males; slightly increased bw in TS treated females during premating period but
reduced 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)

1

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

other: female fertility, parturition

2

Dose descriptor:

NOAEL

Effect level:

> 75 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed in pups at any dose level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of liveborn pups per litter was not affected in low and mid dose groups; 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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).

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross external abnormalities in any pup.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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 effect was seen on the anogenital distance on day 1 post partum. The sex ratio was not altered.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

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.