Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A NOAEL of 19.22 mg/kg bw/day was determined based on read-across data from CAS 60 -24 -2

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to same study
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)
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:
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.
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
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
The nominal concentration deviated in an acceptable range of ± 10% from the actual concentration
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
The nominal concentration deviated in an acceptable range of ± 10% from the actual concentration
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.
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.
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: 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.
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.
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
Dose descriptor:
NOAEL
Effect level:
> 75 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
Remarks on result:
not measured/tested
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

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
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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
19.22 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Read across: 2 -Mercaptoethanol

No studies regarding the reproductive toxicity of sodium 2 -mercaptoethanolate were available. Therefore, data on the structural analogue 2 -mercaptoethanol (CAS: 60 -24 -2) has been used.

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG 422), groups of 10 male and 10 female rats were gavaged once daily with 0, 15, 50, or 75 mg/kg 2-mercaptoethanol. Males were treated 5 weeks before mating, during mating and post-mating period until sacrifice after ~7 weeks. Females were treated 5 weeks before mating, during mating and pregnancy and lactation until day 21 post partum inclusive except at the mid and high dose when treatment was interrupted from days 19 and 20 post coitum until delivery due to toxic effects (see below); all females were sacrificed on day 21 post partum. There were no treatment-related effects on mating and fertility parameters at any dose level. Seminology in males revealed no effects on sperm count, motility and morphology. The estrous cycle, mating and fertility indices, and pre-coital time were not affected, and no treatment-related effects were detected in reproductive organs on macro- or microscopic examination. Deaths of pregnant females occured at 50 and 75 mg/kg bw/day (six dead or sacrificed on post coitum (PC) days 19-23 at 75 mg/kg bw/day, three dead on PC day 21 or day 2 post partum at 50 mg/kg bw/day). Surviving females in the mid- and high-dose groups showed higher body weight gain (52 and 67 %) and food consumption (14 %; statistically significant in the high-dose group) during the premating period and lactation, while those in the top-dose group had reduced body weight gain in last week of pregnancy (-47 % ). Effects on body weight gain and food consumption were considered by the investigators to be treatment related. At 75 mg/kg bw/day the duration of gestation was increased (22.3 days v 21.5 days in controls) and the number of live born pups/litter was significantly decreased due to one dam with only 1 live born pup. The number of females with live-born pups was reduced in the mid- and high-dose groups (7/10 and 4/10 versus 8/9 in controls; statistically significant at the high dose). Gestation indices were given as 78% and 40 % in the mid- and high-dose groups compared to 100% in the low-dose group and the controls. There was no effect on sex ratio. The primary treatment-related effect on reproduction in the TG 422 study was prolonged labor and dystocia at dose levels of 50 and 75 mg/kg/day. Certain aliphatic thiol compounds, including 2-mercaptoethanol, have been shown to act as antagonists to the neuropeptide oxytocin or the oxytocin receptor, blocking the contractile response of oxytocin on the rat uterus in vitro (Martin and Schild, 1965). Oxytocin is secreted primarily by the posterior pituitary gland and is critical to the normal progress of parturition and sustaining sufficient uterine contractions during labor to expel the fetus(es) and to ligate severed blood vessels within the contracted myometrium after the placenta separates, thus preventing hemorrhage. It is possible that oral administration of 2-mercaptoethanol to pregnant rats in the TG 422 study was sufficient to disrupt the normal oxytocin-mediated progression of parturition by diminishing uterine contractions and prolonging labor. However also systemic toxicity classified with STOT RE 2 was observed at these dose levels making the effects on parturition not a selective isolated effect. Slightly higher pup body weights were noted at birth in the 75 mg/kg/day group, probably as a result of the slight increase in the length of pregnancy. However, despite these higher pup weights at birth, mean pup body weight at 75 mg/kg was lower than control values throughout the remainder of the lactation period as a result of significantly lower pup body weight gain beginning on postnatal day 4. The effects on pup body weight and survival at 75 mg/kg are most likely secondary to maternal care issues related to the poor condition of the dams.

Taken together, the substance may reversibly antagonize the oxytocin receptor, rendering it partially inactive. This may contribute to problems observed in particularly sensitive female rats during parturition (death of some females and increased pup mortality due to prolonged parturition or inability to give birth, which can be explained by the inability to induce proper uterus contractions). However, exposure of the substance also leads to significant systemic target organ toxicity at the same dose levels where the effects on parturition in the female rats were observed. The effects on the organs require classification with STOT RE Cat. 2. Based on the currently available data it cannot be ruled out that the observed systemic toxicity at least partially contributed to the effects observed on parturition. The pup mortality (developmental toxicity) is most likely secondary to maternal effects (systemic toxicity/effects on parturition) and does not require classification.

In addition to the effects on parturition and pup body weight, mean live litter size was significantly (p<0.5) lower in the 75 mg/kg/day group compared to the control group (10.0 versus 14.9 pups). The smaller litter size at 75 mg/kg was primarily attributed to one dam (out of 4 surviving dams) that delivered a single pup. This reduction in mean live litter size correlated with higher post-implantation loss and decreased pup survival at the same dose level. However, the small number of surviving dams/litters available for evaluation at 75 mg/kg (n=3 or 4 dams) is a potential major confounding factor in establishing a relationship between these endpoints (live litter size and pup survival) and test article administration. No apparent effects on mating or fertility indices or on male reproductive parameters were observed at any dose level evaluated. As a result of the number of pregnant females found dead or euthanized in extremis at 50 and 75 mg/kg due to dystocia and prolonged labor, the number of pups/litters available for evaluation was limited, especially at 75 mg/kg. To adequately assess the potential developmental effects of the test article on fetal external, visceral and skeletal morphology as well as intrauterine growth and fetal survival (without the confounding effects of parturition-related mortality and morbidity), a prenatal developmental toxicity study in rats (OECD guideline 414) was proposed and conducted in 2013 following ECHA's approval

Reference Martin, P.J., and Schild, H.O. (1965) The antagonism of disulphhide polypeptides by thiols. Brit. J. Pharmacol. 25: 418-431.

Effects on developmental toxicity

Description of key information
A NOAEL of 32 mg/kg bw/day was determined based on read-across data from CAS 60 -24 -2
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug, 1998
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ATOFINA
- Lot/batch No. of test material: 63133756P0
- Appearance: Liquid / colorless, clear
- Expiration date of the lot/batch: 01 May 2014
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and under nitrogen atmosphere. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-13 weeks
- Weight at study initiation:
- Housing: 1 rat per cage
- Diet: Ground Kliba maintenance diet ad libitum
- Water : filtered tap water ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD6), the animals will be accustomed to the environmental conditions and to the diet

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 airchanges per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
other: isotonic saline solution
Details on exposure:
5 mL/kg body weight; for test substance preparation, the specific amount of test substance will be weighed, topped up with cooled isotonic saline solution (fresenius) in a graduated flask and intensely mixed by shaking; all constituents are cooled before and during preparation; preparations are prepared at intervals which guarantee that the test substance concentrations in the vehicle remain stable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in isotonic saline solution (Fresenius) over a period of 9 days in a refrigerator were conducted prior to the start of the study.
Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations.
Details on mating procedure:
Animals paired by the breeder (time-mated animals) were supplied at noon on the day of evidence of mating; this day is referred to as GD0
Duration of treatment / exposure:
Animals are treated once daily from GD6-GD19
Frequency of treatment:
Once daily
Duration of test:
Dams are sacrificed on day 20
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose was selected based on signs of toxicity noted at a dose level of 50 mg/kg bw/d in a previously conducted OECD 422 study (test facility CIT, laboratory study number 24847 RSR, 2004) and in a maternal toxicity range-finding study (BASF project 10R0234/04R034) which preceded this definitive prenatal developmental toxicity study.

In the OECD 422 combined repeated dose with the reproductive/developmental screening test, 2 out of 10 Sprague-Dawley dams in the 50 mg/kg bw/d group died immediately prior to or during parturition with another death occurring during early lactation. Although the OECD Guideline 414 study design does not continue the dose administration through the period immediately prior to and during parturition, the presence of this toxicity was pertinent in the selection of dose levels for the definitive prenatal developmental toxicity study.

In the maternal toxicity range-finding study, 10 pregnant Wistar rats were administered the test substance by oral gavage from gestational day (GD) 6 through GD 19. One dam died on GD 18 and one dam was sacrificed in moribund condition on GD 20, showing signs of piloerection, semiclosed eyelids, apathy, and hypothermia. While it is possible that the mortality observed and the clinical signs were due to gavage errors in administering the test substance (as they were not observed in the OECD 422 study at the 50 mg/kg and higher dose levels), a conservative interpretation is that the toxicity observed was due to the test substance. For these reasons, the dose level of 50 mg/kg bw/d was considered to be potentially lethal to the dams in the OECD 414 study.

The selected high dose for the present study represented half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 86/609/EEC on animal protection.

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily, abnormalities and changes are documented

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Bw is recorded on day 0, 1, 3, 6, 7, 8, 9, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consuption is recoreded from GD0-1, 1-3, 3-6, 6-7, 7-8, 8-9, 9-10, 10-13, 13-15, 15-17, 17-19, 19-20

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day #20
- Organs examined: Adrenal glands, Heart, Kidneys, Liver, Spleen

Ovaries and uterine content:
The ovaries and uterine content was examined after termination:
Examinations included:
- Weight of the unopened uterus
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Site of implantations in the uterus
Fetal examinations:
After removal of fetuses from the uterus, the following examinations have been made:
- Weight of each fetus
- Sex
- Weight of placentas
- Gross pathological examination of fetuses after dissection from uterus
- Half of the fetuses of each dam is skines, fixed in ethyl alcohol and the skeloton and cartilage stained (method by Kimmel and Trammell)
- The other half of the fetuses is fixed in Harrisons fluid and soft tissues examined
Statistics:
Dunnett's test, Fishers exact test and Wilcoxon test
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any females of all test groups
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of the dams in test groups 1-3 (5, 15 or 25 mg/kg bw/d) were generally comparable to the concurrent control group throughout the whole study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1-3 (5, 15 or 25 mg/kg bw/d) were generally comparable to the concurrent control group throughout the whole study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among haematological parameters were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes of behaviour were observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- The mean gravid uterus weights of the animals of test groups 1-3 (5, 15 and 25 mg/kg bw/d) were not influenced by the test substance.
- There were no test substance-related differences between the test groups 0, 1, 2 and 3 (0, 5, 15 and 25 mg/kg bw/d) in absolute and relative organ weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The conception rate reached 100% in all test groups (0, 5, 15 and 25 mg/kg bw/d). There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 5, 15 and 25 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One male control fetus had more than one malformation affecting the head (i.e. mandibular micrognathia, aglossia and microphthalmia). The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One male control fetus had associated external malformations. Furthermore, one male mid-dose fetus (15 mg/kg bw/d) had more than one malformation affecting the skeleton in different areas. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group.
Visceral malformations:
no effects observed
Description (incidence and severity):
Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the control (test groups 0-3; 0, 5, 15 and 25 mg/kg bw/d). The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
Other effects:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
32 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Read across: 2 -Mercaptoethanol

No studies regarding the developmental toxicity of sodium 2 -mercaptoethanolate were available. Therefore, data on the structural analogue 2 -mercaptoethanol (CAS: 60 -24 -2) has been used.

2-Mercaptoethanol was tested for its prenatal developmental toxicity in Wistar rats according to OECD TG 414 and GLP. The test substance was administered as a solution in isotonic saline solution to groups of 25 time-mated female Wistar rats by gavage at doses of 5, 15 and 25 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (isotonic saline solution) in parallel. The high dose was selected based on signs of toxicity noted at a dose level of 50 mg/kg bw/d in a previously conducted OECD 422 study (test facility CIT, laboratory study number 24847 RSR, 2004) and in a maternal toxicity range-finding study (BASF project 10R0234/04R034) which preceded this definitive prenatal developmental toxicity study. In the OECD 422 combined repeated dose with the reproductive/developmental screening test, 2 out of 10 Sprague-Dawley dams in the 50 mg/kg bw/d group died immediately prior to or during parturition with another death occurring during early lactation. Although the OECD Guideline 414 study design does not continue the dose administration through the period immediately prior to and during parturition, the presence of this toxicity was pertinent in the selection of dose levels for the definitive prenatal developmental toxicity study. In the maternal toxicity range-finding study, 10 pregnant Wistar rats were administered the test substance by oral gavage from gestational day (GD) 6 through GD 19. One dam died on GD 18 and one dam was sacrificed in moribund condition on GD 20, showing signs of piloerection, semiclosed eyelids, apathy, and hypothermia. While it is possible that the mortality observed and the clinical signs were due to gavage errors in administering the test substance (as they were not observed in the OECD 422 study at the 50 mg/kg and higher dose levels), a conservative interpretation is that the toxicity observed was due to the test substance. For these reasons, the dose level of 50 mg/kg bw/d was considered to be potentially lethal to the dams in the OECD 414 study. The selected high dose for the present study represented half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive 86/609/EEC on animal protection. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle over a 9-day period in a refrigerator (in the dark). Generally, clinical observations revealed no toxicologically relevant difference between the animals receiving 5, 15 or 25 mg/kg bw/d 2-Mercaptoethanol and controls. Concerning clinical pathology and organ weights of dams no treatment-related, adverse effects were observed up to a dose of 25 mg/kg bw/d. No differences of toxicological relevance between the control and the treated groups (5, 15 or 25 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test compound on sex distribution of the fetuses was noted at any dose. In the assessment of all fetal external, soft tissue and skeletal observations no evidence for toxicologically relevant adverse effects of the test substance was determined on fetal morphology at any dose. Taken togehter, under the conditions of this prenatal developmental toxicity study, the oral administration of 2-Mercaptoethanol to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 5, 15, and 25 mg/kg bw/d caused neither maternal toxicity nor prenatal developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) of 2-Mercaptoethanol for maternal and prenatal developmental toxicity is at least 25 mg/kg bw/d.

Mode of Action Analysis / Human Relevance Framework

Martin and Schild (1965) demonstrated that certain thiols, including 2-mercaptoethanol, act as specific pharmacological antagonists of disulphide polypeptides when tested on isolated preparations of uterus and mammary gland. The observed antagonistic effect was not attributed to chemical destruction since it is also exhibited by a thiol such as thioglycerol which does not inactivate disulphide polypeptides. The effect was not present in other stimulants such as bradykin or acetylcholine. It was therefore concluded, employing a functional definition of receptors, that the effect is exerted at the receptor level. There is evidence that the antagonism between thiols and disulphide polypeptides is not typically competitive. It may be caused by a reversible inactivation of essential disulphide groups in the receptor.

Justification for classification or non-classification

Thiols have been known to produce specific pharmacological antagonism of oxytocin by breaking its disulphide bonds or by reversibly antagonizing the Oxytocin receptor (Martin and Schild, 1965). Oxytocin is a mammalian neuropeptide secreted primarily by the posterior pituitary gland and is critical to the normal progress of parturition and sustaining sufficient uterine contractions during Iabor to expel the fetus(es) and to ligate severed blood vessels within the contracted myometrium after the placenta separates, thus preventing hemorrhage. The dose-related mortality and morbidity in the pregnant female rats after administration of the read across substance 2-mercaptoethanol (≥ 50 mg/kg bw/day) was not a gender-specific effect or the result of other reported treatment-related effects, but most likely a manifestation of delivery related difficulties at the time of parturition. In general, the classification criteria for reproductive toxicity according to Regulation EC 1272/2008 (CLP) are met. Repeated oral administration of the read acorss substance 2-mercaptoethanol resulted in clinical symptoms (excessive salivation, decreased body weight in males) and effects on the liver (organ weight increased and vacuolated liver cells in both genders) and the heart (degenerative cardiomyopathy in females at ≥ 50 mg/kg bw/day, in males at 75 mg/kg bw/day). Thus, the read across substance 2-mercaptoethanol is classified for Specific Target Organ Toxicity (STOT RE cat.2; target organs: liver, heart) based on the effects observed at ≥ 50 mg/kg bw/day. In conclusion, the mortality and morbidity in the pregnant female rats due to delivery related difficulties at the time of parturition is not an isolated effect and was accompanied by additional systemic toxicity which requires classification for Specific Target Organ Toxicity. According to chapter 3.7.2.2.1. of Regulation EC 1272/2008 (CLP), classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction. Therefore, classification with category 2 for reproductive toxicity (H361f) is considered the most appropriate in line with the criteria laid down in Regulation EC 1272/2008 (CLP). The same classification (Repro Cat. 2, H361f) thus also applies to sodium 2 -mercaptoethanolate.

Additional information